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In this study, we aimed to evaluate the effects of exenatide (EXE) treatment on exocrine pancreas of nonhuman primates. To this end, 52 baboons (Papio hamadryas) underwent partial pancreatectomy, followed by continuous infusion of EXE or saline (SAL) for 14 weeks. Histological analysis, immunohistochemistry, Computer Assisted Stereology Toolbox morphometry, and immunofluorescence staining were performed at baseline and after treatment. The EXE treatment did not induce pancreatitis, parenchymal or periductal inflammatory cell accumulation, ductal hyperplasia, or dysplastic lesions/pancreatic intraepithelial neoplasia. At study end, Ki-67-positive (proliferating) acinar cell number did not change, compared with baseline, in either group. Ki-67-positive ductal cells increased after EXE treatment (P = 0.04). However, the change in Ki-67-positive ductal cell number did not differ significantly between the EXE and SAL groups (P = 0.13). M-30-positive (apoptotic) acinar and ductal cell number did not change after SAL or EXE treatment. No changes in ductal density and volume were observed after EXE or SAL. Interestingly, by triple-immunofluorescence staining, we detected c-kit (a marker of cell transdifferentiation) positive ductal cells co-expressing insulin in ducts only in the EXE group at study end, suggesting that EXE may promote the differentiation of ductal cells toward a β-cell phenotype. In conclusion, 14 weeks of EXE treatment did not exert any negative effect on exocrine pancreas, by inducing either pancreatic inflammation or hyperplasia/dysplasia in nonhuman primates., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Microglia/macrophages (M) are major contributors to postinjury inflammation, but they may also promote brain repair in response to specific environmental signals that drive classic (M1) or alternative (M2) polarization. We investigated the activation and functional changes of M in mice with traumatic brain injuries and receiving intracerebroventricular human bone marrow mesenchymal stromal cells (MSCs) or saline infusion. MSCs upregulated Ym1 and Arginase-1 mRNA (p < 0.001), two M2 markers of protective M polarization, at 3 and 7 d postinjury, and increased the number of Ym1(+) cells at 7 d postinjury (p < 0.05). MSCs reduced the presence of the lysosomal activity marker CD68 on the membrane surface of CD11b-positive M (p < 0.05), indicating reduced phagocytosis. MSC-mediated induction of the M2 phenotype in M was associated with early and persistent recovery of neurological functions evaluated up to 35 days postinjury (p < 0.01) and reparative changes of the lesioned microenvironment. In vitro, MSCs directly counteracted the proinflammatory response of primary murine microglia stimulated by tumor necrosis factor-α + interleukin 17 or by tumor necrosis factor-α + interferon-γ and induced M2 proregenerative traits, as indicated by the downregulation of inducible nitric oxide synthase and upregulation of Ym1 and CD206 mRNA (p < 0.01). In conclusion, we found evidence that MSCs can drive the M transcriptional environment and induce the acquisition of an early, persistent M2-beneficial phenotype both in vivo and in vitro. Increased Ym1 expression together with reduced in vivo phagocytosis suggests M selection by MSCs towards the M2a subpopulation, which is involved in growth stimulation and tissue repair.

After brain stroke microglia/macrophages (M/M) undergo rapid activation with dramatic morphological and phenotypic changes that include expression of novel surface antigens and production of mediators that build up and maintain the inflammatory response. Emerging evidence indicates that M/M are highly plastic cells that can assume classic pro-inflammatory (M1) or alternative anti-inflammatory (M2) activation after acute brain injury. However a complete characterization of M/M phenotype marker expression, their colocalization and temporal evolution in the injured brain is still missing. Immunofluorescence protocols specifically staining relevant markers of M/M activation can be performed in the ischemic brain. Here we present immunofluorescence-based protocols followed by three-dimensional confocal analysis as a powerful approach to investigate the pattern of localization and co-expression of M/M phenotype markers such as CD11b, CD68, Ym1, in mouse model of focal ischemia induced by permanent occlusion of the middle cerebral artery (pMCAO). Two-dimensional analysis of the stained area reveals that each marker is associated to a defined M/M morphology and has a given localization in the ischemic lesion. Patterns of M/M phenotype marker co-expression can be assessed by three-dimensional confocal imaging in the ischemic area. Images can be acquired over a defined volume (10 μm z-axis and a 0.23 μm step size, corresponding to a 180 x 135 x 10 μm volume) with a sequential scanning mode to minimize bleed-through effects and avoid wavelength overlapping. Images are then processed to obtain three-dimensional renderings by means of Imaris software. Solid view of three dimensional renderings allows the definition of marker expression in clusters of cells. We show that M/M have the ability to differentiate towards a multitude of phenotypes, depending on the location in the lesion site and time after injury.

The role of tumor necrosis factor (TNF) and its receptors after traumatic brain injury (TBI) remains unclear. We evaluated the effects of genetic deletion of either p55 or p75 TNF receptor on neurobehavioral outcome, histopathology, DNA damage and apoptosis-related cell death/survival gene expression (bcl-2/bax), and microglia/macrophage (M/M) activation in wild-type (WT) and knockout mice after TBI. Injured p55 (-/-) mice showed a significant attenuation while p75 (-/-) mice showed a significant worsening of sensorimotor deficits compared with WT mice over 4 weeks postinjury. At the same time point, contusion volume in p55 (-/-) mice (11.1±3.3 mm(3)) was significantly reduced compared with WT (19.7±3.4 mm(3)) and p75 (-/-) mice (20.9±3.2 mm(3)). At 4 hours postinjury, bcl-2/bax ratio mRNA expression was increased in p55 (-/-) compared with p75 (-/-) mice and was associated with reduced DNA damage terminal deoxynucleotidyl transferaseYmediated dUTP nick end labeling (TUNEL-positivity), reduced CD11b expression and increased Ym1 expression at 24 hours postinjury in p55 (-/-) compared with p75 (-/-) mice, indicative of a protective M/M response. These data suggest that TNF may exacerbate neurobehavioral deficits and tissue damage via p55 TNF receptor whose inhibition may represent a specific therapeutic target after TBI.

The studies on fractalkine and its unique receptor CX3CR1 in neurological disorders yielded contrasting results. We have explored the consequences of CX3CR1 deletion in ischemic (30' MCAo) mice on: (1) brain infarct size; (2) microglia dynamism and morphology; (3) expression of markers of microglia/macrophages (M/M) activation and polarization. We observed smaller infarcts in cx3cr1(-/-) (26.42 ± 7.41 mm(3) , mean ± sd) compared to wild type (36.29 ± 11.57) and cx3cr1(-/+) (34.49 ± 8.91) mice. We longitudinally analyzed microglia by in vivo two-photon microscopy before, 1 and 24 h after transient ischemia. Microglia were stationary in both cx3cr1(-/-) and cx3cr1(-/+) mice throughout the study. In cx3cr1(-/-) mice, they displayed a significantly higher number of ramifications >10 μm at baseline and at 24 h after ischemia compared to cx3cr1(-/+) mice, indicating that CX3CR1 deficiency impaired the development of microglia hypertrophic/amoeboid morphology. At 24 h after ischemia, we performed post mortem quantitative immunohistochemistry for different M/M markers. In cx3cr1(-/-) immunoreactivity for CD11b (M/M activation) and for CD68 (associated with phagocytosis) were decreased, while that for CD45(high) (macrophage and leukocyte recruitment) was increased. In addition, immunoreactivity for Ym1 (M2 polarization) was enhanced, while that for iNOS (M1) was decreased. Our data show that in cx3cr1(-/-) mice protection from ischemia at early time points after injury is associated with a protective inflammatory milieu, characterized by the promotion of M2 polarization markers., (Copyright © 2013 Wiley Periodicals, Inc.)

The discovery of ordered mesoporous materials has opened great opportunities for new applications in heterogeneous catalysis, thanks to their hitherto unprecedented intrinsic structural features. Evidence shows that, however, these materials have not met the researchers' expectations mainly because of the severe limitations related to the strength of acid sites and to the thermal/hydrothermal stability, significantly lower than those of zeolites and due to the amorphous nature of the mesostructured materials. These features are highlighted in the first part of this review, where the peculiarities of mesostructured materials are compared with those of zeolite catalysts in some reactions of industrial interest. New synthesis strategies, especially designed for preparing materials with improved physico-chemical and textural properties, together with the catalytic features of the resulting materials, are described and discussed in the second part of the review.

Erratic regulation of glucose metabolism including hyperglycemia is a common condition in premature infants and is associated with increased morbidity and mortality. The objective of this study was to examine histological and ultrastructural differences in the endocrine pancreas in fetal (throughout gestation) and neonatal baboons. Twelve fetal baboons were delivered at 125 days (d) gestational age (GA), 140d GA, or 175d GA. Eight animals were delivered at term (185d GA); half were fed for 5 days. Seventy-three nondiabetic adult baboons were used for comparison. Pancreatic tissue was studied using light microscopy, confocal imaging, and electron microscopy. The fetal and neonatal endocrine pancreas islet architecture became more organized as GA advanced. The percent areas of α-β-δ-cell type were similar within each fetal and newborn GA (NS) but were higher than the adults (P<0.05) regardless of GA. The ratio of β cells within the islet (whole and core) increased with gestation (P<0.01). Neonatal baboons, which survived for 5 days (feeding), had a 2.5-fold increase in pancreas weight compared with their counterparts killed at birth (P=0.01). Endocrine cells were also found in exocrine ductal and acinar cells in 125, 140 and 175d GA fetuses. Subpopulation of tissue that coexpressed trypsin and glucagon/insulin shows the presence of cells with mixed endo-exocrine lineage in fetuses. In summary, the fetal endocrine pancreas has no prevalence of a α-β-δ-cell type with larger endocrine cell percent areas than adults. Cells with mixed endocrine/exocrine phenotype occur during fetal development. Developmental differences may play a role in glucose homeostasis during the neonatal period and may have long-term implications.

In the present article, we propose the perspective that abnormal glutamate homeostasis might contribute to diabetes pathogenesis. Previous reports and our recent data indicate that chronically high extracellular glutamate levels exert direct and indirect effects that might participate in the progressive loss of β-cells occurring in both T1D and T2D. In addition, abnormal glutamate homeostasis may impact all the three accelerators of the "accelerator hypothesis" and could partially explain the rising frequency of T1D and T2D.

Recent evidence shows that neurotransmitters (e.g., GABA, Ach, adenosine, glutamate) are active on Schwann cells, which form myelin sheaths in the peripheral nervous system under different pathophysiologic conditions. Glutamate, the most important excitatory neurotransmitter, has been recently involved in peripheral neuropathies, thus prevention of its toxic effect is desirable to preserve the integrity of peripheral nervous system and Schwann cells physiology. Removal of glutamate from the extracellular space is accomplished by the high affinity glutamate transporters, so we address our studies to analyze their functional presence in Schwann cells. We first demonstrate that Schwann cells express the EAAC1 transporter in the plasma membrane and in intracellular vesicular compartments of the endocytic recycling pathways. Uptake experiments confirm its presence and functional activity in Schwann cells. Secondly, we demonstrate that the EAAC1 activity can be modulated by exposure to the neurosteroid allopregnanolone 10 nM (a progesterone metabolite proved to support Schwann cells). Transporter up-regulation by allopregnanolone is rapid, does not involve protein neo-synthesis and is prevented by actin depolymerization. Allopregnanolone modulation involves GABA-A receptor and PKC activation, promotes the exocytosis of the EAAC1 transporter from intracellular stores to the Schwann cell membrane, in actin-rich cell tips, and modifies the morphology of cell processes. Finally, we provide evidence that glutamate transporters control the allopregnanolone-mediated effects on cell proliferation. Our findings are the first to demonstrate the presence of a functional glutamate uptake system, which can be dynamically modulated by allopregnanolone in Schwann cells. Glutamate transporters may represent a potential therapeutic target to control Schwann cell physiology., (Copyright © 2011 Wiley Periodicals, Inc.)

Metastable TiO(2) polymorphs are more promising materials than rutile for specific applications such as photocatalysis or catalysis support. This was clearly demonstrated for the anatase phase but still under consideration for brookite, which is difficult to obtain as pure phase. Moreover, the surface doping of anatase with lanthanum ions is known to both increase the thermal stability of the metastable phase and improve its photocatalytic activity. In this study, TiO(2) nanoparticles of almost only the brookite structure were prepared by a simple sol-gel procedure in aqueous solution. The nanoparticles were then doped with lanthanum(III) ions. The thermal stability of the nanoparticles was analyzed by X-ray diffraction and kinetic models were successfully applied to quantify phases evolutions. The presence of surface-sorbed lanthanum(III) ions increased the phase stability of at least 200 °C and this temperature shift was attributed to the selective phase stabilization of metastable TiO(2) polymorphs. Moreover, the combination of the surface doping ions and the thermal treatment induces the vanishing of the secondary anatase phase, and the photocatalytic tests on the doped brookite nanoparticles demonstrated that the doping increased photocatalytic activity and that the extent depended on the duration of the sintering treatment.

We report a case of unusually long-lasting remission of type 1 diabetes (T1D). The patient, a Caucasian man, at the age of 43 years developed a ketotic diabetes, classified as type 1 based on clinical presentation and positivity for islet autoantibodies. Shortly after diabetes onset, oral topiramate was added to preexisting valproic acid for generalized seizures and maintained thereafter. Initial intensive insulin treatment was rapidly reduced to low doses (3 Units/day) maintained for a long time and then discontinued at month 55; fasting glucose and glycosylated hemoglobin were basically normalized at 58 months. An oral glucose tolerance test performed at month 53 showed an impaired fasting glucose (6.0 mmol/l) and a value slightly above the threshold for the diagnosis of diabetes at 2 h (11.2 mmol/l). We hypothesize that this unusually prolonged preservation of β-cell function might be ascribed to the concomitant therapy with topiramate, an antiepileptic agent with demonstrated efficacy as antidiabetic in type 2 diabetes (T2D). Topiramate should be further investigated as candidate agent for the preservation of β-cell function also in T1D.

The chemokine CX3CL1 and its receptor CX3CR1 are constitutively expressed in the nervous system. In this study, we used in vivo murine models of permanent middle cerebral artery occlusion (pMCAO) to investigate the protective potential of CX3CL1. We report that exogenous CX3CL1 reduced ischemia-induced cerebral infarct size, neurological deficits, and caspase-3 activation. CX3CL1-induced neuroprotective effects were long lasting, being observed up to 50 d after pMCAO in rats. The neuroprotective action of CX3CL1 in different models of brain injuries is mediated by its inhibitory activity on microglia and, in vitro, requires the activation of adenosine receptor 1 (A₁R). We show that, in the presence of the A₁R antagonist 1,3-dipropyl-8-cyclopentylxanthine and in A₁R⁻/⁻ mice, the neuroprotective effect of CX3CL1 on pMCAO was abolished, indicating the critical importance of the adenosine system in CX3CL1 protection also in vivo. In apparent contrast with the above reported data but in agreement with previous findings, cx3cl1⁻/⁻ and cx3cr1(GFP/GFP) mice, respectively, deficient in CX3CL1 or CX3CR1, had less severe brain injury on pMCAO, and the administration of exogenous CX3CL1 increased brain damage in cx3cl1⁻/⁻ ischemic mice. We also report that CX3CL1 induced a different phagocytic activity in wild type and cx3cl1⁻/⁻ microglia in vitro during cotreatment with the medium conditioned by neurons damaged by oxygen-glucose deprivation. Together, these data suggest that acute administration of CX3CL1 reduces ischemic damage via an adenosine-dependent mechanism and that the absence of constitutive CX3CL1-CX3CR1 signaling changes the outcome of microglia-mediated effects during CX3CL1 administration to ischemic brain.

A growing body of evidence suggests that oxidative stress plays a key role in the pathogenesis of micro- and macrovascular diabetic complications. The increased oxidative stress in subjects with type 2 diabetes is a consequence of several abnormalities, including hyperglycemia, insulin resistance, hyperinsulinemia, and dyslipidemia, each of which contributes to mitochondrial superoxide overproduction in endothelial cells of large and small vessels as well as the myocardium. The unifying pathophysiological mechanism that underlies diabetic complications could be explained by increased production of reactive oxygen species (ROS) via: (1) the polyol pathway flux, (2) increased formation of advanced glycation end products (AGEs), (3) increased expression of the receptor for AGEs, (4) activation of protein kinase C isoforms, and (5) overactivity of the hexosamine pathway. Furthermore, the effects of oxidative stress in individuals with type 2 diabetes are compounded by the inactivation of two critical anti-atherosclerotic enzymes: endothelial nitric oxide synthase and prostacyclin synthase. Of interest, the results of clinical trials in patients with type 2 diabetes in whom intensive management of all the components of the metabolic syndrome (hyperglycemia, hypercholesterolemia, and essential hypertension) was attempted (with agents that exert a beneficial effect on serum glucose, serum lipid concentrations, and blood pressure, respectively) showed a decrease in adverse cardiovascular end points. The purpose of this review is (1) to examine the mechanisms that link oxidative stress to micro- and macrovascular complications in subjects with type 2 diabetes and (2) to consider the therapeutic opportunities that are presented by currently used therapeutic agents which possess antioxidant properties as well as new potential antioxidant substances.

We investigated the occurrence of endotoxin (lipopolysaccharide, LPS) preconditioning in traumatic brain injury (TBI), evaluating the time window of LPS-induced protection, its persistence, and the associated molecular mechanisms. Mice received 0.1 mg/kg LPS or saline intraperitoneally and subsequently TBI (by controlled cortical impact brain injury) at various time intervals. Mice receiving LPS 3, 5, or 7 days before TBI showed attenuated motor deficits at 1 week after injury compared with mice receiving saline. Those receiving LPS 5 days before injury had also a reduced contusion volume (7.9±1.3 versus 12±2.3 mm(3)) and decreased cell death. One month after injury, the protective effect of LPS on contusion volume (14.5±1.2 versus 18.2±1.2 mm(3)) and neurologic function was still present. Traumatic brain injury increased glial fibrillary acidic protein, CD11b, CD68, tumor necrosis factor-α, interleukin (IL)-10, and IL-6 mRNA expression 24 hours after injury. Lipopolysaccharide administered 5 (but not 9) days before injury increased the expression of CD11b (233%) and of interferon β (500%) in uninjured mice, while it reduced the expression of CD68 (by 46%) and increased that of IL-6 (by 52%) in injured mice. Lipopolysaccharide preconditioning conferred a long-lasting neuroprotection after TBI, which was associated with a modulation of microglia/macrophages activity and cytokine production.

Glutamate is the major excitatory neurotransmitter of the central nervous system (CNS) and may induce cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its extracellular concentration is maintained at physiological concentrations by high affinity glutamate transporters of the solute carrier 1 family (SLC1). Glutamate is also present in islet of Langerhans where it is secreted by the α-cells and acts as a signaling molecule to modulate hormone secretion. Whether glutamate plays a role in islet cell viability is presently unknown. We demonstrate that chronic exposure to glutamate exerts a cytotoxic effect in clonal β-cell lines and human islet β-cells but not in α-cells. In human islets, glutamate-induced β-cell cytotoxicity was associated with increased oxidative stress and led to apoptosis and autophagy. We also provide evidence that the key regulator of extracellular islet glutamate concentration is the glial glutamate transporter 1 (GLT1). GLT1 localizes to the plasma membrane of β-cells, modulates hormone secretion, and prevents glutamate-induced cytotoxicity as shown by the fact that its down-regulation induced β-cell death, whereas GLT1 up-regulation promoted β-cell survival. In conclusion, the present study identifies GLT1 as a new player in glutamate homeostasis and signaling in the islet of Langerhans and demonstrates that β-cells critically depend on its activity to control extracellular glutamate levels and cellular integrity.

This study was designed to test the hypothesis that improved mitochondrial biogenesis could help reducing ischemic cerebral injury. We found that levels of proliferator-activated receptor γ coactivator 1α and nuclear respiratory factor-1, mitochondrial DNA content and other markers of mitochondrial biogenesis and function were reduced in primary mouse cortical neurons under oxygen-glucose deprivation (OGD). The glycogen synthase kinase-3 (GSK-3) inhibitor SB216763 activated an efficient mitochondrial biogenesis program in control cortical neurons and counteracted the OGD-mediated mitochondrial biogenesis impairment. This was accompanied by the activation of an antioxidant response that reduced mitochondrial reactive oxygen species generation and ischemic neuronal damage. The in vitro effects of SB216763 were mimicked by two other structurally unrelated GSK-3 inhibitors. The protective effects of SB216763 on OGD-mediated neuronal damage were abolished in the presence of diverse mitochondrial inhibitors. Finally, when systemically administered in vivo, SB216763 reduced the infarct size and recovered the loss of mitochondrial DNA in mice subjected to permanent middle cerebral artery occlusion. We conclude that GSK-3 inhibition by SB216763 might pave the way of novel promising therapies aimed at stimulating the renewal of functional mitochondria and reducing reactive oxygen species-mediated damage in ischemic stroke., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Insulin resistance, reduced β-cell mass, and hyperglucagonemia are consistent features in type 2 diabetes mellitus (T2DM). We used pancreas and islets from humans with T2DM to examine the regulation of insulin signaling and cell-cycle control of islet cells. We observed reduced β-cell mass and increased α-cell mass in the Type 2 diabetic pancreas. Confocal microscopy, real-time PCR and western blotting analyses revealed increased expression of PCNA and down-regulation of p27-Kip1 and altered expression of insulin receptors, insulin receptor substrate-2 and phosphorylated BAD. To investigate the mechanisms underlying these findings, we examined a mouse model of insulin resistance in β-cells--which also exhibits reduced β-cell mass, the β-cell-specific insulin receptor knockout (βIRKO). Freshly isolated islets and β-cell lines derived from βIRKO mice exhibited poor cell-cycle progression, nuclear restriction of FoxO1 and reduced expression of cell-cycle proteins favoring growth arrest. Re-expression of insulin receptors in βIRKO β-cells reversed the defects and promoted cell cycle progression and proliferation implying a role for insulin-signaling in β-cell growth. These data provide evidence that human β- and α-cells can enter the cell-cycle, but proliferation of β-cells in T2DM fails due to G1-to-S phase arrest secondary to defective insulin signaling. Activation of insulin signaling, FoxO1 and proteins in β-cell-cycle progression are attractive therapeutic targets to enhance β-cell regeneration in the treatment of T2DM.

The glutamate transporter excitatory amino acid carrier (EAAC1/EAAT3) mediates the absorption of dicarboxylic amino acids in epithelial cells as well as the uptake of glutamate from the synaptic cleft. Its cell-surface density is regulated by interaction with accessory proteins which remain to be identified. We detected a consensus sequence for interaction with post-synaptic density-95/Discs large/Zonula occludens (PDZ) proteins (-SQF) and a tyrosine-based internalization signal (-YVNG-) in the C-terminus of EAAC1, and investigated their role in the transporter localization. We demonstrated that PDZ interactions are required for the efficient delivery to and the retention in the plasma membrane of EAAC1 and we identified PDZK1/NHERF3 (Na+/H+-exchanger regulatory factor 3) as a novel EAAC1 interacting protein. Expression of PDZK1 in Madin-Darby canine kidney (MDCK) cells tethered EAAC1 to filopodia and increased its surface activity. Removal of the PDZ-target motif promoted the EAAC1 binding to α-adaptin and clathrin and the transporter internalization in endocytic/degradative compartments. This defect was largely prevented by hypertonic treatment or overexpression of the dominant-negative µ2-W421A-subunit of AP-2 clathrin-adaptor. The rate of transporter endocytosis was attenuated following tyrosine mutagenesis in the internalization signal, thus indicating that this motif can regulate the transporter endocytosis. We suggest that EAAC1 density is controlled by balanced interactions with PDZK1 and adaptor protein 2 (AP2): the former promotes the transporter expression at the cell surface, and the latter mediates its constitutive endocytosis., (© 2010 John Wiley & Sons A/S.)

Several different technologies are available for H(2)S removal from the gas stream of medium capacity. Among them, the most widely used is Locat with more than 120 plants worldwide. In the last decade, many new processes, such as Sulfatreat-DO, Crystasulf, Caltech, and UCSR, were proposed to overcome the drawbacks of the state-of-the-art processes (low sulfur purity, chemical degradation, thiosulfate formation). We have developed a new H(2)S conversion method based on acid ferric nitrate solution, co-catalyzed by a heteropolyacid. H(2)S was converted to pure sulfur (>99.9 %), with no traces of organic compounds. Due to the acid pH of the solution, no chelant or surfactant was needed and iron content in the solution could reach very high levels. Keggin heteropolyacid (H(6)PW(9)V(3)O(40)) catalyzed the reoxidation of reduced ferrous solution with air at mild temperature and at very high reaction rate. The undesired side reaction (NO(x) formation) could be avoided by simply increasing the oxygen partial pressure.

Ac-SDKP (N-acetyl-seryl-aspartyl-lysyl-proline) is a physiological tetrapeptide hydrolysed by ACE (angiotensin-converting enzyme). In experimental models of hypertension, Ac-SDKP has antifibrotic effects in the heart; however, the role of Ac-SDKP in diabetic cardiomyopathy is currently unknown. The aim of the present study was to evaluate the effect of Ac-SDKP on cardiac systolic and diastolic function, and interstitial and perivascular fibrosis in the heart of diabetic rats.Diabetes was induced in 55 Sprague-Dawley rats by streptozotocin injection. Control rats (n=18)underwent only buffer injection.Out of the 55 diabetic rats, 19 were chronically treated with insulin and 13 with the ACEI (ACE inhibitor) ramipril (3 mg x kg(-1 )of body weight x day(-1)). At 2 months after the onset of diabetes, Ac-SDKP (1 mg x kg(-1) of body weight x day(-1)) was administered by osmotic minipumps for 8 weeks to eight control rats, 13 diabetic rats, seven diabetic rats treated with ramipril and nine insulin-treated diabetic rats. Diabetic rats had a significant increase in blood glucose levels. Left ventricular interstitial and perivascular fibrosis, and TGF-beta1 (transforming growth factor-beta1) protein levels were increased in diabetic rats, but not in insulin-treated diabetic rats and ramipril-treated diabetic rats, compared with control rats. Ac-SDKP administration significantly reduced left ventricular interstitial and perivascular fibrosis in diabetic rats and in diabetic rats treated with ramipril. This was accompanied by a significant reduction in active TGF-beta1 and phospho-Smad2/3 protein levels in myocardial tissue of diabetic rats. Echocardiography showed that diabetes was associated with increased end-systolic diameters, and depressed global systolic function and diastolic dysfunction, as assessed by transmitral Doppler velocity profile. These changes were completely reversed by insulin or ramipril treatment. Ac-SDKP treatment partially restored diastolic function in diabetic rats. In conclusion, Ac-SDKP administration in diabetic rats reduces left ventricular interstitial and perivascular fibrosis, active TGF-beta1 and phospho-Smad2/3levels, and improves diastolic function. Taken together, these findings suggest that, by inhibiting theTGF-beta/Smad pathway, Ac-SDKP protects against the development of diabetic cardiomyopathy

The c-Jun N-terminal kinase (JNK) pathway is involved in cell stress and apoptosis. We tested the hypothesis that this pathway plays a role in traumatic brain injury (TBI) by assessing JNK activation in human brain tissues and in brains of mice subjected to controlled cortical impact brain injury. We also assessed the effects of specific inhibition of the JNK pathway by the cell-permeable JNK inhibitor peptide, D-JNKI1, on neurobehavioral function and posttraumatic cell loss in mice. The inhibitor was administered intraperitoneally 10 minutes after injury. The JNK pathway showed robust activation both in human contusion specimens and in injured cortex and hippocampi of TBI-injured mice, 1, 4, and 48 hours after injury. D-JNKI1 treatment significantly improved motor performance at 48 hours and 7 days after injury and reduced the contusion volume compared with saline treatment; the numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells were significantly decreased in the hippocampi of injured mice 48 hours after treatment. Thus, because the JNK pathway is activated after human and experimental TBI and the inhibitor peptide D-JNKI1 affords significant neuroprotection and amelioration of neurobehavioral deficits after experimental TBI, therapeutic targeting of the JNK activation pathway may hold promise for future clinical applications.

beta-Cell dysfunction is an important factor in the development of hyperglycemia of type-2 diabetes mellitus, and pancreatic islet amyloidosis (IA) has been postulated to be one of the main contributors to impaired insulin secretion. The aim of this study was to evaluate the correlation of IA with metabolic parameters and its effect on islets of Langerhans remodeling and relative endocrine-cell volume in baboons. We sequenced the amylin peptide, determined the fibrillogenic propensities, and evaluated pancreatic histology, clinical and biochemical characteristics, and endocrine cell proliferation and apoptosis in 150 baboons with different metabolic status. Amylin sequence in the baboon was 92% similar to humans and showed superimposable fibrillogenic propensities. IA severity correlated with fasting plasma glucose (FPG) (r = 0.662, P < 0.001) and HbA1c (r = 0.726, P < 0.001), as well as with free fatty acid, glucagon values, decreased homeostasis model assessment (HOMA) insulin resistance, and HOMA-B. IA severity was associated with a decreased relative beta-cell volume, and increased relative alpha-cell volume and hyperglucagonemia. These results strongly support the concept that IA and beta-cell apoptosis in concert with alpha-cell proliferation and hypertrophy are key determinants of islets of Langerhans "dysfunctional remodeling" and hyperglycemia in the baboon, a nonhuman primate model of type-2 diabetes mellitus. The most important determinants of IA were age and FPG (R(2) = 0.519, P < 0.0001), and different FPG levels were sensitive and specific to predict IA severity. Finally, a predictive model for islet amyloid severity was generated with age and FPG as required variables.

Background: AQP1 belongs to aquaporins family, water-specific, membrane-channel proteins expressed in diverse tissues. Recent papers showed that during angiogenesis, AQP1 is expressed preferentially by microvessels, favoring angiogenesis via the increase of permeability In particular, in AQP1 null mice, endothelial cell migration is impaired without altering their proliferation or adhesion. Therefore, AQP1 has been proposed as a novel promoter of tumor angiogenesis., Methods/findings: Using targeted silencing of AQP1 gene expression, an impairment in the organization of F-actin and a reduced migration capacity was demonstrated in human endothelial and melanoma cell lines. Interestingly, we showed, for the first time, that AQP1 co-immunoprecipitated with Lin-7. Lin7-GFP experiments confirmed co-immunoprecipitation. In addition, the knock down of AQP1 decreased the level of expression of Lin-7 and beta-catenin and the inhibition of proteasome contrasted partially such a decrease., Conclusions/significance: All together, our findings show that AQP1 plays a role inside the cells through Lin-7/beta-catenin interaction. Such a role of AQP1 is the same in human melanoma and endothelial cells, suggesting that AQP1 plays a global physiological role. A model is presented.

In this study, we examined the role of the L27 [(LIN2-LIN7) domain] and PDZ domain (domain previously found in PSD95-DlgA-ZO-1) for protein-protein interaction of the scaffold protein LIN7 in tight junction (TJ) assembly in Madin-Darby canine kidney (MDCK) cells and found that the stable expression of a LIN7 mutant lacking the L27 domain (DeltaL27 mutant) acts as a dominant interfering protein by inhibiting TJ localization of endogenous LIN7. The loss of LIN7 did not alter the localization of the PALS1 (protein associated with LIN7) partner of the L27 domain but prevented TJ localization of the insulin receptor substrate p53 (IRSp53), a partner of the PDZ domain of LIN7. The function of both L27 and PDZ domains of LIN7 in IRSp53 localization to TJs has been further demonstrated by reducing the expression of LIN7 (LIN7 small hairpin RNA experiments) and by expression of IRSp53 deleted of its motif for PDZ interaction (IRSp53Delta5) or fused to the L27 domain of LIN7 (L27-IRSp53Delta5). Cell lines with decreased localization of LIN7 and IRSp53 to TJs showed defects during assembly of TJs and cyst polarization and failed to activate Rac1, a member of the Rho guanosine triphosphatases family crucially involved in actin organization and orientation of apicobasal polarity. These data therefore indicate that LIN7-IRSp53 association plays a role during assembly of functional TJs and surface polarization in epithelial cells.

Background: Tumor necrosis factor (TNF)-alpha has been suggested to play both a deleterious and beneficial role in neurobehavioral dysfunction and recovery following traumatic brain injury (TBI). The goal of this study was to evaluate the specific role of tumor necrosis factor (TNF) receptors p55 and p75 in mediating cognitive outcome following controlled cortical impact (CCI) brain injury by comparing post-traumatic cognitive function in mice with genetically engineered deletion of the gene for either p55 (-/-) or p75 (-/-) receptors., Method: Male C57B1/6 mice (WT, n=29), and mice genetically engineered to delete p55 TNF (p55 (-/-), n=8) or p75 TNF (p75 (-/-), n=23) receptors were used. They were anesthetized with intraperitoneal (i.p.) administration of sodium pentobarbital (65 mg/kg) and subjected to CCI brain injury of moderate severity. Sham-injured control mice were anesthetized and surgically prepared similarly but they received no impact. Assessment of mRNA expression of inflammatory, proapoptotic and antiapoptotic genes was done by real time-polymerase chain reaction (RT-PCR). Cognitive outcome was evaluated at 4 weeks postinjury using the Morris water maze (MWM)., Findings: mRNA expression of inflammatory, proapoptotic and antiapoptotic genes prior to TBI did not reveal any baseline difference between p55 and p75 (-/-) mice. WT mice showed greater baseline expression of inflammatory genes. The learning ability of p55 (-/-) brain-injured mice was significantly better than that observed in p75 (-/-) brain-injured mice (p < 0.05). Cognitive learning in WT control mice fell between the p55 (-/-) and p75 (-/-) mice., Conclusions: These data suggest that TNF-alpha may both exacerbate cognitive dysfunction via p55 receptor and attenuate it via p75 receptor.

To learn more about nonimmune-mediated islet graft failure, we transplanted different preparations (preps) of isolated human islets under the kidney capsule of streptozotocin (STZ)-diabetic nude mice. One month after the implantation of 1,000 or 2,000 islets, grafts were harvested for morphological, immunohistochemical, and ultrastructural analysis. Only a single islet prep cured the diabetes out of all the recipients, while the remaining preps showed only partial function after the implantation of 2,000 islets. Transplanted mice showed high circulating proinsulin levels but, with the exclusion of those bearing curative grafts, relatively low mature insulin levels. Engrafted beta-cells showed positive carboxypeptidase E (CPE) and prohormone convertase 1 (PC1) staining, while prohormone convertase 2 (PC2) was undetectable. In contrast, PC2 was abundantly expressed by engrafted alpha-cells. Moreover, engrafted beta-cells did not show evidence of replication, and preapoptotic beta-cells, with intra- and extracellular amyloid deposition, were detected with electron microscopy. Cell cycle inhibitors p16(INK4), p21(WAF1), and p27(Kip1) were abundantly expressed in the islet grafts and showed a predominant nuclear localization. In conclusion, diabetic nude mice transplanted with human islets showed disproportionate hyperproinsulinemia and graft evidence of beta-cell restricted PC2 depletion, amyloid deposition and beta-cell death, and lack of beta-cell replication with nuclear translocation of p27(Kip1) and p21(WAF1) that together may contribute to delayed graft failure.

Background: The goal of the study was to evaluate the effects of Cl-inhibitor (C1-INH), an endogenous glycoprotein endowed with multiple anti-inflammatory actions, on cognitive and histological outcome following controlled cortical impact (CCI) brain injury., Methods: Male C57B1/6 mice (n=48) were subjected to CCI brain injury. After brain injury, animals randomly received an intravenous infusion of either C1-INH (15 U either at 10 minutes or 1 hour postinjury) or saline (equal volume, 150 microl at 10 min postinjury). Uninjured control mice received identical surgery and saline injection without brain injury. Cognitive function was evaluated at 4 weeks postinjury using the Morris Water Maze. Mice were subsequently sacrificed, the brains were frozen and serial sections were cut. Traumatic brain lesion was assessed by dividing the area of the ipsilateral hemisphere for the area of the contralateral one at the level of the injured area of the brain., Findings: Brain-injured mice receiving C1-INH at 10 min postinjury showed attenuated cognitive dysfunction compared to brain-injured mice receiving saline (p < 0.01). These mice also showed a significantly reduced traumatic brain lesion compared to mice receiving saline (p < 0.01). Mice receiving C1-INH at 1 hour post injury did not show a significant improvement in either cognitive or histological outcome. Conclusions Our results suggest that administration of C1-INH at 10 minutes postinjury attenuates cognitive deficits and histological damage associated with traumatic brain injury.

The present study was undertaken to evaluate whether in a neonatal model of stroke a prophylactic neuroprotective treatment with simvastatin modulates hypoxia-ischemia-induced inflammatory and apoptotic signaling. Procaspase-3 and cleaved caspase-3 expression showed a peak at 24 h and returned to control values after 5 days. Caspase-3 activity followed the same pattern of caspase-3 proteolytic cleavage. In simvastatin-treated ischemic animals, the expression of these proteins and caspase-3 activity were significantly lower when compared to that of ischemic animals. alpha-Spectrin and protein kinase C-alpha (PKCalpha) cleavages were not affected by the treatment. Poly (ADP-ribose) polymerase fragmentation, caspase-1 activation, and IL-1beta and ICAM-1 mRNA expression were increased by hypoxia-ischemia and significantly reduced in simvastatin-treated animals. The results indicate that simvastatin-induced attenuation of hypoxia-ischemia brain injury in the newborn rat occurs through reduction of the inflammatory response, caspase-3 activation, and apoptotic cell death.

Background: Pseudomonas aeruginosa is responsible for a wide range of infections. In immunocompromised patients with cancer, the emergence of multidrug resistant P. aeruginosa may have grave consequences., Methods: Patients with cancer who were infected with multidrug-resistant P. aeruginosa with polyclonal DNA restriction patterns were used as the case group. Two control groups were used: one group of cancer patients who were infected with multidrug-susceptible P. aeruginosa and another group of cancer patients who had the same underlying disease and the same intensive care unit exposure as patients in the case group but who were not infected or colonized by P. aeruginosa., Results: Risk factors that were associated significantly with multidrug-resistant P. aeruginosa infection were the use of carbapenem for > or = 7 days, a history of P. aeruginosa infection during the preceding year, and a history of chronic obstructive pulmonary disease (P < 0.01)., Conclusions: Carbapenems may need to be used more judiciously as first-line empirical therapy for cancer patients with prior pseudomonal infection or chronic obstructive pulmonary disease who require hospitalization, and alternative, antipseudomonal antibiotic regimens may need to be considered, especially in this patient population.

Brain inflammatory reactions have been described in various neurological disorders, including epilepsy. Although there is clear evidence that cytokines affect neuroglial functions and blood-brain barrier permeability, scarce information is available on the functional consequences of brain inflammation on seizures. We studied the role of tumor necrosis factor-alpha (TNF)-alpha and its p55 and p75 receptors in seizure modulation. We found that intrahippocampal injection of murine recombinant TNF-alpha potently inhibits seizure in mice while human recombinant TNF-alpha, which shows strong specificity for mouse p55 receptors, was ineffective. p75 receptors were detected in mouse hippocampal neurons, whereas p55 receptors were absent. Transgenic mice with a perturbed TNF-alpha system showed profound alterations in seizure susceptibility: astrocytic overexpression of TNF-alpha was associated with reduced seizures, whereas mice lacking TNF-alpha p75 or both p55 and p75, receptors showed prolonged seizures. Mice deficient in p55 receptor only showed reduced seizures; and both p75 and TNF receptor-associated factor 2 protein levels were upregulated in their hippocampi. Our findings show that increased brain levels of TNF-alpha result in significant inhibition of seizures in mice, and this action is mediated by neuronal p75 receptors. This evidence highlights a novel function of TNF-alpha in brain and indicates a new system for anticonvulsive intervention.

Background: Anecdotal evidence suggests a rise in zygomycosis in association with voriconazole (VRC) use in immunosuppressed patients., Methods: We performed prospective surveillance of patients with zygomycosis (group A; n = 27) and compared them with contemporaneous patients with invasive aspergillosis (group B; n = 54) and with matched contemporaneous high-risk patients without fungal infection (group C; n = 54). We also performed molecular typing and in vitro susceptibility testing of Zygomycetes isolates., Results: Nearly all patients with zygomycosis either had leukemia (n = 14) or were allogeneic bone marrow transplant recipients (n = 13). The Zygomycetes isolates (74% of which were of the genus Rhizopus) had different molecular fingerprinting profiles, and all were VRC resistant. In multivariate analysis of groups A and C, VRC prophylaxis (odds ratio [OR], 10.37 [95% confidence interval [CI]], 2.76-38.97]; P = .001), diabetes (OR, 8.39 [95% CI, 2.04-34.35]; P = .003), and malnutrition (OR, 3.70 [95% CI, 1.03-13.27]; P = .045) were found to be independent risk factors for zygomycosis. Between patients with zygomycosis (after excluding 6 patients with mixed mold infections) and patients with aspergillosis, VRC prophylaxis (OR, 20.30 [95% CI, 3.85-108.15]; P = .0001) and sinusitis (OR, 76.72 [95% CI, 6.48-908.15]; P = .001) were the only factors that favored the diagnosis of zygomycosis., Conclusions: Zygomycosis should be considered in immunosuppressed patients who develop sinusitis while receiving VRC prophylaxis, especially those with diabetes and malnutrition.

Purpose: We investigated the activation of microglia and astrocytes, induction of cytokines, and hippocampal neuronal damage, 4 and 24 h after kainic acid-induced status epilepticus (SE) in postnatal day (PN) 9, 15, and 21 rats., Methods: Limbic seizures were induced by systemic injection of kainic acid. Glia activation and neuronal cell loss were studied by using immunocytochemistry and Western blot. Cytokine expression was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) followed by Southern blot quantification., Results: After SE onset, hippocampal glia activation, cytokine expression, and neuronal damage are all age-dependent phenomena. In the hippocampus, neuronal injury occurs only when cytokines are induced in glia, and cytokine synthesis precedes the appearance of degenerating neurons. Neuronal injury is more pronounced when interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are produced in addition to IL-1beta., Conclusions: This study shows that cytokine induction in rat brain after sustained seizures is age dependent, and it is associated with the appearance of cell injury.

It has been suggested that glutamate-induced excitotoxicity plays a central role in the development of motor neuron diseases such as amyotrophic lateral sclerosis (ALS). The GLT-1 isoform of the glutamate transporter gene family is the most important transporter involved in keeping extracellular glutamate concentration below neurotoxic levels. Its loss and an increase in extracellular glutamate has been documented in cases of sporadic and familial ALS, as well as in animal models expressing ALS-linked Cu2+-Zn2+ superoxide dismutase (SOD1) mutations, but the underlying molecular mechanisms are still unclear. We developed and characterised a cell model consisting of polarised epithelial Madin-Darby Canine Kidney (MDCK) cell lines stably expressing wild-type SOD1 or the ALS-linked SOD1 G93A mutant, and analysed the expression of glutamate transporters after transient transfection of the corresponding cDNAs. Like ALS patients and animal models of ALS, the G93A-expressing MDCK cell system showed reduced total glial GLT-1 expression, with no change in the expression of the neuronal EAAC1 glutamate transporter isoform. Morphological analysis revealed the intracellular redistribution of GLT-1 to acidic compartments, whereas the surface distribution of other glutamate transporters (neuronal EAAC1 and glial GLAST) was not affected. Moreover, mutant SOD1 affected the cytosolic tail of GLT-1 because reduced protein expression of EAAC-GLT but not GLT-EAAC chimeras was found in G93A-expressing cell lines. GLT-1 downregulation was greatly induced by inhibition of protein synthesis, and prevented by treatment with chloroquine aimed at inhibiting the activity of acidic degradative compartments. Negligible effect on the protein level or distribution of GLT-1 was observed in cells overexpressing wild-type SOD1. The specific decrease in the GLT-1 isoform of glutamate transporters is therefore recapitulated in G93A-expressing MDCK cell lines, thus suggesting an autonomous cell mechanism underlying the loss of GLT-1 in ALS. Our data indicate that the continuous expression of mutant SOD1 causes the downregulation of GLT-1 by increasing the internalisation and degradation of the surface transporter, and suggest that the cytosolic tail of GLT-1 is required to target the transporter to degradation.

Background: Invasive aspergillosis (IA) has emerged as a common cause of morbidity and mortality among immunocompromised patients. At The University of Texas M. D. Anderson Cancer Center (Houston, TX), Aspergillus terreus is second to A. fumigatus as the most common cause of IA. In the current study, the authors compared the risk factors and outcomes associated with IA caused by A. terreus and IA caused by A. fumigatus., Methods: The authors retrospectively reviewed the medical records of 300 patients who received care at our institution between 1995 and 2001 and who had cultures that were positive for Aspergillus infection, including 90 patients whose cultures were positive for A. fumigatus and 70 patients whose cultures were positive for A. terreus., Results: Thirty-two patients with IA caused by A. terreus and 33 patients with IA caused by A. fumigatus were evaluated. The two groups were comparable in terms of age, gender, and underlying disease. Leukemia was the most common underlying malignancy (84%). More than 40% of patients in each group had undergone bone marrow transplantation. There was a trend toward a higher frequency of neutropenia among patients with IA caused by A. terreus (P = 0.12). IA caused by A. terreus was considered to be nosocomial in origin significantly more frequently compared with IA caused by A. fumigatus (P = 0.03). In vitro, A. terreus was found to be more resistant to amphotericin B (minimal inhibitory concentration [MIC90], 4.0 microg/mL) than to antifungal therapy (MIC90, 1.0 Hg/mL) in the isolates that were tested (< 50% of all isolates). The overall rate of response to antifungal therapy was 39% for patients with A. fumigatus infection, compared with 28% for patients with A. terreus infection (P = 0.43)., Conclusions: Despite the decreased in vitro susceptibility of A. terreus (relative to A. fumigatus) to amphotericin B, the two groups within the current patient population had comparably poor responses to amphotericin B preparation and somewhat improved responses to posaconazole., ((c) 2004 American Cancer Society.)

Anti-inflammatory properties of pentoxifylline (PTX) have recently been described. Spontaneously hypertensive stroke-prone rats (SHRSP) constitute an animal model that develops an inflammatory condition that precedes the appearance of brain abnormalities. The aim of the present investigation was to assess: 1) the efficacy of PTX treatment in protecting the neural system in SHRSP, and 2) how its anti-inflammatory properties might be involved in this effect. Male SHRSP fed with a permissive diet received no drug or PTX (100 or 200 mg/kg/day). Brain abnormalities detected by magnetic resonance imaging developed spontaneously in control rats after 42 +/- 3 days, whereas in rats treated with 100 mg/kg/day PTX, abnormalities developed in only 80% of the animals and only after 70 to 80 days. Treatment with a higher dose of PTX (200 mg/kg/day) completely protected the brain from abnormal development. The drug treatment prevented the accumulation of macrophages or CD4+ positive cells, the activation of glia in brain tissues, and the appearance of inflammatory proteins and thiobarbituric acid-reactive substances in body fluids. PTX treatment did induce a greater increase of serum tumor necrosis factor-alpha (TNF-alpha), but not of interleukin (IL)-1beta and IL-6 induced by in vivo administration of lipopolysaccharide (LPS), which suggests a protective role for TNF-alpha. PTX also exerted protective effects when it was administered after the first occurrence of proteinuria (>40 mg/day). These data indicate that PTX treatment dose-dependently prevents the occurrence of spontaneous brain damage by reducing inflammatory events. We also hypothesize that the increase of TNF-alpha by PTX treatment represents a protective mechanism in SHRSP.

Objective: To determine the impact of stool surveillance cultures of critically ill patients on controlling vancomycin-resistant enterococci (VRE) outbreak bacteremia., Design: Stool surveillance cultures were performed on patients who had hematologic malignancy or were critically ill at the time of hospital admission to identify those colonized with VRE. Hence, contact isolation was initiated., Setting: A tertiary-care cancer center with a high prevalence of VRE., Participants: All patients with hematologic malignancy who were admitted to the hospital as well as all of those admitted to the intensive care unit were eligible., Results: Active stool surveillance cultures performed between 1997 and 2001 decreased the incidence density of VRE bacteremias eightfold while vancomycin use remained constant. In fiscal year (FY) 1997 and FY 1998, there were five and three VRE outbreak bacteremias, respectively. The outbreak clones were responsible for infection in 69% of those patients with VRE bacteremia. However, the stool surveillance program resulted in the complete control of VRE bacteremia by FY 1999 until the end of the study., Conclusion: Despite the steady use of vancomycin, the active surveillance program among high-risk patients with hematologic malignancy and those who were critically ill resulted in the complete control of VRE outbreak bacteremia at our institution.

We investigated the effect of long-term, peripheral treatment with enoxaparin, a low molecular weight heparin, in transgenic mice overexpressing human amyloid precursor protein(751). Enoxaparin (6 IU per mouse intraperitoneally, three times a week for 6 months) significantly lowered the number and the area occupied by cortical beta-amyloid deposits and the total beta-amyloid (1-40) cortical concentration. Immunocytochemical analysis of glial fibrillary acid protein-positive cells showed that enoxaparin markedly reduced the number of activated astrocytes surrounding beta-amyloid deposits. In vitro, the drug dose-dependently attenuated the toxic effect of beta-amyloid on neuronal cells. Enoxaparin dose-dependently reduced the ability of beta-amyloid to activate complement and contact systems, two powerful effectors of inflammatory response in AD brain. By reducing the beta-amyloid load and cytotoxicity and proinflammatory activity, enoxaparin offers promise as a tool for slowing the progression of Alzheimer's disease.

Recent evidence has shown that proinflammatory and anti-inflammatory molecules are synthesized during epileptic activity in glial cells in CNS regions where seizures initiate and spread. These molecules are released and interact with specific receptors on neurons. Since various cytokines have been shown to affect neuronal excitability, this led to the hypothesis that they may have a role in altering synaptic transmission in epileptic conditions. Indeed, intracerebral application of IL-1beta enhances epileptic activity in experimental models while its naturally occurring receptor antagonist (IL-1Ra) mediates anticonvulsant actions. Transgenic mice overexpressing IL-1Ra in astrocytes are less susceptible to seizures, indicating that endogenous IL-1 has proconvulsant activity. Several studies indicate a central role of IL-1beta for the exacerbation of brain damage after ischemic, traumatic or excitotoxic insults, suggesting that it may also contribute to neuronal cell injury associated with seizures. Finally, a functional polymorphism in the IL-1beta gene promoter, possibly associated with enhanced ability to produce this cytokine, has been specifically found in temporal lobe epilepsy patients with hippocampal sclerosis and in children with febrile seizures. Thus, the IL-1 system may represent a novel target for controlling seizure activity and/or the associated long-term sequelae. Furthermore, these studies suggest that other inflammatory and anti-inflammatory molecules produced in the CNS may have a role in the pathophysiology of seizure disorders.

In this retrospective evaluation of the 4-year clinical use of minocycline and rifampin-impregnated catheters in bone marrow transplantation (BMT) patients, we report low risk of development of staphylococcal resistance to the antibiotics coating the catheters and efficacy in preventing primary staphylococcal bloodstream infections.

In adult rats, status epilepticus (SE) induces cytokine production by glia especially when seizures are associated with neuronal injury. This suggests that cytokines may play a role in seizure-induced neuronal damage. As SE-induced injury is age-specific, we used rats of different ages (with distinct susceptibilities to seizure-induced neuronal injury) to elucidate the role of cytokines in this process. Thus, we investigated the activation of microglia and astrocytes, induction of cytokines, and hippocampal neuronal injury 4 and 24 h following kainic acid-induced SE in postnatal day (PN) 9, 15, and 21 rats. At PN9, there was little activation of microglia and astrocytes at any time point studied. Interleukin-1beta (IL), tumor necrosis factor-alpha (TNF), and IL-6 or the naturally occurring IL-1 receptor antagonist (Ra) mRNA expression did not increase. No evidence of cell injury has been detected. At PN15, immunostaining of microglia and astrocytes was enhanced, but only IL-1beta mRNA expression was increased. These changes were observed 4 h after SE. Scattered injured neurons in CA3 and subiculum, but not in any other region, were present 24 h following SE. At PN21, immunostaining of microglia and astrocytes and the mRNA expression of all cytokines studied was significantly increased already 4 h after SE. At 24 h, many injured neurons were present in CA1 and CA3 regions and in 40% of rats in other forebrain areas. These data show that (i) the pattern of glia activation and cytokine gene transcription induced by SE is age-dependent and (ii) neuronal injury in the hippocampus occurs only when cytokines are induced and their synthesis precedes the appearance of neuronal damage. Thus, cytokine expression in immature brain is associated specifically with cell injury rather than with seizures per se, suggesting that proinflammatory cytokines may contribute to the occurence of SE-induced hippocampal damage.

KAAT1 is a neutral amino acid transporter activated by K+ or by Na+ (9). The protein shows significant homology with members of the Na+/Cl--dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20-30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of Vmax and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na+ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1.

Medullary thyroid carcinoma (MTC) responds very poorly to chemotherapy. Mutations in the RET gene are critical for MTC pathogenesis. RET therefore represents a rational target for the development of novel MTC therapies. The accumulation of evidence from laboratory studies strongly suggests that PP1 inhibitor is a cytostatic agent for cells expressing RET oncoproteins. PP1 functions as a potent and selective inhibitor of RET oncoprotein phosphorylation, promoting its proteasomal degradation.

Background and Purpose: Prophylactic administration of simvastatin has been shown to protect against brain damage and its long-lasting behavioral consequences in neonatal rats. To establish the drug treatment window, we evaluated the effectiveness of simvastatin administered at different intervals before and after stroke. Furthermore, we determined whether simvastatin affected endothelial nitric oxide synthase (eNOS) or inflammatory cytokines in brain tissue or cholesterol levels in serum., Methods: On postnatal day 7, male rats were subjected to hypoxia-ischemia (HI). The experiment included sham-operated controls and HI animals receiving daily saline or activated simvastatin (20 mg/kg) injections from postnatal day 1 to day 7 (HI-simvastatin 1-7 group), from postnatal day 4 to day 11 (HI-simvastatin 4-11 group), or from postnatal day 7 to day 14 (HI-simvastatin 7-14 group). The neuroprotective effect of simvastatin was evaluated at adulthood by means of behavioral and histological analyses. Cytokines and eNOS expression were assessed by reverse transcriptase-polymerase chain reaction and Western blotting., Results: Animals in both the HI-simvastatin 1-7 and HI-simvastatin 4-11 groups performed better than HI rats in either the T-maze or the circular water maze and showed significantly attenuated brain damage. Expression of interleukin-1beta and tumor necrosis factor-alpha mRNA in cortex was significantly increased in HI but not in HI-simvastatin 1-7 animals. In the same brain area, simvastatin treatment did not affect the increase of eNOS expression observed after HI., Conclusions: These findings indicate that prophylactic but not delayed administration of simvastatin improves functional outcome in neonatal rat stroke. The reduced induction of cytokines suggests that the neuroprotective effect of simvastatin may be related to a dampening of the inflammatory response.

Recent evidence suggests that insulin signaling through the insulin receptor A type (Ex11-), regulates insulin gene transcription. Because chronic hyperglycemia negatively affects insulin receptor function and regulates alternative splicing of the insulin receptor, we inquired whether chronic exposure of pancreatic beta-cells to high glucose results in alterations in insulin signaling due to changes in insulin receptor expression and relative abundance of its spliced isoforms. Our results demonstrate that the insulin receptor is localized in insulin secretory vescicles in human pancreatic beta-cells. Furthermore, we find that alterations in insulin expression and secretion caused by chronic exposure to high glucose are paralleled by decreased insulin receptor expression and increased relative abundance of the Ex11+ isoform in both human islets and RIN beta-cells. PDX-1 and HMGI(Y) transcription factors are down-regulated by high glucose. These changes are associated with defects in insulin signaling involving insulin receptor-associated PI 3-kinase/Akt/PHAS-I pathway in RIN beta-cells. Re-expression in RIN beta-cells chronically exposed to high glucose of the Ex11-, but not the Ex11+, isoform restored insulin mRNA expression. These data suggest that changes in early steps of insulin receptor signaling may play a role in determining beta-cell dysfunction caused by chronic hyperglycemia.

RET tyrosine kinase oncoproteins are potential targets for anticancer therapy. We show here that along with the inhibition of RET tyrosine phosphorylation, the pyrazolo-pyrimidine inhibitor PP1 induces RETMEN2A and RETMEN2B oncoprotein destruction. In fact, as a consequence of PP1 treatment, RET oncoproteins translocate from the outer limiting membrane to inner cellular compartments and are rapidly addressed to the degradative pathway. The cleavage of RET oncoproteins is associated with an impairment of RET mitogenic signaling pathways that causes a reversion of the oncogenic transformation and establishes a long-term cytostatic effect. By using specific inhibitors of both the proteosome and the lysosome, we assessed that PP1 targets RET oncoproteins to proteosomal, rather than lysosomal, degradation. In this context of studies, we interestingly demonstrated that RETMEN2A and RETMEN2B receptors are constitutively ubiquitinated and interact with the ubiquitin ligase c-Cbl. Moreover, PP1 does not modify these interactions, although it indeed causes RET dephosphorylation. Therefore, even if the degradative pathway stimulated by the inhibitor appears to be mediated by the proteosome, PP1 does not seem to enhance nor promote receptor ubiquitination. These observations lead us to favor two models for PP1-induced RET oncoprotein degradation: either PP1-mediated RET dephosphorylation per se targets the oncoproteins for destruction or alternatively, PP1 insertion in the RET ATP-binding pocket promotes a mechanism for fast stress-induced degradation. The use of PP1, which therefore acts as a degradation-inducing factor, may represent a promising new strategy to selectively target RET oncogenic products for destruction and holds promise for future medullary thyroid cancer therapy.

The receptor tyrosine kinase RET, with a known role in embryonic development and in human pathologies, is alternatively spliced to yield at least two functional isoforms, which differ only in their carboxyl terminal. Enigma protein is a member of the PDZ-LIM family and is known to interact with the short isoform of RET/PTC2, a chimeric oncoprotein isolated from papillary thyroid carcinoma. Here, we show that Enigma also interacts in intact cells with the short isoform of RET-wt and of its pathologic mutants associated to MEN2 syndromes, RET-C634R and RET-M918T. In contrast, Enigma binds all the corresponding RET long isoforms very poorly and colocalizes with short but not long RET/PTC2 isoforms. The RET docking tyrosine for Enigma is the last but one before the divergence between the two isoforms and we demonstrated that short-isoform-specific amino acid residues +2 to +4 to this tyrosine are required for the interaction of RET/PTC2 with Enigma.

As little is known about the role of cadherin-mediated cell-cell adhesion in astrocytes and its alteration in migrating and invasive glioblastomas, we investigated its molecular composition and organisation in primary cultured astrocytes and the T98G and U373MG glioblastoma cell lines. Biochemical and morphological analysis indicated that all three cell types express all of the structural components of the adhesion system, including the LIN-7 PDZ protein, a novel component involved in the organisation of the junctional domain in epithelia and neurons. However, only the astrocytes and T98G cells generated and maintained mature adhesive junctional domains to which LIN-7 was recruited. Alterations in the junctional domain of U373MG cells were associated with higher motility in a poly-L-lysine migration assay. When the T98G cells were cultured on Matrigel matrix, they acquired invasive properties but, despite unchanged cadherin adhesion system protein levels, the invasive T98G cell-cell contacts failed to accumulate LIN-7 and failed to mature. These results identify the LIN-7 PDZ protein as a marker of cell adhesion maturity and cell invasion and indicate that instability and disorganisation of cadherin-mediated junctions rather than reduced expression of cadherin-catenin system components are required to promote migration and invasiveness in glioblastoma cell lines.

nPKC delta is a phospholipid-dependent and calcium-independent PKC isoform, whose over expression in BL6T murine melanoma cells, modifies their proliferative and metastatic potential in vivo. We focus here on the possible relationship between the subcellular localisation of nPKC delta and distinct phase of the cell cycle. Our findings show a dynamic localisation of nPKC delta in dependence of the phase of the cell cycle. Actually, this isoform is preferentially localised to the cytoplasm in serum-starved cells, shifting to the nucleus during the S-phase and becoming peri-nuclear, associated to the Golgi apparatus, in G2-M phase. Therefore, taken together our findings demonstrate that the subcellular localisation of nPKC delta changes dynamically during the cell cycle in dependence of the requirement of the enzyme at a particular place of the cell.