Examining the gateway to the limbic system with diffusion tensor imaging: the perforant pathway in dementia

Current treatments for Alzheimer's disease (AD) are only able to slow the progression of mental deterioration, making early and reliable diagnosis an essential part of any promising therapeutic strategy. In the initial stages of AD, the first neuropathological alterations occur in the perforant pathway (PP), a large neuronal fiber tract located at the entrance to the limbic system. However, to date, there is no sensitive diagnostic tool for performing in vivo assessments of this structure. In the present bimodal magnetic resonance imaging (MRI) study, we examined 10 elderly controls, 10 subjects suffering from mild cognitive impairment (MCI), and 10 AD patients in order to evaluate the sensitivity of diffusion tensor imaging (DTI), a new MRI technique, for detecting changes in the PP. Furthermore, the diagnostic explanatory power of DTI data of the PP should be compared to high-resolution MRI volumetry and intervoxel coherences (COH) of the hippocampus and the entorhinal cortex, two limbic regions also involved in the pathophysiology of early AD. DTI revealed a marked decrease in COH values in the PP region of MCI (right side: 26%, left side: 29%, as compared to controls) and AD patients (right side: 37%, left side: 43%, as compared to controls). Reductions in COH values of the PP region were significantly correlated with cognitive impairment. DTI data of the PP zone were the only parameter differing significantly between control subjects and MCI patients, while the volumetric measures and the COH values of the hippocampus and the entorhinal cortex did not. DTI of medial temporal brain regions is a promising non-invasive tool for the in vivo diagnosis of the early/preclinical stages of AD.

Reorganization of CA3 area of the mouse hippocampus after pilocarpine induced temporal lobe epilepsy with special reference to the CA3-septum pathway

We showed that when CA3 pyramidal neurons in the caudal 80% of the dorsal hippocampus had almost disappeared completely, the efferent pathway of CA3 was rarely detectable. We used the mouse pilocarpine model of temporal lobe epilepsy (TLE), and injected iontophoretically the anterograde tracer phaseolus vulgaris leucoagglutinin (PHA-L) into gliotic CA3, medial septum and the nucleus of diagonal band of Broca, median raphe, and lateral supramammillary nuclei, or the retrograde tracer cholera toxin B subunit (CTB) into gliotic CA3 area of hippocampus. In the afferent pathway, the number of neurons projecting to CA3 from medial septum and the nucleus of diagonal band of Broca, median raphe, and lateral supramammillary nuclei increased significantly. In the hippocampus, where CA3 pyramidal neurons were partially lost, calbindin, calretinin, parvalbumin immunopositive back-projection neurons from CA1-CA3 area were observed. Sprouting of Schaffer collaterals with increased number of large boutons in both sides of CA1 area, particularly in the stratum pyramidale, was found. When CA3 pyramidal neurons in caudal 80% of the dorsal hippocampus have almost disappeared completely, surviving CA3 neurons in the rostral 20% of the dorsal hippocampus may play an important role in transmitting hyperactivity of granule cells to surviving CA1 neurons or to dorsal part of the lateral septum. We concluded that reorganization of CA3 area with its downstream or upstream nuclei may be involved in the occurrence of epilepsy.

Regulation of endothelial cell cytoskeletal reorganization by a secreted frizzled-related protein-1 and frizzled 4- and frizzled 7-dependent pathway: role in neovessel formation

Consistent with findings of Wnt pathway members involved in vascular cells, a role for Wnt/Frizzled signaling has recently emerged in vascular cell development. Among the few Wnt family members implicated in vessel formation in adult, Wnt7b and Frizzled 4 have been shown as involved in vessel formation in the lung and in the retina, respectively. Our previous work has shown a role for secreted Frizzled-related protein-1 (sFRP-1), a proposed Wnt signaling inhibitor, in neovascularization after an ischemic event and demonstrated its role as a potent angiogenic factor. However the mechanisms involved have not been investigated. Here, we show that sFRP-1 treatment increases endothelial cell spreading on extracellular matrix as revealed by actin stress fiber reorganization in an integrin-dependent manner. We demonstrate that sFRP-1 can interact with Wnt receptors Frizzled 4 and 7 on endothelial cells to transduce downstream to cellular machineries requiring Rac-1 activity in cooperation with GSK-3beta. sFRP-1 overexpression in endothelium specifically reversed the inactivation of GSK-3 beta and increased neovascularization in ischemia-induced angiogenesis in mouse hindlimb. This study illustrates a regulated pathway by sFRP-1 involving GSK-3beta and Rac-1 in endothelial cell cytoskeletal reorganization and in neovessel formation.

Ligand binding induces Cbl-dependent EphB1 receptor degradation through the lysosomal pathway

Eph receptor tyrosine kinases play a critical role in embryonic patterning and angiogenesis. In the adult, they are involved in carcinogenesis and pathological neovascularization. However, the mechanisms underlying their role in tumor formation and metastasis remain to be defined. Here, we demonstrated that stimulation of EphB1 with ephrinB1/Fc led to a marked downregulation of EphB1 protein, a process blocked by the lysosomal inhibitor bafilomycin. Following ephrinB1 stimulation, the ubiquitin ligase Cbl was recruited by EphB1 and then phosphorylated. Both Cbl phosphorylation and EphB1 ubiquitination were blocked by the Src inhibitor PP2. Overexpression of wild-type Cbl, but not of 70Z mutant lacking ligase activity, enhanced EphB1 ubiquitination and degradation. This negative regulation required the tyrosine kinase activity of EphB1 as kinase-dead EphB1-K652R was resistant to Cbl. Glutathione S-transferase binding experiments showed that Cbl bound to EphB1 through its tyrosine kinase-binding domain. In aggregate, we demonstrated that Cbl induces the ubiquitination and lysosomal degradation of activated EphB1, a process requiring EphB1 and Src kinase activity. To our knowledge, this is the first study dissecting the molecular mechanisms leading to EphB1 downregulation, thus paving the way to new means of modulating their angiogenic and tumorigenic properties.

Isolated mediotegmental lesion causing narcolepsy and rapid eye movement sleep behaviour disorder: a case evidencing a common pathway in narcolepsy and rapid eye movement sleep behaviour disorder

Narcolepsy is usually an idiopathic disorder, often with a genetic predisposition. Symptomatic cases have been described repeatedly, often as a consequence of hypothalamic lesions. Conversely, REM (rapid eye movement) sleep behaviour disorder (RBD) is usually a secondary disorder, often due to degenerative brain stem disorders or narcolepsy. The case of a hitherto healthy man is presented, who simultaneously developed narcolepsy and RBD as the result of an acute focal inflammatory lesion in the dorsomedial pontine tegmentum in the presence of normal cerebrospinal fluid hypocretin-1 levels and in the absence of human lymphocyte antigen haplotypes typically associated with narcolepsy and RBD (DQB1*0602, DQB1*05). This first observation of symptomatic narcolepsy with RBD underlines the importance of the mediotegmental pontine area in the pathophysiology of both disorders, even in the absence of a detectable hypocretin deficiency and a genetic predisposition.

Matrix metalloproteinase-9 in pneumococcal meningitis: activation via an oxidative pathway

In experimental bacterial meningitis, matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) contribute to brain damage. MMP-9 increases in cerebrospinal fluid (CSF) during bacterial meningitis and is associated with the brain damage that is a consequence of the disease. This study assesses the origin of MMP-9 in bacterial meningitis and how ROS modulate its activity. Rat brain-slice cultures and rat polymorphonuclear cells (PMNs) that had been challenged with capsule-deficient heat-inactivated Streptococcus pneumoniae R6 (hiR6) released MMP-9. Coincubation with either catalase, with the myeloperoxidase inhibitor azide, or with the hypochlorous acid scavenger methionine almost completely prevented activation, but not the release, of MMP-9, in supernatants of human PMNs stimulated with hiR6. Thus, in bacterial meningitis, both brain-resident cells and invading PMNs may act as sources of MMP-9, and stimulated PMNs may activate MMP-9 via an ROS-dependent pathway. MMP-9 activation by ROS may represent a target for therapeutic intervention in bacterial meningitis.

Inhibition by interferon-gamma of human mononuclear cell-mediated low density lipoprotein oxidation. Participation of tryptophan metabolism along the kynurenine pathway

In this study we examined the potential inhibition by interferon-gamma (IFN gamma) of the early stages of low density lipoprotein (LDL) oxidation mediated by human peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM) in Ham's F-10 medium supplemented with physiological amounts of L-tryptophan (Trp). We assessed LDL oxidation by measuring the consumption of LDL's major antioxidant (i.e., alpha-tocopherol) and targets for oxidation (cholesteryllinoleate and cholesterylarachidonate), together with the accumulation of cholesterylester hydroperoxides and the increase in relative electrophoretic mobility of the lipoprotein particle. Exposure of PBMC or MDM to IFN gamma induced the degradation of extracellular Trp with concomitant accumulation of kynurenine, anthranilic and 3-hydroxyanthranilic acid (3HAA) in the culture medium. Formation of 3HAA, but neither Trp degradation nor formation of kynurenine and anthranilic acid, was inhibited by low amounts of diphenylene iodonium (DPI) in a concentration-dependent manner. In contrast to oxidative Trp metabolism, exposure of human PBMC or MDM to IFN gamma failed to induce degradation of arginine, and nitrite was not detected in the cell supernatant, indicating that nitric oxide synthase was not induced under these conditions. Incubation of LDL in Trp-supplemented F-10 medium resulted in a time-dependent oxidation of the lipoprotein that was accelerated in the presence of PBMC or MDM but inhibited strongly in the presence of both cells and IFN gamma, i.e., when Trp degradation and formation of 3HAA were induced. In contrast, when IFN gamma was added to PBMC or MDM in F-10 medium that was virtually devoid of Trp, inhibition of cell-accelerated LDL oxidation was not observed. Exogenous 3HAA added to PBMC or purified monocytes in the absence of IFN gamma also strongly and in a concentration-dependent manner inhibited LDL oxidation. Selective inhibition of IFN gamma-induced formation of 3HAA by DPI caused reversion of the inhibitory action of this cytokine on both PBMC- and MDM-mediated LDL oxidation. These results show that IFN gamma treatment of human PBMC or MDM in vitro attenuates the extent of LDL oxidation caused by these cells, and indicate that Trp degradation with formation of 3HAA is a major contributing factor to this inhibitory activity.

[Hyperinsulinemia as a consequence of systemic venous drainage in patients with pancreas transplantation]

To evaluate the metabolic consequences of pancreatic transplantation with systemic venous drainage on beta cell function, we examined insulin and C-peptide responses to arginine and secretin in type I diabetic recipients of pancreas transplantation (n = 16), and normal controls (n = 28). Basal insulin levels were 24 +/- 3 microU/l in pancreas recipients, and 7 +/- 1 microU/l in controls (p less than 0.001). Stimulated insulin levels following arginine (MANOVA, p less than 0.001), and secretin (MANOVA, p less than 0.001) were 1.5 to 3 fold elevated compared to controls. In contrast, integrated C-peptide responses following stimulation with arginine or secretin did not differ significantly between the two groups. We conclude that recipients of pancreas allografts with systemic venous drainage have elevated basal and stimulated insulin levels and that these alterations are primarily due to alterations of first pass hepatic insulin clearance although insulin resistance secondary to immunosuppressive therapy (including prednisone) may also play a contributing role. To avoid hyperinsulinemia and its possible long term adverse consequences, transplantation of pancreas allografts in sites with portal rather than systemic venous drainage may be preferable.

Systemic venous drainage of pancreas allografts as independent cause of hyperinsulinemia in type I diabetic recipients

To evaluate the metabolic consequences of pancreas transplantation with systemic venous drainage on beta-cell function, we examined insulin and C-peptide responses to glucose and arginine in type I (insulin-dependent) diabetic pancreas recipients (n = 30), nondiabetic kidney recipients (n = 8), and nondiabetic control subjects (n = 28). Basal insulin levels were 66 +/- 5 pM in control subjects, 204 +/- 18 pM in pancreas recipients (P less than 0.0001 vs. control), and 77 +/- 17 pM in kidney recipients. Acute insulin responses to glucose were 416 +/- 44 pM in control subjects, 763 +/- 91 pM in pancreas recipients (P less than 0.01 vs. control), and 589 +/- 113 pM in kidney recipients (NS vs. control). Basal and stimulated insulin levels in two pancreas recipients with portal venous drainage were normal. Integrated acute C-peptide responses were not statistically different (25.3 +/- 4.3 nM/min in pancreas recipients, 34.2 +/- 5.5 nM/min in kidney recipients, and 23.7 +/- 2.1 nM/min in control subjects). Similar insulin and C-peptide results were obtained with arginine stimulation, and both basal and glucose-stimulated insulin-C-peptide ratios in pancreas recipients were significantly greater than in control subjects. We conclude that recipients of pancreas allografts with systemic venous drainage have elevated basal and stimulated insulin levels and that these alterations are primarily due to alterations of first-pass hepatic insulin clearance, although insulin resistance secondary to immunosuppressive therapy (including prednisone) probably plays a contributing role. To avoid hyperinsulinemia and its possible long-term adverse consequences, transplantation of pancreas allografts into sites with portal rather than systemic venous drainage should be considered.

Different migration of vascular smooth muscle cells from human coronary artery bypass vessels. Role of Rho/ROCK pathway

BACKGROUND: We examined whether vascular smooth muscle (VSMC) or endothelial cell (EC) migration from internal mammary artery (MA) differed from VSMC or EC migration from saphenous vein (SV). METHODS AND RESULTS: Migration to PDGF-BB (1-10 ng/ml) was lower in VSMC from MA than SV; however, attachment, movement without chemokine, and chemokinesis were identical. Unlike VSMC, migration of EC was similar in response to several mediators. Expression of PDGF receptor-beta was lower in VSMC from MA than SV, while alpha-receptor expression was higher. PDGF-BB-induced RhoA activity was lower in MA than SV, while basal activity was identical. Rosuvastatin and hydroxyfasudil impaired PDGF-BB-induced migration of VSMC from MA and SV. Mevalonate and geranylgeranylpyrophosphate rescued inhibition by rosuvastatin. PDGF-BB induced less stress fiber formation in VSMC from MA than SV. A dominant negative RhoA mutant inhibited stress fiber formation to PDGF-BB, while a constitutively active mutant resulted in maximal stress fiber formation in MA and SV. Rosuvastatin and hydroxyfasudil impaired PDGF-BB-induced stress fiber formation in MA and SV. CONCLUSIONS: VSMC migration to PDGF-BB is lower in MA than SV, which is at least in part related to lower activity of the Rho/ROCK pathway.

Roux-en-Y drainage of the pancreatic stump decreases pancreatic fistula after distal pancreatic resection

Clinically relevant fistula after distal pancreatic resection occurs in 5-30% of patients, prolonging recovery and considerably increasing in-hospital stay and costs. We tested whether routine drainage of the pancreatic stump into a Roux-en-Y limb after distal pancreatic resection decreased the incidence of fistula. From October 2001, data of all patients undergoing pancreatic distal resection were entered in a prospective database. From June 2003 after resection, the main pancreatic duct and the pancreatic stump were oversewn, and in addition, anastomosed into a jejunal Roux-en-Y limb by a single-layer suture (n = 23). A drain was placed near the anastomosis, and all patients received octreotide for 5-7 days postoperatively. The volume of the drained fluid was registered daily, and concentration of amylase was measured and recorded every other day. Patient demographics, hospital stay, pancreatic fistula incidence (> or =30 ml amylase-rich fluid/day on/after postoperative day 10), perioperative morbidity, and follow-up after discharge were compared with our initial series of patients (treated October 2001-May 2003) who underwent oversewing only (n = 20). Indications, patient demographics, blood loss, and tolerance of an oral diet were similar. There were four (20%) pancreatic fistulas in the "oversewn" group and none in the anastomosis group (p < 0.05). Nonsurgical morbidity, in-hospital stay, and follow-up were comparable in both groups.

Biosynthesis and expression of VE-cadherin is regulated by the PI3K/mTOR signaling pathway

Vascular endothelial (VE)-cadherin is an essential protein of adherens junctions of endothelial cells and plays a pivotal role in vascular homeostasis. Mammalian target of rapamycin complex 2 (mTORC2) deficient mice display defects in fetal vascular development. Blocking mTOR or the upstream kinase phosphoinositide 3-kinase (PI3K) led to a dose-dependently decrease of the VE-cadherin mRNA and protein expression. Immunofluorescent staining showed a strongly decreased expression of VE-cadherin at the interface of human umbilical endothelial cells (HUVECs) followed by intercellular gap formation. Herewith, we demonstrated that the expression of VE-cadherin is dependent on mTOR and PI3K signaling.

IL-3 induces a Pim1-dependent antiapoptotic pathway in primary human basophils

The contribution of basophils in allergic disease and other Th2-type immune responses depends on their persistence at sites of inflammation, but the ligands and molecular pathways supporting basophil survival are largely unknown. The comparison of rates of apoptosis and of the expression of antiapoptotic proteins in different human granulocyte types revealed that basophils have a considerably longer spontaneous life span than neutrophils and eosinophils consistent with high levels of constitutive Bcl-2 expression. Interleukin-3 (IL-3) is the only ligand that efficiently protects basophils from apoptosis as evidenced by screening a large number of stimuli. IL-3 up-regulates the expression of the antiapoptotic proteins cIAP2, Mcl-1, and Bcl-X(L) and induces a rapid and sustained de novo expression of the serine/threonine kinase Pim1 that closely correlates with cytokine-enhanced survival. Inhibitor studies and protein transduction of primary basophils using wild-type and kinase-dead Pim1-Tat fusion-proteins demonstrate the functional importance of Pim1 induction in the IL-3-enhanced survival. Our data further indicate that the antiapoptotic Pim1-mediated pathway operates independently of PI3-kinase but involves the activation of p38 MAPK. The induction of Pim1 leading to PI3-kinase-independent survival as described here for basophils may also be a relevant antiapoptotic mechanism in other terminally differentiated leukocyte types.

Regulation of Id1 expression by SRC: implications for targeting of the bone morphogenetic protein pathway in cancer

Deregulated activation of the Src tyrosine kinase and heightened Id1 expression are independent mediators of aggressive tumor biology. The present report implicates Src signaling as a critical regulator of Id1 gene expression. Microarray analyses showed that Id family genes were among the most highly down-regulated by incubation of A549 lung carcinoma cells with the small-molecule Src inhibitor AZD0530. Id1 transcript and protein levels were potently reduced in a dose-dependent manner concomitantly with the reduction of activated Src levels. These effects were conserved across a panel of lung, breast, prostate, and colon cancer cell lines and confirmed by the ability of PP2, Src siRNA, and Src-blocking peptides to suppress Id1 expression. PP2, AZD0530, and dominant-negative Src abrogated Id1 promoter activity, which was induced by constitutively active Src. The Src-responsive region of the Id1 promoter was mapped to a region 1,199 to 1,360 bps upstream of the translation start site and contained a Smad-binding element. Src was also required for bone morphogenetic protein-2 (BMP-2)-induced Id1 expression and promoter activity, was moderately activated by BMP-2, and complexed with Smad1/5. Conversely, Src inhibitors blocked Smad1/5 nuclear translocation and binding to the Src-responsive region of the Id1 promoter. Consistent with a role for Src and Id1 in cancer cell invasion, Src inhibitors and Id1 siRNA decreased cancer cell invasion, which was increased by Id1 overexpression. Taken together, these results reveal that Src positively interacts with the BMP-Smad-Id pathway and provide new ways for targeted inhibition of Id1.