Ketoprofeenin biologinen käytettävyys ja farmakokinetiikka sioilla eri antotavoilla ja ketoprofeeniyliannoksen farmakokinetiikka ja vaikutus munuaisiin lampailla

Ketoprofeeni on yleisesti käytetty ei-steroidinen tulehduskipulääke (NSAID) lampaiden ja sikojen kivunlievityksessä. Tietoa ketoprofeenin oikeista annosmääristä eri eläinlajeilla on saatavilla rajallisesti. Oikeaa lääkeainemäärää ei voida luotettavasti ekstrapoloida toisten eläinlajien tai ihmisten perusteella. Epäillyissä tulehduskipulääkemyrkytyksissä ongelmana on tietää, oliko eläimen saama lääkeannos toksinen. Lampailla tehdyn tutkimuksen tavoitteena oli selvittää, muuttuuko ketoprofeenin kinetiikka kymmenkertaisella yliannoksella, tutkia yliannoksen vaikutusta munuaisiin ja löytää yksinkertainen tapa diagnosoida yliannos virtsasta. Sioilla tehdyn tutkimuksen tavoitteena oli selvittää ketoprofeenin biologista käytettävyyttä ja ketoprofeenin farmakokinetiikkaa sioilla intravaskulaarisella, intramuskulaarisella ja peroraalisella annolla. Keskeiset tutkimuksessa määritettävät muuttujat olivat AUC0-_, Cmax ja tmax. Hyötyosuus laskettiin i.v. -annon perusteella. Kuudelle lampaalle annettiin 30 mg/kg i.v. -ketoprofeenia. Ketoprofeenin pitoisuuksia seurattiin 24 tunnin ajan plasmanäytteillä, joiden perusteella määritettiin farmakokineettiset parametrit. Veri- ja virtsanäytteistä tutkittiin muun muassa mahdollisesta munuaisvauriosta kertovia entsyymejä. 24 tunnin kuluttua lääkkeenannosta lampaat lopetettiin ja munuaiset tutkittiin histologisesti. Tutkittaville kahdeksalle sialle annosteltiin 3 mg/kg intravaskulaarista, intramuskulaarista ja oraalista ketoprofeenia sekä 6 mg/kg oraalista ketoprofeenia. Tutkimus suoritettiin satunnaistettuna vaihtovuorotutkimuksena. Ketoprofeenin pitoisuuksia seurattiin plasmanäytteillä 48 tunnin ajan lääkkeenannosta ja kaikille antotavoille laskettiin farmakokineettiset parametrit. Lisäksi tutkittiin valmisteiden biologinen samanarvoisuus. Molempien tutkimusten in vivo -kokeet suoritettiin Eläinlääketieteellisessä tiedekunnassa. Samoin munuaisten histologinen tutkimus ja virtsasta ja verestä tehdyt määritykset, lukuun ottamatta ketoprofeeninpitoisuuden analysointia. Plasman ketoprofeenipitoisuus analysoitiin korkean erotuskyvyn nestekromatografialla (HPLC). Ketoprofeenimääritykset ja farmakokineettinen analyysi suoritettiin Farmasian tiedekunnassa. Lampaiden kymmenkertainen ketoprofeeniyliannos oli toksinen. Seerumin urea- ja kreatiniinipitoisuus nousivat ja histologisissa näytteissä näkyi akuutti munuaistiehyen vaurio. Useiden entsyymien pitoisuus nousi virtsassa. Selvimmin ja nopeimmin nousi virtsan laktaattidehydrogenaasipitoisuus, jonka määrittäminen vaikuttaa potentiaaliselta tavalta diagnosoida ketoprofeenin toksinen annos. Ketoprofeenin eliminaation puoliintumisaika toksisella annoksella oli samaa suuruusluokkaa kuin aiemmissa tutkimuksissa terapeuttisella annoksella, joten yliannos ei muuttanut ketoprofeenin kinetiikkaa. AUC- ja Cmax -arvot olivat suhteessa suurempia kuin terapeuttisella annoksella, joten tutkimuksen perusteella kyseiset arvot eivät nousseet lineaarisesti annoksen noustessa toksiseksi. Sioille annetut ketoprofeenivalmisteet eivät olleet biologisesti samanarvoisia keskenään. Hyötyosuus oli erittäin hyvä kaikilla antotavoilla. tmax oli kaikilla antotavoilla hieman yli tunnin kuluttua lääkkeenannosta. Oraalisen 3 mg/kg -annoksen Cmax oli 5,1 mg/l ja AUC 32 mg l-1 h ja intramuskulaarisen vastaavat arvot olivat 7,6 mg/l ja 37 mg l-1 h. Oraalisen ketoprofeenin annostasojen AUC- ja Cmax -arvot korreloivat keskenään, joten ketoprofeenin kinetiikka oli lineaarista. Intravaskulaarisen ja oraalisen annon puoliintumisajoissa oli tilastollisesti merkitsevä ero. Ketoprofeenin jakautumistilavuudessa ja puhdistumassa ei ollut tilastollisesti merkitsevää eroa eri antotapojen välillä.

Moonlighting Proteins of Lactobacillus crispatus : Extracellular Localization, Cell Wall Anchoring and Interactions with the Host

Moonlighting functions have been described for several proteins previously thought to localize exclusively in the cytoplasm of bacterial or eukaryotic cells. Moonlighting proteins usually perform conserved functions, e. g. in glycolysis or as chaperonins, and their traditional and moonlighting function(s) usually localize to different cell compartments. The most characterized moonlighting proteins in Grampositive bacteria are the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which function in bacteria-host interactions, e. g. as adhesins or plasminogen receptors. Research on bacterial moonlighting proteins has focused on Gram-positive bacterial pathogens, where many of their functions have been associated with bacterial virulence. In this thesis work I show that also species of the genus Lactobacillus have moonlighting proteins that carry out functions earlier associated with bacterial virulence only. I identified enolase, GAPDH, glutamine synthetase (GS), and glucose-6-phosphate isomerase (GPI) as moonlighting proteins of Lactobacillus crispatus strain ST1 and demonstrated that they are associated with cell surface and easily released from the cell surface into incubation buffer. I also showed that these lactobacillar proteins moonlight either as adhesins with affinity for basement membrane and extracellular matrix proteins or as plasminogen receptors. The mechanisms of surface translocation and anchoring of bacterial moonlighting proteins have remained enigmatic. In this work, the surface localization of enolase, GAPDH, GS and GPI was shown to depend on environmental factors. The members of the genus Lactobacillus are fermentative organisms that lower the ambient pH by producing lactic acid. At acidic pH enolase, GAPDH, GS and GPI were associated with the cell surface, whereas at neutral pH they were released into the buffer. The release did not involve de novo protein synthesis. I showed that purified recombinant His6-enolase, His6-GAPDH, His6-GS and His6-GPI reassociate with cell wall and bind in vitro to lipoteichoic acids at acidic pH. The in-vitro binding of these proteins localizes to cell division septa and cell poles. I also show that the release of moonlighting proteins is enhanced in the presence of cathelicidin LL- 37, which is an antimicrobial peptide and a central part of the innate immunity defence. I found that the LL-37-induced detachment of moonlighting proteins from cell surface is associated with cell wall permeabilization by LL-37. The results in this thesis work are compatible with the hypothesis that the moonlighting proteins of L. crispatus associate to the cell wall via electrostatic or ionic interactions and that they are released into surroundings in stress conditions. Their surface translocation is, at least in part, a result from their release from dead or permeabilized cells and subsequent reassociation onto the cell wall. The results of this thesis show that lactobacillar cells rapidly change their surface architecture in response to environmental factors and that these changes influence bacterial interactions with the host.

Production of Carcinogenic Acetaldehyde by Oral Microbiome

Oral cancer is the seventh most common cancer worldwide and its incidence is increasing. The most important risk factors for oral cancer are chronic alcohol consumption and tobacco smoking, up to 80 % of oral carcinomas are estimated to be caused by alcohol and tobacco. They both trigger an increased level of salivary acetaldehyde, during and after consumption, which is believed to lead to carcinogenesis. Acetaldehyde has multiple mutagenic features and it has recently been classified as a Group 1 carcinogen for humans by the International Agency for Research on Cancer. Acetaldehyde is metabolized from ethanol by microbes of oral microbiota. Some oral microbes possess alcohol dehydrogenase enzyme (ADH) activity, which is the main enzyme in acetaldehyde production. Many microbes are also capable of acetaldehyde production via alcohol fermentation from glucose. However, metabolism of ethanol into acetaldehyde leads to production of high levels of this carcinogen. Acetaldehyde is found in saliva during and after alcohol consumption. In fact, rather low ethanol concentrations (2-20mM) derived from blood to saliva are enough for microbial acetaldehyde production. The high acetaldehyde levels in saliva after alcohol challenge are explained by the lack of oral microbiota and mucosa to detoxify acetaldehyde by metabolizing it into acetate and acetyl coenzymeA. The aim of this thesis project was to specify the role of oral microbes in the in vitro production of acetaldehyde in the presence of ethanol. In addition, it was sought to establish whether microbial metabolism could also produce acetaldehyde from glucose. Furthermore, the potential of xylitol to inhibit ethanol metabolism and acetaldehyde production was explored. Isolates of oral microbes were used in the first three studies. Acetaldehyde production was analyzed after ethanol, glucose and fructose incubation with gas chromatography measurement. In studies I and III, the ADH enzyme activity of some microbes was measured by fluorescence. The effect of xylitol was analyzed by incubating microbes with ethanol and xylitol. The fourth study was made ex vivo and microbial samples obtained from different patient groups were analyzed. This work has demonstrated that isolates of oral microbiota are able to produce acetaldehyde in the presence of clinically relevant ethanol and glucose concentrations. Significant differences were found between microbial species and isolates from different patient groups. In particular, the ability of candidal isolates from APECED patients to produce significantly more acetaldehyde in glucose incubation compared to healthy and cancer patient isolates is an interesting observation. Moreover, xylitol was found to reduce their acetaldehyde production significantly. Significant ADH enzyme activity was found in the analyzed high acetaldehyde producing streptococci and candida isolates. In addition, xylitol was found to reduce the ADH enzyme activity of C. albicans. Some results from the ex vivo study were controversial, since acetaldehyde production did not correlate as expected with the amount of microbes in the samples. Nevertheless, the samples isolated from patients did produce significant amounts of acetaldehyde with a clinically relevant ethanol concentration.