The ammonium sulphate precipitation method was used for the extraction of cystatin from bovine lungs, pancreas and a mixture of lungs and pancreas. The main aim of this study was to determine the highest cystatin yield from each of the samples and make recommendations to the industry as to whether a separation of these animal wastes are needed during the slaughtering and rendering processes. The effects of incubation time (15, 30, 45, 60 mins), pH (6.5, 7.0, 7.5, 8.0) and ammonium sulphate saturations (25-45% and 45-65%) on total protein (TP), total activity (TA), specific activity (SA) and yield (Y) were studied. The optimum conditions for total activity (TA), specific activity (SA) and yield (Y) of cystatin during ammonium sulphate extraction were an incubation time of 30 min, a pH of 7.5 and an ammonium sulphate saturation of 45-65%. Among the three samples, the lung produced the high yield of 60.07% at optimum conditions. Under these optimum conditions, the bovine pancreas and the mixture of lung and pancreas produced 39.57% and 51.89%, respectively. The effects of pH and temperature on the maximal inhibitory activity of cystatin obtained from the three samples were evaluated. The optimal pH and temperature for all the three samples were 7.5 and 50 ºC, respectively. The bovine lung sample exhibited a maximal inhibitory activity of 56.8 % at the optimal pH of 7.5 and temperature of 50ºC. Under the same conditions of pH and temperature, the bovine pancreas and the mixture of lung and pancreas samples showed an inhibitory activity of 25.8% and 43.8%, respectively. The bovine lung sample produced the highest values of TP, TA, SA, and Y of cystatin. During the slaughtering process, the lung should be separated from the pancreas in order to recover better cystatin yield.

Thesis (Ph. D.)--University of Chicago, Department of Neurobiology, Pharmacology, and Physiology, 2001. / Includes bibliographical references. Also available on the Internet.

Ticks belong to the ectoparasites which are dangerous for the human beings because of the transmission of bacterial, viral, and protozoan pathogens. The development of a vaccine against tick is very important. Cystatins play important role in tick digestion and they have an immunomodulation effects. For this reason cystatins are possible candidates for this vaccines. This thesis focuses on experimental mice vaccination with recombinant cystatin from Ixodes ricinus.

Trichomoniasis is a common STD caused by the parasitic protozoan Trichomonas vaginalis. The parasite is estimated to have infected roughly 3.7 million Americans. Complications from trichomoniasis can lead to cervical cancer in women and prostate cancer in men. One of the mechanisms of the parasite employs is using cysteine proteases to break down the cellular matrix of its host. However, three endogenous phytocystatin-like protease inhibitors have been found within the parasite’s genome. By recombinantly expressing these cystatins we have been able to test their ability to inhibit cysteine proteases such as papain and those found in T. vaginalis to find their effectiveness. By characterizing these inhibitors, it appears that they are effective at reducing the ability of T. vaginalis cysteine proteases and thus could be useful against the pathogenicity of the parasite.

No description available.

Rice bacterial leaf streak disease caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important rice bacterial diseases. Xanthomonas type III effector gene avrRxo1 is conserved in diverse Xoc strains and its homologues have been identified from several other gram-negative bacteria species such as Burkholderia and Acidovorax. In this research, we studied the protein structure of AvrRxo1 and illustrated its virulence mechanism.We determined the three-dimensional structure of the complex of AvrRxo1 and its cognate chaperone Arc1 (AvrRxo1 required chaperone 1). The AvrRxo1: Arc1 complex is structurally similar to the Zeta-epsilon family of toxin: antitoxin systems from the human bacterial pathogen Streptococcus pyogenes. AvrRxo1 and Arc1 have toxin: antitoxin-like activity in bacteria, and the toxin activity of AvrRxo1 is required for its virulence function in planta. These findings suggest that AvrRxo1 evolved from an endogenous bacterial toxin-antitoxin system.Furthermore, AvrRxo1 was shown to have virulence functions in diverse host plants including Arabidopsis thaliana. The ectopic expression of wild type avrRxo1 in Arabidopsis suppresses plant basal defense. AtVOZ (Arabidopsis vascular one zinc-finger transcription factor), which has two homologues in the Arabidopsis genome, VOZ1 and VOZ2, was identified as one of AvrRxo1 candidate interactor. The knockout of voz1/voz2 renders the plants more susceptible to the virulent pathogen Pseudomonas syringae pv. tomato (Pst) DC3000, but compromises the virulence function of AvrRxo1. The expression profiling of transgenic Arabidopsis plants expressing the avrRxo1 gene allowed us to identify Arabidopsis genes regulated by AvrRxo1 and VOZ1/2. AvrRxo1 interacts with and stabilizes VOZ2 in vivo and directly binds to the promoter region of AtCYS2 (Arabidopsis phytoCYStatin 2) to induce its expression. The overexpression of CYS2 in increased stomatal aperture size, and enhanced plant susceptibility to Pst. Therefore one of AvrRxo1 virulent functions is to regulate the expression of CYS2 by manipulating VOZ2, resulting in increased stomatal aperture. Presumably, this renders the host leaf more susceptible to colonization via the stomata. Another component of my dissertation was based on a genome-wide survey of Arabidopsis papin-like cysteine protease genes (PLCPs). The Arabidopsis genome has 31 PLCP and 7 cystatin genes, and they often worked in pairs to regulate signaling pathways in response to biotic and abiotic stress. The coordinated transcriptional regulation of all Arabidopsis PLCP and cystatin genes has never been systematically investigated. In order to unveil the mechanism of stomata-related plant immunity regulated by CYS2, we analyzed the expression patterns of 28 PLCPs and 7 cystatins in Arabidopsis in response to biotic or abiotic stress, by reprocessing and integrating microarray data from the AtGenExpress database. We also performed enzyme assays and evaluated the inhibition specificity of seven cystatins to the five most abundant PLCPs in Arabidopsis. Finally, we utilized the SVMs (support vector machines) package in R software to predict a functional network of PLCP-cystatin interplay in Arabidopsis. We identified the PLCP protein PAP4 as one of the putative targets of CYS2. The co-expression profiling indicated that the expression patterns of PAP4 and CYS2 were strongly correlated during virulent bacterial infection, and weakly correlated under drought stress. Therefore, PAP4 was determined to be a promising gene in regulating stomatal aperture size. Further research on the interplay of PAP4-CYS2 could be important for understanding AvrRxo1's virulence mechanism and regulation of plant stomatal immunity. / Ph. D.

Parasitische Würmer wie Filarien können das Immunsystem ihres Wirtes modulieren. Diese Immunmodulation schützt nicht nur den Parasiten, sondern kann auch den Wirt vor allergischen Reaktionen schützen. In der vorliegenden Arbeit konnte belegt werden, dass Filariencystatin, ein sezernierter Proteinaseinhibitor des Fadenwurms Acanthocheilonema viteae Th2-assoziierte Entzündungen und die daraus resultierende Atemwegserkrankung im murinen Asthmamodell supprimiert. Diese Effekte von Cystatin beruhen wahrscheinlich auf der Induktion von IL-10 sezernierenden Makrophagen. Dafür spricht, dass sowohl die Depletion von Makrophagen, als auch die Blockierung von IL-10 den schützenden Effekt von Cystatin aufhoben. In einem murinen Dermatitismodell konnte durch Cystatinapplikation die Entzündung der Haut ebenfalls unterbunden werden. Die Verbesserung der atopischen Dermatitis führte zu einem Anstieg von TGF-Beta in den Ekzemregionen. Zusammenfassend belegen die Daten, dass ein einzelnes rekombinantes Helminthenprotein den anti-allergischen Effekt einer Gesamtwurminfektion nachahmen kann. Des weiteren wurde untersucht, ob die Immunmodulation von Helminthen an die Biologie des Wurmes gebunden ist. Der Einfluss einer gastrointestinalen Wurminfektion mit Heligmosomoides polygyrus wurde in Modellen mucosa-assoziierter allergischer Atemwegsentzündung und nicht-mukosa-assoziierter, kutanen Dermatitis untersucht. Im Asthmamodell konnte eine Reduzierung der Inflammation beobachtet werden, im Dermatitis Modell keinerlei Verbesserung des klinischen Bildes. Die Wurminfektion führte zu erhöhten Werten von Foxp3+ regulatorischen T-Zellen (Treg) in peribronchialen Lymphknoten, im Gegensatz dazu konnten keine Treg Zellen in ekzematöser Haut nachgewiesen werden. Diese Ergebnisse sind ein wichtiger Aspekt beim Einsatz von Helminthen zur Behandlung allergischer Erkrankungen, da die Lokalisation des Wurmes ein entscheidender Faktor in Richtung Verbesserung oder Verschlimmerung allergischer Erkrankungen zu sein scheint. / Parasitic worms, like filariae, have the intriguing capacity to modulate immune responses directed against them. This immunomodulation protects the parasite and was found to mediate a negative correlation between parasite infections and the prevalence of allergic disease. In the present study filarial cystatin (Av17), a secreted protease inhibitor of the filarial nematode Acanthocheilonema viteae, was found to suppress Th2-related inflammation and allergic airway responsiveness in mice. These effects of Av17 on allergic airway inflammation were probably due to induction of IL-10 secreting macrophages. Depletion of macrophages as well as blocking of IL-10 by application of anti-IL-10 receptor antibodies, prevented the curative effects and restored allergic airway inflammation. Treatment with cystatin in a model of allergic skin disease, suppressed inflammation, too. The protective effect on atopic dermatitis was accompanied by an increase of TGF-Beta in the eczema region. Hence, the data demonstrate that treatment with a single recombinant helminth protein can exert the anti-allergic effects of helminth infections. Furthermore this study investigated if immunomodulation by helminths is dependent on the biology of the worm. Infection with the gastrointestinal nematode Heligmosomoides polygyrus, was analyzed in models of mucosa-associated airway disease and non-mucosal, cutaneous atopic dermatitis. Infected mice showed reduced signs of allergic airway inflammation but no differences in phenotypical signs of dermatitis. Worm infection was associated with elevated numbers of Foxp3+ regulatory T cells (Treg) in peribronchial lymph nodes in the asthma model, whereas Treg cells were basically absent in eczematous skin. These findings might be an important aspect for future considerations of helminths for treatment of allergic diseases, as localisation of the parasites might be a crucial factor leading to amelioration or aggravation of allergic disease.

Introduktion: På senare tid har en ny generation av antikoagulantia (NOAK) uppkommit. NOAK kommer med fördelar då tidigare antikoagulantia behandling krävt täta läkarkontroller, reglering av dosering och hänsyn till kost. Tidigare antikoagulantia som warafarin och hepariner inhiberar flera koagulationsfaktorer. Medan Edoxaban som tillhör NOAK endast hämmar fria faktor Xa och protrombinaktivitet. Som följd av detta har Edoxaban en bättre effekt och ger ett mer säkert resultat vid korrekt ordination. Eftersom Edoxaban elimineras via njurar är det viktigt med njurfunktionskontroller som görs via estimerat glomerulär filtrationshastighet (eGFR). Kreatininbaserad eGFR gjordes med Lund-Malmö-formeln (LM) som är anpassad för den svenska populationen. Cystatin-C-baserad eGFR beräknas via Caucasian-Asian-pediatric-and-adult-cohorts-formeln (CAPA) där endast ålder tas hänsyn, till skillnad från LM-formeln där fler faktorer spelar roll. Syftet: Undersöka korrelationen mellan kreatininbaserade eGFR formeln LM och cystatin-C-baserad eGFR formeln CAPA. Metod: Blodprov togs från 43 Edoxabanbehandlade patienter i Karlstad Centralsjukhus. Patienternas eGFR beräknades via CAPA- respektive LM-formel. Faktorer som vikt, längd och ålder togs till hänsyn. Resultat: LM- och CAPA-eGFR visar en stark korrelation. Slutsats: LM-eGFR påverkas av fler faktorer och som följd fungerar bäst för patienter med balanserad längd och vikt samt stabil kost. Medan CAPA-eGFR, som påverkas av färre faktorer är anpassade för patienter som är över- och underviktiga med mycket varierad kost. / Introduction: A new generation of anticouagulants has emerged called NOAC. NOAC comes with benefits that have previously been problematic for patients treated with anticouagulants such as regular check-ups and dose regulation. Previous anticouagulants, warafarin and heparins, inhibits multiple coagulation factors. While NOAC only inhibits select few factors. For example, Edoxaban only inhibits free factor Xa and prothrombin activity. As result, Edoxaban has a better effect with proper prescription. Edoxaban eliminates through the kidneys which makes renal function tests important – eGFR. Creatinine-based eGFR is calculated via LM-formula which is adapted for the Swedish population. Cystatin-C-based eGFR is calculated via CAPA formula where only age is taken into account whereas the LM-formula relies on multiple factors. Aim: The purpose of the study is to understand the correlation between creatinine- and cystatin-C-based eGFR. Method: blood samples were taken from 43 patient treated with Edoxaban at Karlstad Hospital. eGFR were calculated via CAPA and LM formula. Weight, age and height were taken into account.  Result: A strong correlation shows in LM- and CAPA-eGFR. Conclusion: LM-eGFR is affected by several factors therefore works best for patients with balanced height and weight as well as a stable diet. Patients who are overweight, underweight or has a very varied diet is best fit for CAPA-eGFR which is unaffected by these factors.

Bakgrund: För estimering av glomerulär filtrationshastighet (eGFR) behövs ett blodprov, såsom kreatinin eller cystatin C. En av röntgensjuksköterskans arbetsuppgifter är skattning av GFR innan jodkontrastmedel administreras, på grund av risken för njurbiverkningar. Hos äldre patienter är känsligheten för läkemedel påtaglig på grund av förändrad farmakokinetik. Osäkerhet råder om vilket blodprov som ger noggrannast eGFR.  Syfte: Syftet med studien var att genom litteraturgenomgång undersöka hur markörerna kreatinin respektive cystatin C skattar eGFR hos äldre patienter. Metod: Syftet besvarades av en litteraturstudie med resultat från åtta kvantitativa studier. De ingående studierna analyserades och sammanställdes. Databassökningarna utfördes i PubMed och Cinahl. Resultat: Resultatet visade att skattningarna av eGFR kunde skilja sig signifikant mellan kreatinin och cystatin C, medan andra gånger var de mer liknande. Vid stigande ålder ökade skillnaden mellan skattningarna av eGFR med de två markörerna. Konklusion:  Varken kreatinin eller cystatin C är optimalt. Delar av resultatet pekade mot att kombinationen kan vara en fördel. Mer forskning behövs inom området. / Background: When estimating glomerular filtration rate (eGFR), a blood test is required, such as creatinine or cystatin C. One of the radiographers' assignments is to estimate GFR before administering iodinated contrast agents, due to the risk of renal side effects. In elderly patients, the sensitivity to drugs is palpable due to altered pharmacokinetics. There is uncertainty about which blood test produces the most accurate eGFR. Aim: The aim was to investigate through a literature review how the markers creatinine and cystatin C estimate eGFR in elderly patients. Methods: The aim was answered by a literature study with results from eight quantitative studies, that were found in the databases PubMed and Cinahl. All included studies were analyzed and compiled. Results: The results showed that the estimates of eGFR could differ significantly between creatinine and cystatin C, while other times they were more similar. With an increasing age the two markers showed a larger difference in estimation of GFR. Conclusion: Neither creatinine nor cystatin C is optimal. Parts of the results indicated that the combination may be an advantage. More research is needed in this area.

Inledning: Undersökningar som inkluderar kontrastmedel har ökat på datortomografin (DT). Inför kontrastmedelsundersökningar ska den glomerulära filtrationshastigheten (GFR) estimeras för att få ett mått på patientens njurfunktion. I nuläget finns det två olika markörer som kan användas till detta, kreatinin och cystatin C. Det är röntgensjuksköterskans ansvar att skatta GFR för att kunna göra en bedömning om patienten kan utföra undersökningen. Syfte: Syftet med studien var att sammanställa studier som jämför kreatinin och cystatin C vid skattning av GFR. Metod: Den här studien genomfördes som en allmän litteraturöversikt. Litteratursökningen genomfördes i databaserna CINAHL, PubMed och SveMed+. Tio kvantitativa vetenskapliga artiklar lokaliserades och gick vidare till analys. Resultat: Resultatet visade att cystatin C i många fall var en bättre indikator för att estimera GFR. Slutsats: För äldre och njursjuka ansågs cystatin C vara en bättre markör för njurfunktionen. Dock anser författarna att det krävs vidare forskning inom ämnet och dess påverkande faktorer för att kunna introducera cystatin C som ny njurfunktionsmarkör.