Novel conserved amino acid variations of papaya cystatin (PC) were investigated by amino acid substitutions using oryzacystatin-I (OCI) as a model plant cystatin for comparison. These amino acid residues in the conserved motifs are involved in binding with cysteine proteases, these include the GG (Gly-Gly) in the N-terminal region for both OCI and PC, the (Q)QVVAG (Gln-Val-Val-Ala-Gly) motif for OCI and (Q)AVVEG (Ala-Val-Val-Glu-Gly) motif for PC in the first inhibitory loop, and the PW (Pro-Trp) motif for OCI and LW (Leu-Trp) motif for PC in the second inhibitory loop. Recombinant OCI and PC mutant proteins were expressed in Escherichia coli and were tested for altered inhibitory activity against commercial cysteine proteases (papain and cathepsin L) and extracts from Colorado potato beetle (Leptinotarsa decemlineata) larvae, from banana weevil larvae (Cosmopolites sordidus) and tobacco leaf extracts (Nicotiana benthamiana). In all tests higher amounts of PC had to be used to obtain similar inhibition levels as OCI. Changing the amino acid Q at position 52 to E in OCI in the first inhibitory loop, had lowered the Ki value of the mutant against the commercial proteases. Concurrently the same amino acid string (EQ) in PC had resulted in a significantly decreased Ki value compared to PC wild-type and other mutants. All other OCI mutants were less efficient than the wild-type OCI, whereas all PC first inhibitory loop mutants had improved inhibitory activity against protease activity with the highest improvement against the protease extracts was found for the substitution of E with A at position 55. This study has shown the importance of the three conserved motifs and that it is possible to improve the binding capacity of a plant cystatins to cysteine protease activity by amino acid substitution using site-directed mutagenesis. By mutating individual amino acid residues in the first binding loop of the relatively “weak” papaya cystatin to amino acid residues found in OCI caused a significant improvement in inhibitory potency of PC. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Plant Science / unrestricted

Background. This study aims to establish age- and gender-specific cystatin C (CysC) reference values for healthy infants, children, and adolescents and to relate them to pubertal stage, height, weight, and body mass index (BMI). Methods. Serum CysC and creatinine levels of 6217 fasting, morning venous blood samples from 2803 healthy participants of the LIFE Child study (age 3 months to 18 years) were analyzed by an immunoassay. Recruitment started in 2011; 1636 participants provided at least one follow-up measurement. Percentiles for CysC were calculated. Age- and gender-related effects of height, weight, BMI, and puberty status were assessed through linear regression models. Results. Over the first 2 years of life, median CysC levels decrease depending on height (ß = − 0.010 mg/l/cm, p < 0.001) and weight (ß = − 0.033 mg/l/kg, p < 0.001) from 1.06 to 0.88 mg/l for males and from 1.04 to 0.87 mg/l for females. Following the second year of age, the levels remain stable for eight years. From 11 to 14 years of age, there is an increase of median CysC levels in males to 0.98 mg/l and a decrease in females to 0.86 mg/l. The change is associated with puberty (ß = 0.105 mg/l/Tanner stage, p < 0.001 in males and ß = − 0.093 mg/l/Tanner stage, p < 0.01 in females) and in males with height (ß = 0.003 mg/l/cm, p < 0.001). Conclusions. CysC levels depend on age, gender, and height, especially during infancy and puberty. We recommend the use of age- and gender-specific reference values for CysC serum levels for estimating kidney function in clinical practice.:1 Contents ........................................................................................................... 1 2 Introduction ...................................................................................................... 2 GFR measurement ............................................................................................. 2 Technically advanced methods ........................................................................ 2 Serum creatinine................................................................................................ 3 Serum Cystatin C............................................................................................... 3 Current state of research..................................................................................... 5 Reference values of Cystatin C ......................................................................... 5 Cystatin C in healthy test persons .................................................................... 6 Validity of Cystatin C for kidney diseases.......................................................... 6 Post-transplant validity of Cystatin C ............................................................... 7 Validity of Cystatin C in extra-renal diseases .................................................... 7 GFR-equations................................................................................................... 8 Confounders ...................................................................................................... 9 Relevance of the topic ......................................................................................... 11 Hypothesis ........................................................................................................... 13 3 Publication – manuscript................................................................................... 14 4 Summary and interpretation ............................................................................. 16 5 References ........................................................................................................ 20 6 Appendix ........................................................................................................... I 7 Description of the own contributions ................................................................ VII 8 Erklärung über die eigenständige Abfassung der Arbeit .................................. VIII 9 Curriculum vitae ................................................................................................ IX 10 Scientific publications and presentations ....................................................... XI 11 Acknowledgements.......................................................................................... XII

Acute Kidney Injury (AKI) is a prevalent systemic disorder that has an extremely high rate of mortality even after detection. Historically, the diagnosis and treatment of AKI was marred by the lack of universally accepted criteria defining AKI. Therefore, reports of incidence and mortality varied widely depending on location and the criteria used at the time, but all reports indicated a poor prognosis for the patient. Until recently, the only modes of detecting AKI were primarily through measurements of three clinical findings: serum creatinine concentration, blood urea nitrogen concentration, and urine output. While these measurements are still widely used as standard practice, they have limitations in their utility because their values can fluctuate depending on a person's age, gender, race, diet, and other comorbid conditions. Nevertheless, as these were the only universally accepted units of measurement for kidney function, the Acute Dialysis Quality Initiative (ADQI) used them to create the Risk, Injury, Failure, Loss, and End stage kidney disease (RIFLE) criteria to classify the severity of kidney injury across clinical settings. Eventually, modifications were made by the Acute Kidney Injury Network (AKIN) to increase the sensitivity of AKI diagnosis. It was not until the last decade that new biomarkers of kidney injury began to be researched that provided earlier detection of physical kidney injury before functional manifestations would present themselves. Some of these new biomarkers include cystatin C, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase associated lipocalin (NGAL). This study will investigate how the properties of these new biomarkers are superior when compared to those of serum creatinine in early detection of AKI and specification as to the local site of injury within the nephron. The conclusion is that cystatin C has the potential to indicate damage to glomerular filtration while KIM-1 and NGAL have the ability to indicate damage to the proximal tubule. Along with the ability to provide information as to the specific site of renal injury, the levels of cystatin C, KIM-1, and NGAL increase much more rapidly and to a much higher value than serum creatinine once physical renal damage has occurred. These characteristics along with future research will allow for earlier detection of AKI, more personalized treatment plans, and an overall better prognosis for the patient.

Cardiovascular diseases (CVD) are the number one cause of death worldwide. About one fifth of those who survived a myocardial infarction will suffer a recurrent cardiovascular event (CVE). Given the low participation in recommended cardiac rehabilitation, there is interest in early risk stratification after a primary CVE. This dissertation evaluates leisure time physical activity (LTPA), N-Terminal pro-Brain Natriuretic Peptide (NT-proBNP) and cystatin C as predictors of a secondary CVE in a German cohort of cardiac rehabilitation patients with stable coronary heart disease followed from 1999 to 2008. Study 1 evaluated self-reported LTPA at one-year follow-up. Those reporting seldom/never practice of LTPA showed a higher risk (Hazard Ratio (HR) 1.30 [95% Confidence Interval (CI) 0.62, 2.69]), while those reporting LTPA at least 5-6 times/week had a reduced risk (HR 0.88 [95% CI 0.54, 1.43]) for a subsequent CVE, when compared to the reference group (1-4 times/month). Study 2 examined LTPA trajectories during the age period 20-49 years. Compared to those with a gradual decline of LTPA, the highest risk was observed among those with a steeper decrease of LTPA (HR 1.59 [95% CI 0.97, 2.62]). A continuous increase of LTPA was associated with a risk reduction (HR 0.71 [95% CI 0.41, 1.22]) with respect to a recurrent CVE. Studies 3 and 4 evaluated the prognostic value of two novel biomarkers, when added to a model containing well-established CVD risk factors. In Study 3, NT-proBNP levels at one-year follow-up and a 10% increase in the slope of a NT-proBNP three-year trajectory were associated with a subsequent CVE ,with HRs of 1.63 [95% CI 1.17, 2.27] and 1.24 [95% CI 1.12, 1.37], respectively. One-year, but not baseline, levels of NT-proBNP showed an improvement in risk reclassification. Study 4 examined cystatin C versus creatinine. Although both were associated with a recurrent CVE, only the addition of cystatin C improved model performance, discrimination and reclassification. In conclusion, in patients with stable coronary heart disease, LTPA, NT-proBNP, and cystatin C might help to identify individuals at high risk for a recurrent CVE. Further research is needed to evaluate treatment modalities for secondary prevention in this group.

No description available.

Cysteinproteaset cathepsin B har en trolig roll i vävnadsnedbrytningen vid kronisk parodontit då det visats kunna bryta ner extracellulära komponenter. Cystatin C är en fysiologisk inhibitor av cathepsin B och båda har kopplats till parodontal sjukdom. Syftet med studien var att undersöka närvaro av samt lokalisera cathepsin B och cystatin C i gingivala biopsier från patienter med kronisk parodontit. Närvaro och lokalisation av cathepsin Bs och cystatin Cs demonstrerades genom immunohistokemisk metod av kryostatsnittad vävnad. Polyklonal kanin anti-human primär antikropp för både enzym och inhibitor användes. För identifiering av primära antikroppar användes Dako REAL™ EnVision™ Detection System. Granskning skedde i ljusmikroskop. Studien visar på en närvaro av både enzym och inhibitor i epitel och bindväv i parodontalt sjuk vävnad. Cathepsin B lokaliserades i stor utsträckning till bindväven, främst perivaskulärt. I epitelet lokaliserades endast ett fåtal infärgade celler. Cystatin C hade i bindväven en diffus utbredning vilket gjorde lokalisation till specifika cellpopulationer svår. I epitelet sågs en tydlig infärgning kring/i celler med dendritiskt utseende. Dessa celler identifierades som sannolika langerhanska celler. / The cysteine protease cathepsin B has been shown to have a possible role in tissue destruction in chronic periodontitis, as it has been shown to degrade extracellular components. Cystatin C is a physiological inhibitor of cathepsin B and both are associated with periodontal disease. The aim with this study was to investigate the presence and localization of cathepsin B and cystatin C in gingival biopsies from patients with chronic periodontitis. Cathepsin B's and cystatin C’s presence and localization was demonstrated by immunohistochemical method of cryostat sectioned tissue from patients with chronic periodontitis. Polyclonal rabbit anti-human primary antibody for both the enzyme and the inhibitor were used. Identification of the primary antibodies were confirmed with Dako REAL ™ EnVision ™ Detection System. The sectioned tissue was analysed by using light microscopy. Our study indicates a presence of both enzyme and inhibitor in epithelial and connective tissue from patients with periodontal disease. Cathepsin B was localized mainly to the connective tissue, primarily to the perivascular space. In the epithelium only a few stained cells were identified. In the connective tissue, cystatin C had a diffuse distribution. Therefor it was difficult to distinguish the specific cell populations associated with the inhibitor. The epithelium showed a clear staining around/in dendritic cells. These cells were identified as probable Langerhans cells.

Filarien persistieren bis zu 15 Jahren in ihren Wirten. Als eine Ursache dieser Persistenz diskutiert man die Fähigkeit der Filarien, die Immunantwort des Wirtes gezielt zu modulieren und eine zelluläre Hyporeaktivität zu induzieren. In der vorliegenden Arbeit wurde untersucht, ob ein sezernierter Cysteinproteasen-Inhibitor (Onchocystatin) der humanpathogenen Filarie Onchocerca volvulus immunmodulierende Eigenschaften besitzt und an der Herausbildung eines hyporeaktiven Immunstatus des Wirtes beteiligt ist. Für die Untersuchungen wurde Onchocystatin als full length Molekül (rOv17) und als verkürztes Molekül (trOv17) rekombinant hergestellt. Das verkürzte trOv17 besitzt aufgrund des Fehlens des N-terminalen Bereiches eine verminderte proteaseinhibitorische Aktivität. Die in vitro Studien mit den rekombinant hergestellten O. volvulus Cystatinen verdeutlichen, daß rOv17 und auch trOv17 potente Immunmodulatoren sind, die sowohl die antigenspezifische als auch die polyklonal-stimulierte Proliferation von humanen PBMC inhibieren. Die zelluläre Hyporeaktivität ist dabei auf die Modulation von Monozytenfunktionen zurückzuführen. rOv17 und trOv17 modulieren die Antigenpräsentation, die Zytokinproduktion und die Expression kostimulatorischer Signale von humanen Monozyten. So konnte gezeigt werden, daß rOv17 und trOv17 die Aktivität von humanem Cathepsin L und S inhibieren und die Expression von HLA-DR, CD40 und CD86 vermindern. Die Modulation der Zytokinproduktion durch rOv17 und trOv17 ist durch eine verstärkte TNF-alpha und IL-10 Produktion und durch eine verminderte IL-12 Produktion charakterisiert. Desweiteren konnte in Neutralisationsstudien mit anti-IL-10 Ak gezeigt werden, daß die verminderte Expression von HLA-DR, CD40 und CD86 Folge der durch rOv17 und trOv17 induzierten verstärkten IL-10 Produktion ist. Im Gegensatz dazu bleibt die verminderte IL-12 Produktion und die verminderte polyklonal-stimulierte Proliferation humaner PBMC auch nach der Neutralisation von IL-10 bestehen. Die Studien mit den rekombinant hergestellten O. volvulus Cystatinen zeigen, daß rOv17 und trOv17 ihr immunologisches Umfeld auf vielfältige Art und Weise modulieren. Dabei spielt vermutlich die Inhibition der Aktivität einer Wirtscysteinprotease, aber auch ein von der Funktion als Cysteinproteasen-Inhibitor unabhängiger Mechanismus eine Rolle. Der Cysteinproteasen-Inhibitor der Filarie O. volvulus besitzt somit die Eigenschaft, die Immunantwort des Wirtes zu modifizieren, und ist vermutlich eine wesentliche Komponente, die dem Parasiten eine lange Persistenz im Wirt ermöglicht. / Immune responses of individuals infected with filarial nematodes are characterized by a marked cellular hyporesponsiveness. The establishment of this hyporesponsiveness is considered as an important mechanism to avoid host immune responses which could eliminate the parasites. The present study is investigating the immunomodulatory potential of a 17 kD secreted cysteine protease inhibitor (onchocystatin) of the human pathogenic filaria Onchocerca volvulus. In vitro studies using recombinant onchocystatin (rOv17) identified this inhibitor as a potent immunomodulator. rOv17 suppresses the antigen-driven and the polyclonally-stimulated proliferation of human PBMC. This cellular hyporeactivity is due to the modulation of monocytic function by rOv17, comprising the modulation of antigenpresentation, the expression of costimulatory molecules and the production of cytokines. Thus rOv17 strongly inhibits the activity of human cathepsin L and S and reduces the expression of MHC class II molecules as well as the expression of CD40 and CD86 on human monocytes. The modulation of cytokine production by rOv17 is characterized by an initial increase of TNF-alpha which is followed by an increase of IL-10 and a decrease of IL-12. By neutralization studies it was shown that the suppression of MHC class II molecules and of CD86 and CD40 is mediated by the rOv17 induced increase of IL-10. In contrast cellular hyporeactivity and the reduced IL-12 production remain unaffected by neutralization of IL-10. In comparison to rOv17 a truncated onchocystatin (trOv17) with lowered protease inhibitory activity was investigated. Surprisingly even trOv17 is immunomodulatory active suggesting that immunomodulation by onchocystatin is mediated by both an inhibitor-dependent and an inhibitor-independent mechanism. These data demonstrate that onchocystatin is a potent immunomodulator of host immune responses and in consequence is an essential component that enables the parasites a long persistence within their hosts.

Thesis (PhD (Genetics))--University of Stellenbosch, 2011. / Includes bibliography. / Please refer to full text to view abstract.

Les maladies cardiovasculaires apparaissent précocement au cours de l'insuffisance rénale chronique (IRC) et représentent la première cause de mortalité. La 1ère étape pour apprécier la relation entre risque cardiovasculaire et progression de l'IRC consiste à améliorer l'estimation du débit de filtration glomérulaire (DFG). Nous avons donc évalué l'impact des conditions analytiques de mesure de la créatininémie et de la cystatinémie sur l'estimation du DFG. Les créatinines IDMS traçables (enzymatique et Jaffe compensé) améliorent l'estimation du DFG. Cependant, les méthodes enzymatiques non sensibles aux pseudochromogènes doivent être préférées. Concernant la cystatine C, nos résultats soulignent l'absence de standardisation du dosage. Chez des patients IRC non dialysés (stade I à V), nous avons identifié l'ostéoprotégérine (OPG) comme marqueur biologique de la présence de calcifications vasculaires. In vitro, nous avons démontré que le stress oxydant, majoré en présence de sérum urémique, jouait un rôle clé dans la transdifférenciation des cellules musculaires lisses vasculaires en ost oblastes. La mortalité en dialyse reste élevée et est largement dépendante des maladies cardiovasculaires. Il nous a donc paru nécessaire de rechercher les marqueurs pronostics et/ou d'en suivre l'évolution en transplantation. En dialyse, malgré une épuration significative par hémodiafiltration, les peptides natriurétiques sont des marqueurs du remodelage ventriculaire. La combinaison "NT-proBNP-CRP" est un puissant facteur pronostic de mortalité cardiovasculaire en hémodialyse. Après transplantation rénale, les calcifications vasculaires se stabilisent chez la majorité des patients et les taux d'OPG diminuent précocement. Les taux d'OPG sont significativement plus élevés chez les patients dont les calcifications progressent. Toutefois, seule l'intensité des calcifications avant transplantation permet de prédire la progression / Cardiovascular disease occurs in the early stage of chronic kidney disease (CKD) and is the leading cause of death. The first step, to appreciate the link between cardiovascular risk and CKD progression, is to improve glomerular filtration rate (GFR) estimation. We have therefore evaluated the impact of analytical conditions for creatinine and cystatin C measurement on estimated GFR. New creatinine ID-MS traceable methods (enzymatic and compensated Jaffe) improved estimation of GFR by predictive equations. However, enzymatic methods that are much less susceptible to interfere with non-creatinine chromogens may provide more reliable estimations of GFR. Regarding cystatin C, our results highlighted the lack of standardization. In non dialyzed CKD patients (stage I to V), we identified osteoprotegerin (OPG) as a biomarker for the presence of vascular calcification. In vitro, we demonstrated that oxidative stress, increased in the presence of uremi c serum, played a key role in the transdifferentiation of vascular smooth muscle cells into osteoblast-like cells. In dialysis, mortality is high and largely dependant on cardiovascular disease. We have therefore investigated prognostic markers and/or followed their evolution after transplantation. In dialysis, despite their removal by hemodiafiltration, natriuretic peptides could be potential markers of left ventricular remodelling. In addition, the combination of high CRP and circulating NT-proBNP dramatically impaired the hemodialysis survival rate. After renal transplantation, stabilization of vascular calcification was observed in the majority of patients and OPG levels are dramatically reduced. Despite a higher baseline OPG level in progressors vs. non-progressors patients, post transplant vascular calcification progression was only predicted by baseline score.

It is suggested that Alzheimer’s disease (AD) is caused by an imbalance between production, degradation and clearance of the amyloid-β (Aβ) protein. This imbalance leads to aggregation of Aβ and tau proteins and neurodegeneration in the brain. Today there is increasing evidence that the balance between the protease cathepsin B and the protease inhibitor cystatin C affects the tendency for Aβ to aggregate. The primary aim of this thesis was to investigate Aβ, cystatin C and cathepsin B levels in blood and cerebro-spinal fluid (CSF) in relation to the risk of AD. Studies I &amp; II were based on the re-examinations of participants, at ages 70 and 77, in the Uppsala Longitudinal Study of Adult Men (ULSAM), a community-based prospective study initiated in 1970 (participants then being 50 years of age). In ULSAM, low plasma Aβ1-40 (Study I) and low serum cystatin C levels (Study II) were associated with a higher risk of AD. Studies III &amp; IV were based on a cross-sectional sample of people with AD, mild cognitive impairment and healthy controls, recruited at three Swedish Memory Disorder units: Uppsala University Hospital, Uppsala, Skåne University Hospital, Malmö, and Karolinska University Hospital, Huddinge, Stockholm. In Study III, CSF cystatin C levels were positively correlated with both Aβ1-42 and tau levels. In Study IV, individuals with AD had higher mean plasma cathepsin B levels than healthy controls. In conclusion, low plasma Aβ1-40 and low serum cystatin C levels may precede clinically manifest AD in elderly men, cystatin C levels are positively correlated with Aβ1-42 and tau levels in CSF, and mean plasma cathepsin B levels are higher in people with AD compared to healthy controls. In addition to Aβ1-42 and tau levels in CSF, Aβ1-40, cystatin C and cathepsin B levels in blood may reflect the risk of AD.