Der Einfluss der löslichen Spaltprodukte des Amyloid-beta-Vorläuferproteins auf die Proteinhomöostase

Die Alzheimer’sche Erkrankung (AD) ist die am häufigsten vorkommende Form der Demenz. Die Spaltung des APP scheint eine große Rolle in der Pathologie der Erkrankung zu spielen. APP kann auf zwei Wegen prozessiert werden. Dem amyloidogenen Weg, bei dem neben einem löslichen extrazellulären Fragment (sAPPβ) und der APP Intrazellulären Domäne (AICD) auch Aβ entsteht. Auf dem nicht-amyloidogenen Weg entsteht sAPPα, p3 und die AICD. Dem sAPPα werden neuroprotektiv Eigenschaften zugeschrieben. rnEs konnte gezeigt werden, dass sAPPα in jungen IMR90 Zellen, den durch proteasomalen Stress ausgelösten Anstieg der Bag3 und Hsp70 Proteinlevel senkt. Gleichzeitig konnte gezeigt werden, dass sAPPα die Zellviabilität nach proteasomalen Stress erhöht und weniger Aggresomen gebildet werden. Die Analyse der proteasomalen Aktivität zeigte, dass sAPPα die proteasomale Aktivität gestresster junger Zellen erhöhen kann. In alten IMR90 Zellen konnte keine Beeinflussung der Autophagie und der proteasomalen Aktivität festgestellt werden. Das ist ein Anhaltspunkt dafür, dass im Alter das Proteasom zu stark geschädigt ist, um durch sAPPα aktiviert zu werden. Das bei der amyloidogenen Prozessierung von APP entstehende sAPPβ zeigte eine ähnliche protektive Eigenschaft. rnInsgesamt konnte ein protektiver Einfluss von sAPPα und sAPPβ unter proteotoxischen Bedingungen in jungen und klonalen Zellen gezeigt werden, wodurch die Zellviabilität verbessert wird. rn

Untersuchungen zur Lokalisation und Expression des respiratorischen Proteins Neuroglobin bei Säugetieren

In der vorliegenden Arbeit wurde Neuroglobin (Ngb), ein evolutiv altes und in Metazoen konserviertes respiratorisches Protein, funktionell untersucht. Mittels des induzierbaren Tet on / Tet off Systems wurde Ngb ektopisch in der murinen Leber und im Gehirn überexprimiert. Die Transkriptome von Leber und Gehirnregionen Ngb-transgener Mäuse wurden mittels Microarrays und RNA-Seq im Vergleich zum Wildtyp analysiert, um Auswirkungen der Ngb-Überexpression zu ermitteln. Die Transkriptom-Analyse in Leber und Gehirn zeigte eine nur geringe Anzahl differenziell regulierter Gene und Stoffwechselwege nach Ngb-Überexpression. Ngb transgene Mäuse wurden CCl4-induziertem ROS-Stress ausgesetzt und die Leberfunktion untersucht. Zudem wurden primäre Hepatozyten-Kulturen etabliert und in diesen in vitro die extrinsische Apoptose induziert. Die Stressversuche zeigten: (i) Die Ngb-Überexpression hat keine protektive Wirkung in der Leber in vivo. (ii) In Leberzellen in vitro hingegen verminderte eine Ngb-Überexpression effizient die Aktivierung der apoptotischen Kaskade. Eine protektive Wirkung von Ngb ist vermutlich von betrachtetem Gewebe und dem verwendeten Stressor abhängig und keine generelle, selektierte Funktion des Proteins.rnWeiterhin wurde eine Ngb-KnockOut-Mauslinie mit einem LacZ-KnockIn-Genotyp etabliert. Hierbei zeigten die KO-Mäuse keinen offensichtlichen Phänotyp in ihrer Entwicklung, Fortpflanzung und Retina-Funktion. Unter Verwendung des LacZ-Knockin-Konstrukts konnten kontrovers diskutierte Ngb-Expressionsorte im adulten Mausgehirn (Hippocampus, Cortex und Cerebellum) sowie in Testes experimentell bestätigt werden. Parallel wurden öffentlich verfügbare RNA-Seq Datensätze ausgewertet, um die regionale Ngb-Expression systematisch ohne Antikörper-assoziierte Spezifitätsprobleme zu charakterisieren. Eine basale Ngb-Expression (RPKM: ~1-5) wurde im Hippocampus, Cortex und Cerebellum, sowie in Retina und Testes ermittelt. Eine 20-40fach höhere, starke Expression (RPKM: ~160) wurde im Hypothalamus bzw. im Hirnstamm nachgewiesen. Die „digitale“ Expressionsuntersuchung wurde mittels qRT-PCR und Western Blot bestätigt. Dieses Expressionsprofil von Ngb in der Maus weist auf eine besondere funktionelle Bedeutung von Ngb im Hypothalamus hin. Eine Funktion von Ngb in der Sauerstoffversorgung der Retina und eine generelle Funktion von Ngb in der Protektion von Neuronen sind mit dem beobachteten Expressionsspektrum weniger gut vereinbar.

Dynamics of small interfering RNA - FRET based integrity measurements of siRNA in the cuvette & inside cells

RNAi ist ein bedeutendes Werkzeug zur Funktionsanalyse von Genen und hat großes Potential für den Einsatz in der Therapie. Obwohl effiziente Knockdowns in der Zellkultur erzielt werden, erweist sich eine in vivo Anwendung als schwierig. Die großen Hürden sind dabei der Transport der siRNA ins Zielgewebe und deren voranschreitende Degradierung.rnMarkierte siRNA kann sowohl zur eigenen Integritätsmessung als auch zur Lokalisierung verwendet werden. Zwei Farbstoffe an den jeweiligen 3’- bzw. -5’-Enden des Sense- bzw. Antisense-Stranges erzeugen ein robustes FRET-System (Hirsch et al. 2012). Das Verhältnis von FRET- zu Donor-Signal, das R/G-Ratio, dient zur sensitiven Klassifizierung des Integritätslevels einer siRNA Probe (Järve et al. 2007; Hirsch et al. 2011; Kim et al. 2010). Mit diesem System kann eine Degradierung von weniger als 5 % in der Küvette und in Zellen nachgewiesen werden.rnDie vorliegende Arbeit beschäftigt sich mit der Evaluierung von potentiellen FRET Farbstoffpaaren hinsichtlich deren Eignung für in vitro und in vivo Anwendung. Verschiedenste FRET-Paare, die das gesamte sichtbare Spektrum abdecken, wurden evaluiert und ermöglichen nun die Auswahl eines geeigneten Paares für die jeweilige Anwendung oder Kombination mit anderen Farbstoffen.rnMit Hilfe von Alexa555/Atto647N siRNA wurde ein erfolgreicher Einschluss von siRNA in Liposomen beobachtet. Eine anschließende Evaluierung der RNase-Protektion ergab für Liposomen, Nanohydrogele und kationische Peptide hervorragende protektive Eigenschaften. Basierend auf den Ergebnisse können diese und andere Transportsysteme nun für eine zelluläre Aufnahme optimiert werden.rnAtto488/Atto590 zeigte die besten Eigenschaften für Echtzeit-Integritätsmessungen in der Lebendzellmikroskopie. Verringerte Bleicheigenschaften und minimaler spektraler “Cross-Talk” ermöglichten es, transfizierte Zellen über einen Zeitraum von bis zu 8 Stunden zu beobachten. Mittels Atto488/Atto590 siRNA wurde die Einschleusung und Freisetzung in Zellen in Echtzeit untersucht. Dabei konnten Freisetzung und Verteilung in einzelnen Zellen beobachtet und analysiert werden. rnAuf eine anfängliche Phase mit hoher Freisetzungsrate folgte eine Phase mit geringerer Rate für den restlichen Beobachtungszeitraum. Die durchschnittliche Verweildauer im Zytosol betrug 24 und 58 Minuten, wobei zwischen lang- und kurzanhaltenden Ereignissen unterschieden werden konnte. Obwohl ein Import von siRNA in den Zellkern beobachtet wurde, konnte kein Schema bzw. genauer Zeitpunkt, in Bezug auf den Transfektionszeitraum für diese Ereignisse bestimmt werden. Die beobachteten Freisetzungsprozesse fanden sporadisch statt und Änderungen in der zellulären Verteilung geschahen innerhalb von wenigen Minuten. Einmal freigesetzte siRNA verschwand mit der Zeit wieder aus dem Zytosol und es blieben nur kleine Aggregate von siRNA mit immer noch geringer Integrität zurück.rn

The physiological role of APP in the activation of neuroprotective signaling mechanisms

Accumulating evidence indicates that loss of physiological amyloid precursor protein (APP) function leads to enhanced susceptibility of neurons to cellular stress during brain aging. This study investigated the neuroprotective function of the soluble APP ectodomain sAPPα. Recombinant sAPPα protected primary hippocampal neurons and neuroblastoma cells from cell death induced by trophic factor deprivation. This protective effect was abrogated in APP-depleted neurons, but not in APLP1-, APLP2- or IGF1-R-deficient cells, indicating that expression of holo-APP is required for sAPPα-dependent neuroprotection. Strikingly, recombinant sAPPα, APP-E1 domain and the copper-binding growth factor-like domain (GFLD) of APP were able to stimulate PI3K/Akt survival signaling in different wildtype cell models, but failed in APP-deficient cells. An ADAM10 inhibitor blocking endogenous sAPPα secretion exacerbated neuron death in organotypic hippocampal slices subjected to metabolic stress, which could be rescued by exogenous sAPPα. Interestingly, sAPPα-dependent neuroprotection was unaffected in neurons of APP-ΔCT15 mice which lack the intracellular C-terminal YENPTY motif of APP. In contrast, sAPPα-dependent Akt signaling was completely abolished in APP mutant cells lacking the C-terminal G-protein interaction motif and by specifically blocking Gi/o-dependent signaling with pertussis toxin. Collectively, the present thesis provides new mechanistic insights into the physiological role of APP: the data suggest that cell surface APP mediates sAPPα-induced neuroprotection via Go-protein-coupled activation of the Akt pathway.

GnRH analogs do not protect ovaries from chemotherapy-induced ultrastructural injury in Hodgkin's lymphoma patients

To determine the protective effect of gonadotropin-releasing hormone analogs (GnRHa) using several ultrasound and endocrine markers to detect ultrastructural ovarian damage in Hodgkin's lymphoma patients.

Paracrine factors secreted by endothelial progenitor cells prevent oxidative stress-induced apoptosis of mature endothelial cells

Endothelial progenitor cells (EPC) play a fundamental role in tissue regeneration and vascular repair. Current research suggests that EPC are more resistant to oxidative stress as compared to differentiated endothelial cells. Here we hypothesized that EPC not only possess the ability to protect themselves against oxidative stress but also confer this protection upon differentiated endothelial cells by release of paracrine factors. To test this hypothesis, HUVEC incubated with conditioned medium obtained from early EPC cultures (EPC-CM) were exposed to H2O2 to assess the accumulation of intracellular ROS, extent of apoptosis and endothelial cell functionality. Under oxidative stress conditions HUVEC treated with EPC-CM exhibited substantially lower levels of intracellular oxidative stress (0.2+/-0.02 vs. 0.4+/-0.03 relative fluorescence units, p<0.05) compared to control medium. Moreover, the incubation with EPC-CM elevated the expression level of antioxidant enzymes in HUVEC (catalase: 2.6+/-0.4; copper/zinc superoxide dismutase (Cu/ZnSOD): 1.6+/-0.1; manganese superoxide dismutase (MnSOD): 1.4+/-0.1-fold increase compared to control, all p<0.05). Furthermore, EPC-CM had the distinct potential to reverse the functional impairment of HUVEC as measured by their capability to form tubular structures in vitro. Finally, incubation of HUVEC with EPC-CM resulted in a significant reduction of apoptosis (0.34+/-0.01 vs. 1.52+/-0.12 relative fluorescence units, p<0.01) accompanied by an increased expression ratio of the anti/pro-apoptotic factors Bcl-2/Bax to 2.9+/-0.7-fold (compared to control, p<0.05). Most importantly, neutralization of selected cytokines such as VEGF, HGF, IL-8 and MMP-9 did not significantly reverse the cyto-protective effect of EPC-CM (p>0.05), suggesting that soluble factors secreted by EPC, possibly via broad synergistic actions, exert strong cyto-protective properties on differentiated endothelium through modulation of intracellular antioxidant defensive mechanisms and pro-survival signals.

Glucocorticoids protect renal mesangial cells from apoptosis by increasing cellular sphingosine-1-phosphate

Neutral ceramidase (NCDase) and sphingosine kinases (SphKs) are key enzymes regulating cellular sphingosine-1-phosphate (S1P) levels. In this study we found that stress factor-induced apoptosis of rat renal mesangial cells was significantly reduced by dexamethasone treatment. Concomitantly, dexamethasone increased cellular S1P levels, suggesting an activation of sphingolipid-metabolizing enzymes. The cell-protective effect of glucocorticoids was reversed by a SphK inhibitor, was completely absent in SphK1-deficient cells, and was associated with upregulated mRNA and protein expression of NCDase and SphK1. Additionally, in vivo experiments in mice showed that dexamethasone also upregulated SphK1 mRNA and activity, and NCDase protein expression in the kidney. Fragments (2285, 1724, and 1126 bp) of the rat NCDase promoter linked to a luciferase reporter were transfected into rat kidney fibroblasts and mesangial cells. There was enhanced NCDase promoter activity upon glucocorticoids treatment that was abolished by the glucocorticoid receptor antagonist RU-486. Single and double mutations of the two putative glucocorticoid response element sites within the promoter reduced the dexamethasone effect, suggesting that both glucocorticoid response elements are functionally active and required for induction. Our study shows that glucocorticoids exert a protective effect on stress-induced mesangial cell apoptosis in vitro and in vivo by upregulating NCDase and SphK1 expression and activity, resulting in enhanced levels of the protective lipid second messenger S1P.

Matrix metalloproteinases and angiogenic factors: predictors of survival after radical prostatectomy for clinically organ-confined prostate cancer?

The aim of the present study was to investigate whether biomarkers improve the prediction of recurrence-free, disease-specific, and overall survival in patients with clinically localized prostate cancer. A tissue microarray was constructed from prostate specimens of 278 patients who underwent open radical retropubic prostatectomy for clinically localized prostate cancer. For immunohistochemical studies, antibodies were used against matrix metalloproteinase (MMP)-2, MMP-3, MMP-7, MMP-9, MMP-13, and MMP-19, as well as against vascular endothelial growth factor, hypoxia-induced factor 1 , basic fibroblast growth factor, and cluster of differentiation 31. Univariate and multivariable analyses were performed to evaluate the potential predictors of overall, disease-specific, and recurrence-free survival. In univariate analysis of patients with clinically organ-confined prostate cancer, only higher expression levels of MMP-9 (hazard ratio [0.6], 95% CI 0.45-0.8) had a protective effect in terms of overall survival. This positive effect of high MMP-9 expression was also observed for recurrence-free (HR 0.88, 95% CI 0.78-0.99) and disease-specific survival (HR 0.5, 95% CI 0.36-0.73). In multivariable analysis, none of these potential markers was found to be an independent prognostic factor of survival. Of all MMPs and angiogenic factors tested, MMP-9 expression has the potential as a prognostic marker in patients undergoing radical prostatectomy for clinically organ-confined cases of prostate cancer.

Religion and assisted and non-assisted suicide in Switzerland: National Cohort Study

Background In the 19th century, eminent French sociologist Emile Durkheim found suicide rates to be higher in the Protestant compared with the Catholic cantons of Switzerland. We examined religious affiliation and suicide in modern Switzerland, where assisted suicide is legal. Methods The 2000 census records of 1 722 456 (46.0%) Catholics, 1 565 452 (41.8%) Protestants and 454 397 (12.2%) individuals with no affiliation were linked to mortality records up to December 2005. The association between religious affiliation and suicide, with the Protestant faith serving as the reference category, was examined in Cox regression models. Hazard ratios (HRs) with 95% confidence intervals (CIs) were adjusted for age, marital status, education, type of household, language and degree of urbanization. Results Suicide rates per 100 000 inhabitants were 19.7 in Catholics (1664 suicides), 28.5 in Protestants (2158 suicides) and 39.0 in those with no affiliation (882 suicides). Associations with religion were modified by age and gender (P < 0.0001). Compared with Protestant men aged 35–64 years, HRs (95% CI) for all suicides were 0.80 (0.73–0.88) in Catholic men and 1.09 (0.98–1.22) in men with no affiliation; and 0.60 (0.53–0.67) and 1.96 (1.69–2.27), respectively, in men aged 65–94 years. Corresponding HRs in women aged 35–64 years were 0.90 (0.80–1.03) and 1.46 (1.25–1.72); and 0.67 (0.59–0.77) and 2.63 (2.22–3.12) in women aged 65–94 years. The association was strongest for suicides by poisoning in the 65–94-year-old age group, the majority of which was assisted: HRs were 0.45 (0.35–0.59) for Catholic men and 3.01 (2.37–3.82) for men with no affiliation; 0.44 (0.36–0.55) for Catholic women and 3.14 (2.51–3.94) for women with no affiliation. Conclusions In Switzerland, the protective effect of a religious affiliation appears to be stronger in Catholics than in Protestants, stronger in older than in younger people, stronger in women than in men, and particularly strong for assisted suicides.

Endogenous estrogens, through estrogen receptor , constrain autoimmune inflammation in female mice by limiting CD4(+) T-cell homing into the CNS

Sex hormones influence immune responses and the development of autoimmune diseases including MS and its animal model, EAE. Although it has been previously reported that ovariectomy could worsen EAE, the mechanisms implicated in the protective action of endogenous ovarian hormones have not been addressed. In this report, we now show that endogenous estrogens limit EAE development and CNS inflammation in adult female mice through estrogen receptor expression in the host non-hematopoietic tissues. We provide evidence that the enhancing effect of gonadectomy on EAE development was due to quantitative rather than qualitative changes in effector Th1 or Th17 cell recruitment into the CNS. Consistent with this observation, adoptive transfer of myelin oligodendrocyte glycoprotein-specific encephalitogenic CD4(+) T lymphocytes induced more severe EAE in ovariectomized mice as compared to normal female mice. Finally, we show that gonadectomy accelerated the early recruitment of inflammatory cells into the CNS upon adoptive transfer of encephalitogenic CD4(+) T cells. Altogether, these data show that endogenous estrogens, through estrogen receptor , exert a protective effect on EAE by limiting the recruitment of blood-derived inflammatory cells into the CNS.

Preventing erosion with novel agents

Objectives: This in vitro study aimed to investigate the protective effect of four commercial novel agents against erosion. Methods: Ninety human molars were distributed into 9 groups, and after incubation in human saliva for 2 h, a pellicle was formed. Subsequently, the specimens were submitted to demineralization (orange juice, pH 3.6, 3 min) and remineralization (paste slurry containing one of the tested novel agents, 3 min) cycles, two times per day, for 4 days. The tested agents were: (1) DenShield Tooth; active ingredient: 7.5% W/W NovaMin® (calcium sodium phosphosilicate); (2) Nanosensitive hca; active ingredient: 7.5% W/W NovaMin®; (3) GC Tooth Mousse; active ingredient: 10% Recaldent™ (CPP-ACP); (4) GC MI Paste Plus; active ingredients: 10% Recaldent™, 900 ppm fluoride. Two experimental procedures were performed: in procedure 1, the tested agents were applied prior to the erosive attack, and in procedure 2 after the erosive attack. A control group receiving no prophylactic treatment was included. Surface nanohardness (SNH) of enamel specimens was measured after pellicle formation and after completion of daily cyclic treatment. Results: SNH significantly decreased at the end of the experiment for all groups (p < 0.05). In both procedures, there was no statistically significant difference between the control group and those treated with paste slurries (p > 0.05). In addition, the changes in SNH (ΔSNH = SNHbaseline − SNHfinal) did not show statistically significant difference between both procedures (p > 0.05). Conclusion: Tooth erosion cannot be prevented or repaired by these novel agents, regardless of fluoride content.

Estrogen Receptor α Signaling in T Lymphocytes Is Required for Estradiol-Mediated Inhibition of Th1 and Th17 Cell Differentiation and Protection against Experimental Autoimmune Encephalomyelitis

Estrogen treatment exerts a protective effect on experimental autoimmune encephalomyelitis (EAE) and is under clinical trial for multiple sclerosis therapy. Estrogens have been suspected to protect from CNS autoimmunity through their capacity to exert anti-inflammatory as well as neuroprotective effects. Despite the obvious impacts of estrogens on the pathophysiology of multiple sclerosis and EAE, the dominant cellular target that orchestrates the anti-inflammatory effect of 17β-estradiol (E2) in EAE is still ill defined. Using conditional estrogen receptor (ER) α-deficient mice and bone marrow chimera experiments, we show that expression of ERα is critical in hematopoietic cells but not in endothelial ones to mediate the E2 inhibitory effect on Th1 and Th17 cell priming, resulting in EAE protection. Furthermore, using newly created cell type-specific ERα-deficient mice, we demonstrate that ERα is required in T lymphocytes, but neither in macrophages nor dendritic cells, for E2-mediated inhibition of Th1/Th17 cell differentiation and protection from EAE. Lastly, in absence of ERα in host nonhematopoietic tissues, we further show that ERα signaling in T cells is necessary and sufficient to mediate the inhibitory effect of E2 on EAE development. These data uncover T lymphocytes as a major and nonredundant cellular target responsible for the anti-inflammatory effects of E2 in Th17 cell-driven CNS autoimmunity.

Pentraxins and atherosclerosis: the role of PTX3

Pentraxins are a family of evolutionarily conserved multifunctional pattern-recognition proteins characterized by a cyclic multimeric structure. Based on the primary structure of the subunit, the pentraxins are divided into two groups: short pentraxins and long pentraxins. C-reactive protein (CRP) and serum amyloid P-component (SAP) are the two short pentraxins. The prototype protein of the long pentraxin group is pentraxin 3 (PTX3). CRP and SAP are produced primarily in the liver in response to IL-6, while PTX3 is produced by a variety of tissues and cells and in particular by innate immunity cells in response to proinflammatory signals and Toll-like receptor (TLR) engagement. PTX3 interacts with several ligands, including growth factors, extracellular matrix components and selected pathogens, playing a role in complement activation and facilitating pathogen recognition by phagocytes, acting as a predecessor of antibodies. In addition, PTX3 is essential in female fertility by acting as a nodal point for the assembly of the cumulus oophorus hyaluronan-rich extracellular matrix. Here we will concisely review the general properties of PTX3 in the context of the pentraxin superfamily and discuss recent data suggesting that PTX3 plays a cardiovascular protective effect. PTX3 may represent a new marker in vascular pathology which correlates with the risk of developing vascular events.

Lipid profiles and outcome in patients treated by intravenous thrombolysis for cerebral ischemia

OBJECTIVE:To determine whether low low-density lipoprotein cholesterol (LDL-C) but not high-density lipoprotein cholesterol (HDL-C) and triglyceride concentrations are associated with worse outcome in a large cohort of ischemic stroke patients treated with IV thrombolysis. METHODS:Observational multicenter post hoc analysis of prospectively collected data in stroke thrombolysis registries. Because of collinearity between total cholesterol (TC) and LDL-C, we used 2 different models with TC (model 1) and with LDL-C (model 2). RESULTS:Of the 2,485 consecutive patients, 1,847 (74%) had detailed lipid profiles available. Independent predictors of 3-month mortality were lower serum HDL-C (adjusted odds ratio [(adj)OR] 0.531, 95% confidence interval [CI] 0.321-0.877 in model 1; (adj)OR 0.570, 95% CI 0.348-0.933 in model 2), lower serum triglyceride levels ((adj)OR 0.549, 95% CI 0.341-0.883 in model 1; (adj)OR 0.560, 95% CI 0.353-0.888 in model 2), symptomatic ICH, and increasing NIH Stroke Scale score, age, C-reactive protein, and serum creatinine. TC, LDL-C, HDL-C, and triglycerides were not independently associated with symptomatic ICH. Increased HDL-C was associated with an excellent outcome (modified Rankin Scale score 0-1) in model 1 ((adj)OR 1.390, 95% CI 1.040-1.860). CONCLUSION:Lower HDL-C and triglycerides were independently associated with mortality. These findings were not due to an association of lipid concentrations with symptomatic ICH and may reflect differences in baseline comorbidities, nutritional state, or a protective effect of triglycerides and HDL-C on mortality following acute ischemic stroke.

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.