Early and late neurodegeneration and memory disruption after intracerebroventricular streptozotocin

Glucose metabolism and insulin signaling disruptions in the brain have been proposed as a likely etiology of Alzheimer's disease. The aim of the present study was to investigate the time course of cognitive impairments induced by intracerebroventricular injection of streptozotocin (STZ) in rats and correlate them with the ensuing neurodegenerative process. Early and late effects of STZ were evaluated by using the reference and working memory versions of the Morris' water maze task and the evaluation of neurodegenerative markers by immunoblotting and the Fluoro-jade C histochemistry. The results revealed different types of behavioral and neurodegenerative responses, with distinct time courses. We observed an early disruption on the working memory as early as 3 h after STZ injections, which was followed by degenerative processes in the hippocampus at 1 and 15 days after STZ injections. Memory disruption increases over time and culminates with significant changes in amyloid-beta peptide and hyperphosphorylated Tau protein levels in distinct brain structures. These findings add information on the Alzheimer's disease-like STZ animal model and on the mechanisms underlying neurodegenerative processes. (C) 2012 Elsevier Inc. All rights reserved.

Transgenic mice as a tool for depression research: examples from the endocannabinoid and the corticotropin-releasing hormone system

Major depression belongs to the most serious and widespread psychiatric disorders in today’s society. There is a great need for the delineation of the underlying molecular mechanisms as well as for the identification of novel targets for its treatment. In this thesis, transgenic mice of the endocannabinoid and the corticotropin-releasing hormone (CRH) system were investigated to determine the putative role of these systems for depression-like phenotypes in mice. In the first part of the thesis, we found that the endocannabinoid system was prominently involved in a brain region-specific and temporally controlled manner in acute as well as in chronic stress processing. Genetic deletion in combination with pharmacological intervention revealed the importance of a fully functional endocannabinoid system for efficient neuroendocrine and behavioral stress coping. Accordingly, cannabinoid type 1 (CB1) receptor-deficient mice displayed several depression-like symptoms and molecular alterations, including “behavioral despair”, stress hormone hypersecretion and decreased glucocorticoid receptor and brain-derived neurotrophic factor expression in the hippocampus. However, the endocannabinoid system was dispensable for the efficacy of currently used antidepressant drugs. To facilitate future endocannabinoid research, a transgenic mouse was generated, which overexpressed the CB1 receptor protein fused to a fluorescent protein. In the second part of the thesis, conditional brain region-specific CRH overexpressing mice were evaluated as a model for pathological chronic CRH hyperactivation. Mutant mice showed aberrant neuroendocrine and behavioral stress coping and hyperarousal due to CRH-induced activation of the noradrenergic system in the brain. Mutant mice appeared to share similarities with naturally occurring endogenous CRH activation in wild-type mice and were sensitive to acute pharmacological blockade of CRH receptor type 1 (CRH-R1). Thus, CRH overexpressing mice serve as an ideal in vivo tool to evaluate the efficacy of novel CRH-R1 antagonists. Together, these findings highlight the potential of transgenic mice for the understanding of certain endo-phenotypes (isolated symptoms) of depression and their molecular correlates.

Die Rolle der humanen Enzyme Paraoxonase-2 und -3 bei Krebserkrankungen und Pseudomonas-Infektionen

Die vorliegende Arbeit befasste sich mit der Charakterisierung molekularer Funktionen humaner Paraoxonase (PON) Enzyme, insbesondere die der Proteine PON2 und PON3 im Hinblick auf medizinisch-relevante Fragestellungen. Zum einen wurde die Rolle von PON3 in der Tumorgenese und zum anderen eine mögliche Schutzfunktion von PON2 und PON3 gegenüber P. aeruginosa Infektionen untersucht. Bereits seit dem Jahr 2000 ist die anti-oxidative Eigenschaft von PON3 bekannt, jedoch war der zugrundeliegende Mechanismus bisher ungeklärt. Im Rahmen dieser Arbeit wurde gezeigt, dass PON3 die Superoxid-Entstehung in den Mitochondrien abschwächt, wobei sie ihre anti-oxidative Eigenschaft vermutlich durch eine direkte Coenzym Q10-Interaktion in der inneren mitochondrialen Membran vermittelt. Dies führt zu weniger oxidativen Stress, zur Abschwächung mitochondrial-induzierter apoptotischer Signalwege und zur erhöhten Resistenz gegenüber Chemotherapeutika. Gleichzeitig wurde demonstriert, dass sich Tumorzellen diese anti-oxidative Eigenschaft zu Nutze machen. PON3 war in zahlreichen Tumorgeweben überexprimiert. Es konnte eine mögliche Funktion von PON3 als Tumormarker und Angriffspunkt in der Krebstherapie aufgezeigt werden. Die hier erlangten Daten liefern wertvolle Hinweise auf die Rolle von PON3 in Krebserkrankungen, welche eine Basis für zukünftige Analysen darstellen, die der Entwicklung neuer Krebstherapien dienen könnten. Ein weiterer Teil der Arbeit befasste sich mit der gegenseitigen Beeinflussung der Enzyme PON2 / PON3 und der für P.aeruginosa essentiellen Virulenzfaktoren Pyocyanin (PCN) und dem Lacton 3OC12. Erstmalig wurde gezeigt, dass PON3 zellschädigende PCN-Effekte abschwächen kann, nämlich die PCN-induzierte Superoxid-Produktion, NF-kB-Aktivierung und IL-8-Sekretion. PON2 schützt in gleicher Weise gegen PCN und hydrolysiert zugleich noch das Lacton 3OC12. Folglich sind PON2 und PON3 wichtige Bestandteile der angeborenen Immunität, werden jedoch durch eine 3OC12-induzierte Ca2+-Mobilisation inaktiviert. Weitere Analysen ergaben, dass die PON2-Inaktivierung wahrscheinlich über einen Ca2+ / Calcineurin / Calmodulin-abhängigen Signalweg erfolgt, welcher eine offenbar regulative Serin311-Dephosphorylierung in PON2 vermittelt. Ähnliches könnte für PON3 gelten und wird derzeit erforscht, da eine Stabilisierung der enzymatischen Aktivitäten von PON2 und PON3 der bakteriellen Virulenz entscheidend entgegen wirken könnte.

Comparison of gingival crevicular fluid sampling methods in patients with severe chronic periodontitis

The analysis of samplings from periodontal pockets is important in the diagnosis and therapy of periodontitis. In this study, three different sampling techniques were compared to determine whether one method yielded samples suitable for the reproducible and simultaneous determination of bacterial load, cytokines, neutrophil elastase, and arginine-specific gingipains (Rgps). Rgps are an important virulence factor of Porphyromonas gingivalis, the exact concentration of which in gingival crevicular fluid (GCF) has not been quantified.

A metalloproteinase karilysin present in the majority of Tannerella forsythia isolates inhibits all pathways of the complement system

Tannerella forsythia is a poorly studied pathogen despite being one of the main causes of periodontitis, which is an inflammatory disease of the supporting structures of the teeth. We found that despite being recognized by all complement pathways, T. forsythia is resistant to killing by human complement, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with karilysin, a metalloproteinase of T. forsythia, resulted in a decrease in bactericidal activity of the serum. T. forsythia strains expressing karilysin at higher levels were more resistant than low-expressing strains. Furthermore, the low-expressing strain was significantly more opsonized with activated complement factor 3 and membrane attack complex from serum compared with the other strains. The high-expressing strain was more resistant to killing in human blood. The protective effect of karilysin against serum bactericidal activity was attributable to its ability to inhibit complement at several stages. The classical and lectin complement pathways were inhibited because of the efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3, and C4 by karilysin, whereas inhibition of the terminal pathway was caused by degradation of C5. Interestingly, karilysin was able to release biologically active C5a peptide in human plasma and induce migration of neutrophils. Importantly, we detected the karilysin gene in >90% of gingival crevicular fluid samples containing T. forsythia obtained from patients with periodontitis. Taken together, the newly characterized karilysin appears to be an important virulence factor of T. forsythia and might have several important implications for immune evasion.

Putative functions of extracellular matrix glycoproteins in secondary palate morphogenesis

Cleft palate is a common birth defect in humans. Elevation and fusion of paired palatal shelves are coordinated by growth and transcription factors, and mutations in these can cause malformations. Among the effector genes for growth factor signaling are extracellular matrix (ECM) glycoproteins. These provide substrates for cell adhesion (e.g., fibronectin, tenascins), but also regulate growth factor availability (e.g., fibrillins). Cleft palate in Bmp7 null mouse embryos is caused by a delay in palatal shelf elevation. In contrast, palatal shelves of Tgf-β3 knockout mice elevate normally, but a cleft develops due to their failure to fuse. However, nothing is known about a possible functional interaction between specific ECM proteins and Tgf-β/Bmp family members in palatogenesis. To start addressing this question, we studied the mRNA and protein distribution of relevant ECM components during secondary palate development, and compared it to growth factor expression in wildtypewild type and mutant mice. We found that fibrillin-2 (but not fibrillin-1) mRNA appeared in the mesenchyme of elevated palatal shelves adjacent to the midline epithelial cells, which were positive for Tgf-β3 mRNA. Moreover, midline epithelial cells started expressing fibronectin upon contact of the two palatal shelves. These findings support the hypothesis that fibrillin-2 and fibronectin are involved in regulating the activity of Tgf-β3 at the fusing midline. In addition, we observed that tenascin-W (but not tenascin-C) was misexpressed in palatal shelves of Bmp7-deficient mouse embryos. In contrast to tenascin-C, tenascin-W secretion was strongly induced by Bmp7 in embryonic cranial fibroblasts in vitro. These results are consistent with a putative function for tenascin-W as a target of Bmp7 signaling during palate elevation. Our results indicate that distinct ECM proteins are important for morphogenesis of the secondary palate, both as downstream effectors and as regulators of Tgf-β/Bmp activity.

Glycoprotein D of bovine herpesvirus 5 (BoHV-5) confers an extended host range to BoHV-1 but does not contribute to invasion of the brain

Bovine herpesvirus 1 (BoHV-1) and BoHV-5 are closely related pathogens of cattle, but only BoHV-5 is considered a neuropathogen. We engineered intertypic gD exchange mutants with BoHV-1 and BoHV-5 backbones in order to address their in vitro and in vivo host ranges, with particular interest in invasion of the brain. The new viruses replicated in cell culture with similar dynamics and to titers comparable to those of their wild-type parents. However, gD of BoHV-5 (gD5) was able to interact with a surprisingly broad range of nectins. In vivo, gD5 provided a virulent phenotype to BoHV-1 in AR129 mice, featuring a high incidence of neurological symptoms and early onset of disease. However, only virus with the BoHV-5 backbone, independent of the gD type, was detected in the brain by immunohistology. Thus, gD of BoHV-5 confers an extended cellular host range to BoHV-1 and may be considered a virulence factor but does not contribute to the invasion of the brain.

Two domains of the V protein of virulent canine distemper virus selectively inhibit STAT1 and STAT2 nuclear import

Canine distemper virus (CDV) causes in dogs a severe systemic infection, with a high frequency of demyelinating encephalitis. Among the six genes transcribed by CDV, the P gene encodes the polymerase cofactor protein (P) as well as two additional nonstructural proteins, C and V; of these V was shown to act as a virulence factor. We investigated the molecular mechanisms by which the P gene products of the neurovirulent CDV A75/17 strain disrupt type I interferon (IFN-alpha/beta)-induced signaling that results in the establishment of the antiviral state. Using recombinant knockout A75/17 viruses, the V protein was identified as the main antagonist of IFN-alpha/beta-mediated signaling. Importantly, immunofluorescence analysis illustrated that the inhibition of IFN-alpha/beta-mediated signaling correlated with impaired STAT1/STAT2 nuclear import, whereas the phosphorylation state of these proteins was not affected. Coimmunoprecipitation assays identified the N-terminal region of V (VNT) responsible for STAT1 targeting, which correlated with its ability to inhibit the activity of the IFN-alpha/beta-mediated antiviral state. Conversely, while the C-terminal domain of V (VCT) could not function autonomously, when fused to VNT it optimally interacted with STAT2 and subsequently efficiently suppressed the IFN-alpha/beta-mediated signaling pathway. The latter result was further supported by a single mutation at position 110 within the VNT domain of CDV V protein, resulting in a mutant that lost STAT1 binding while retaining a partial STAT2 association. Taken together, our results identified the CDV VNT and VCT as two essential modules that complement each other to interfere with the antiviral state induced by IFN-alpha/beta-mediated signaling. Hence, our experiments reveal a novel mechanism of IFN-alpha/beta evasion among the morbilliviruses.

Cyclooxygenase-2 inhibition selectively attenuates bone morphogenetic protein-6 synthesis and bone formation during guided tissue regeneration in a rat model

OBJECTIVES: Bone formation during guided tissue regeneration is a tightly regulated process involving cells, extracellular matrix and growth factors. The aims of this study were (i) to examine the expression of cyclooxygenase-2 (COX-2) during bone regeneration and (ii) the effects of selective COX-2 inhibition on osseous regeneration and growth factor expression in the rodent femur model. MATERIAL AND METHODS: A standardized transcortical defect of 5 x 1.5 mm was prepared in the femur of 12 male rats and a closed half-cylindrical titanium chamber was placed over the defect. The expression of COX-2 and of platelet-derived growth factor-B (PDGF-B), bone morphogenetic protein-6 (BMP-6) and insulin-like growth factor-I/II (IGF-I/II) was analyzed at Days 3, 7, 21 and 28 semiquantitatively by reverse transcriptase-polymerase chain reaction and immunohistochemistry. The effects of COX-2 inhibition by intraperitoneal injection of NS-398 (3 mg/kg/day) were analyzed in five additional animals sacrificed at Day 14. RESULTS: Histomorphometry revealed that new bone formation occurred in the cortical defect area as well as in the supracortical region, i.e. region within the chamber by Day 7 and increased through Day 28. Immunohistochemical evidence of COX-2 and PDGF-B levels were observed early (i.e. Day 3) and decreased rapidly by Day 7. BMP-6 expression was maximal at Day 3 and slowly declined by Day 28. In contrast, IGF-I/II expression gradually increased during the 28-day period. Systemic administration NS-398 caused a statistically significant reduction (P<0.05) in new bone formation (25-30%) and was associated with a statistically significant reduction in BMP-6 protein and mRNA expression (50% and 65% at P<0.05 and P<0.01, respectively). PDGF-B mRNA or protein expression was not affected by NS-398 treatment. CONCLUSION: COX-2 inhibition resulted in reduced BMP-6 expression and impaired osseous regeneration suggesting an important role for COX-2-induced signaling in BMP synthesis and new bone formation.

Beta-D-glucoside utilization by Mycoplasma mycoides subsp. mycoides SC: possible involvement in the control of cytotoxicity towards bovine lung cells

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC) is among the most serious threats for livestock producers in Africa. Glycerol metabolism-associated H2O2 production seems to play a crucial role in virulence of this mycoplasma. A wide number of attenuated strains of M. mycoides subsp. mycoides SC are currently used in Africa as live vaccines. Glycerol metabolism is not affected in these vaccine strains and therefore it does not seem to be the determinant of their attenuation. A non-synonymous single nucleotide polymorphism (SNP) in the bgl gene coding for the 6-phospho-beta-glucosidase (Bgl) has been described recently. The SNP differentiates virulent African strains isolated from outbreaks with severe CBPP, which express the Bgl isoform Val204, from strains to be considered less virulent isolated from CBPP outbreaks with low mortality and vaccine strains, which express the Bgl isoform Ala204. RESULTS: Strains of M. mycoides subsp. mycoides SC considered virulent and possessing the Bgl isoform Val204, but not strains with the Bgl isoform Ala204, do trigger elevated levels of damage to embryonic bovine lung (EBL) cells upon incubation with the disaccharides (i.e., beta-D-glucosides) sucrose and lactose. However, strains expressing the Bgl isoform Val204 show a lower hydrolysing activity on the chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside (pNPbG) when compared to strains that possess the Bgl isoform Ala204. Defective activity of Bgl in M. mycoides subsp. mycoides SC does not lead to H2O2 production. Rather, the viability during addition of beta-D-glucosides in medium-free buffers is higher for strains harbouring the Bgl isoform Val204 than for those with the isoform Ala204. CONCLUSION: Our results indicate that the studied SNP in the bgl gene is one possible cause of the difference in bacterial virulence among strains of M. mycoides subsp. mycoides SC. Bgl does not act as a direct virulence factor, but strains possessing the Bgl isoform Val204 with low hydrolysing activity are more prone to survive in environments that contain high levels of beta-D-glucosides, thus contributing in some extent to mycoplasmaemia.

Aeromonas exoenzyme T of Aeromonas salmonicida is a bifunctional protein that targets the host cytoskeleton

Type III protein secretion has been shown recently to be important in the virulence of the fish pathogen Aeromonas salmonicida. The ADP-ribosylating toxin Aeromonas exoenzyme T (AexT) is one effector protein targeted for secretion via this system. In this study, we identified muscular and nonmuscular actin as substrates of the ADP-ribosylating activity of AexT. Furthermore, we show that AexT also functions as a GTPase-activating protein (GAP), displaying GAP activity against monomeric GTPases of the Rho family, specifically Rho, Rac, and Cdc42. Transfection of fish cells with wild type AexT resulted in depolymerization of the actin cytoskeleton and cell rounding. Point mutations within either the GAP or the ADP-ribosylating active sites of AexT (Arg-143 as well as Glu-398 and Glu-401, respectively) abolished enzymatic activity, yet did not prevent actin filament depolymerization. However, inactivation of the two catalytic sites simultaneously did. These results suggest that both the GAP and ADP-ribosylating domains of AexT contribute to its biological activity. This is the first bacterial virulence factor to be described that has a specific actin ADP-ribosylation activity and GAP activity toward Rho, Rac, and Cdc42, both enzymatic activities contributing to actin filament depolymerization.

Drug-eluting stent and coronary thrombosis: biological mechanisms and clinical implications

Although rare, stent thrombosis remains a severe complication after stent implantation owing to its high morbidity and mortality. Since the introduction of drug-eluting stents (DES), most interventional centers have noted stent thrombosis up to 3 years after implantation, a complication rarely seen with bare-metal stents. Some data from large registries and meta-analyses of randomized trials indicate a higher risk for DES thrombosis, whereas others suggest an absence of such a risk. Several factors are associated with an increased risk of stent thrombosis, including the procedure itself (stent malapposition and/or underexpansion, number of implanted stents, stent length, persistent slow coronary blood flow, and dissections), patient and lesion characteristics, stent design, and premature cessation of antiplatelet drugs. Drugs released from DES exert distinct biological effects, such as activation of signal transduction pathways and inhibition of cell proliferation. As a result, although primarily aimed at preventing vascular smooth muscle cell proliferation and migration (ie, key factors in the development of restenosis), they also impair reendothelialization, which leads to delayed arterial healing, and induce tissue factor expression, which results in a prothrombogenic environment. In the same way, polymers used to load these drugs have been associated with DES thrombosis. Finally, DES impair endothelial function of the coronary artery distal to the stent, which potentially promotes the risk of ischemia and coronary occlusion. Although several reports raise the possibility of a substantially higher risk of stent thrombosis in DES, evidence remains inconclusive; as a consequence, both large-scale and long-term clinical trials, as well as further mechanistic studies, are needed. The present review focuses on the pathophysiological mechanisms and pathological findings of stent thrombosis in DES.

Attenuation of myocardial reperfusion injury in pigs by Mirococept, a membrane-targeted complement inhibitor derived from human CR1

OBJECTIVES: Membrane-targeted application of complement inhibitors may ameliorate ischemia/reperfusion (I/R) injury by directly targeting damaged cells. We investigated whether Mirococept, a membrane-targeted, myristoylated peptidyl construct derived from complement receptor 1 (CR1) could attenuate I/R injury following acute myocardial infarction in pigs. METHODS: In a closed-chest pig model of acute myocardial infarction, Mirococept, the non-tailed derivative APT154, or vehicle was administered intracoronarily into the area at risk 5 min pre-reperfusion. Infarct size, cardiac function and inflammatory status were evaluated. RESULTS: Mirococept targeted damaged vasculature and myocardium, significantly decreasing infarct size compared to vehicle, whereas APT154 had no effect. Cardioprotection correlated with reduced serum troponin I and was paralleled by attenuated local myocardial complement deposition and tissue factor expression. Myocardial apoptosis (TUNEL-positivity) was also reduced with the use of Mirococept. Local modulation of the pro-inflammatory and pro-coagulant phenotype translated to improved left ventricular end-diastolic pressure, ejection fraction and regional wall motion post-reperfusion. CONCLUSIONS: Local modification of a pro-inflammatory and pro-coagulant environment after regional I/R injury by site-specific application of a membrane-targeted complement regulatory protein may offer novel possibilities and insights into potential treatment strategies of reperfusion-induced injury.

Mechanistic and therapeutic implications of angiogenesis in endometriosis

Like tumor metastases, endometriotic implants require neovascularization to proliferate and invade into ectopic sites within the host. Endometrial tissue, with its robust stem cell populations and remarkable regenerative capabilities, is a rich source of proangiogenic factors. Among the most potent and extensively studied of these proteins, vascular endothelial growth factor has emerged as a critical vasculogenic regulator in endometriosis. Accordingly, angiogenesis of the nascent endometriotic lesion has become an attractive target for novel medical therapeutics and strategies to inhibit vascular endothelial growth factor action. Vascular endothelial growth factor gene regulation in endometrial and endometriosis cells by nuclear receptors, other transcription factors, and also by infiltrating immune cells is emphasized. New data showing that oxidative and endoplasmic reticulum stress increase vascular endothelial growth factor expression are provided. Finally, we review the clinical implications of angiogenesis in this condition and propose potential antiangiogenic therapies that may become useful in the control or eradication of endometriotic lesions.

Cattle immunized against the pathogenic L-α-glycerol-3-phosphate oxidase of Mycoplasma mycoides subs. mycoides fail to generate neutralizing antibodies and succumb to disease on challenge

The membrane-associated enzyme L-α-glycerol-3-phosphate oxidase (GlpO) of Mycoplasma mycoides subs. mycoides (Mmm), the causal agent of contagious bovine pleuropneumonia (CBPP) has been identified as a virulence factor responsible for the release of toxic by-products such as H2O2 that mediate host cell injury. Since CBPP pathogenesis is based on host inflammatory reactions, we have determined the capacity of recombinant GlpO to generate in vivo protective responses against challenge in immunized cattle. We also investigated whether sera raised against recombinant GlpO in cattle and mice inhibit production of H2O2 by Mmm. Immunization of cattle with recombinant GlpO did not protect against challenge with a virulent strain of Mmm. Further, although both murine and bovine antisera raised against recombinant GlpO detected recombinant and native forms of GlpO in immunoblot assays with similar titres, only murine antibodies could neutralize GlpO enzymatic function. The data raise the possibility that Mmm has adapted to evade potential detrimental antibody responses in its definitive host.