Biomarkers - regulatory and toxicological perspectives

Referaatti kongressiesitelmästä.

Novel Cellular Luminescence Probes for Immunological and Toxicological Assessments

Escherichia coli K-12 (pEGFPluxABCDEAmp) (E. coli-lux), constitutively emitting bioluminescence (BL), was constructed and its BL emitting properties tested in different growth and killing conditions. The BL emission directly correlated with the number of viable E. coli-lux cells, and when subjected to the antimicrobial agent, the diminishment of the BL signal was linked directly to the number of killed bacterial cells. The method provided a very convenient application, especially when compared to conventional plate counting assays. This novel real-time based method was utilized in both immunological and toxicological assessments. The parameters such as the activation phase, the lytic phase and the capacity of the killing of the serum complement system were specified not only in humans but also in other species. E. coli-lux was also successfully used to study the antimicrobial activities of insect haemolymph. The mechanisms of neutrophil activity, like that of a myeloperoxidase (MPO)-H2O2-halide system, were studied using the E. coli-lux approach. The fundamental role of MPO was challenged, since during the actual killing in described circumstances in phagolysosome the MPO system was inactivated and chlorination halted. The toxicological test system, assessing indoor air total toxicity, particularly suitable for suspected mold damages, was designed based on the E. coli-lux method. Susceptibility to the vast number of various toxins, both pure chemicals and dust samples from the buildings and extracts from molds, were investigated. The E. coli-lux application was found to possess high sensitivity and specificity attributes. Alongside the analysis system, the sampling kit for indoor dust was engineered based on the swipe stick and the container. The combination of practical specimen collector and convenient analysis system provided accurate toxic data from the dust sample within hours. Neutrophils are good indicators of the pathophysiological state of the individual, and they can be utilized as a toxicological probe due to their ability to emit chemiluminescence (CL). Neutrophils can either be used as probe cells, directly exposed to the agent studied, or they can act as indicators of the whole biological system exposed to the agent. Human neutrophils were exposed to the same toxins as tested with the E. coli-lux system and measured as luminol amplified CL emission. The influence of the toxins on the individuals was investigated by exposing rats with moniliniformin, the mycotoxin commonly present in Finnish grains. The activity of the rat neutrophils was found to decrease significantly during the 28 days of exposure.

Unsaturated Polyester Coatings containing Biocides

Vesistöissä laivojen pintaan tarttuvat eliöt ovat sekä taloudellinen että kosmeettinen ongelma. Kontrolloimattoman eliöiden kiinnittymisen seurauksena aiheutuu kitkaa, joka puolestaan hidastaa laivan nopeutta ja aiheuttaa polttoaineen kulutuksen kasvua. Tavallisesti eliöiden kiinnittymistä ehkäistään kiinnittymisenestomaalien avulla. Niiden toiminta perustuu biosidien liukenemiseen, jolloin veden ja pinnoitteen väliselle rajapinnalle muodostuu korkea biosidipitoisuus, joka estää eliöiden kiinnittymistä pinnalle. Maailmanlaajuinen orgaanisten tinayhdisteiden käyttökielto kiinnittymisen-estomaaleissa tulee voimaan vuoden 2003 alusta. Tällä hetkellä 70 % maailman laivastoista on suojattu orgaanista tinayhdistettä sisältävällä kiinnittymisenestomaalilla. Nyt onkin kasvava tarve kehittää uusia ympäristöystävällisempiä kiinnittymisenesto-pinnoitteita. Todennäköisesti tinayhdisteet tullaan korvaamaan synteettisillä orgaanisilla yhdisteillä käytettyinä yhdessä kuparin kanssa. Työn tarkoituksena oli valmistaa ympäristöystävällisempi tyydyttämätön polyesteripinnoite, joka itsessään ehkäisisi eliöiden kiinnittymistä. Kirjallisuusosassa tutustuttiin markkinoilla oleviin biosideihin, niiden myrkyllisyyteen ja vaikutuksiin ympäristölle sekä muuttuvaan lainsäädäntöön. Työssä tarkasteltiin myös tällä hetkellä markkinoilla olevia pinnoitteita ja niiden toimintamekanismeja sekä myrkyttömiä vaihtoehtopinnoitteita kiinnittymisenestoon. Kokeellinen osa koostui kahdesta osasta. Ensimmäisessä osassa tutkittiin biosidien sopivuutta käytettäväksi yhdessä tyydyttymättömän polyesterin kanssa. Yhteensopivuutta määritettiin applikaatiotesteillä ja pinnoitteen käyttäytymisen perusteella. Toinen vaihe oli selvittää pinnoitteen tehokkuus leväntarttumista vastaan. Tyydyttymätön polyesteri gel coat kiinnittymisenesto-ominaisuuksilla valmistettiin dispergoimalla biosideja tyydyttymättömään polyesterigeeliin. Yhteensopivuustestien tulosten perusteella huomattiin, ettei biosidien lisääminen geeliin vaikuta mainittavasti applikaatio-ominaisuuksien huononemiseen. Brookfield viskositeetin stabiilisuus jopa paranee ja yksi työssä käytetyistä biosideista parantaa pinnoitteen säänkestoominaisuuksia. Tässä työssä ei pystytty määrittämään eri biosidien välisiä eroja tehokkuudessa levää vastaan.

Thermoplastic bioactive composite – with special reference to dissolution behaviour and tissue response

Bioactive glasses are surface-active ceramic materials which support and accelerate bone growth in the body. During the healing of a bone fracture or a large bone defect, fixation is often needed. The aim of this thesis was to determine the dissolution behaviour and biocompatibility of a composite consisting of poly(ε-caprolactone-co-DL-lactide) and bioactive glass (S53P4). In addition the applicability as an injectable material straight to a bone defect was assessed. In in vitro tests the dissolution behaviour of plain copolymer and composites containing bioactive glass granules was evaluated, as well as surface reactivity and the material’s capability to form apatite in simulated body fluid (SBF). The human fibroblast proliferation was tested on materials in cell culture. In in vivo experiments, toxicological tests, material degradation and tissue reactions were tested both in subcutaneous space and in experimental bone defects. The composites containing bioactive glass formed a unified layer of apatite on their surface in SBF. The size and amount of glass granules affected the degradation of polymer matrix, as well the material’s surface reactivity. In cell culture on the test materials the human gingival fibroblasts proliferated and matured faster compared with control materials. In in vitro tests a connective tissue capsule was formed around the specimens, and became thinner in the course of time. Foreign body cell reactions in toxicological tests were mild. In experimental bone defects the specimens with a high concentration of small bioactive glass granules (<45 μm) formed a dense apatite surface layer that restricted the bone ingrowth to material. The range of large glass granules (90-315 μm) with high concentrations formed the best bonding with bone, but slow degradation on the copolymer restricted the bone growth only in the superficial layers. In these studies, the handling properties of the material proved to be good and tissue reactions were mild. The reactivity of bioactive glass was retained inside the copolymer matrix, thus enabling bone conductivity with composites. However, the copolymer was noticed to degradate too slowly compared with the bone healing. Therefore, the porosity of the material should be increased in order to improve tissue healing.

NIR-Vis Up-Conversion Luminescence in the Yb3+,Er3+ Doped Y2O2S, ZrO2, and NaYF4 Nanomaterials

Since the discovery of the up-conversion phenomenon, there has been an ever increasing interest in up-converting phosphors in which the absorption of two or more low energy photons is followed by emission of a higher energy photon. Most up-conversion luminescence materials operate by using a combination of a trivalent rare earth (lanthanide) sensitizer (e.g. Yb or Er) and an activator (e.g. Er, Ho, Tm or Pr) ion in a crystal lattice. Up-converting phosphors have a variety of potential applications as lasers and displays as well as inks for security printing (e.g. bank notes and bonds). One of the most sophisticated applications of lanthanide up-conversion luminescence is probably in medical diagnostics. However, there are some major problems in the use of photoluminescence based on the direct UV excitation in immunoassays. Human blood absorbs strongly UV radiation as well as the emission of the phosphor in the visible. A promising way to overcome the problems arising from the blood absorption is to use a long wavelength excitation and benefit from the up-conversion luminescence. Since there is practically no absorption by the whole-blood in the near IR region, it has no capability for up-conversion in the excitation wavelength region of the conventional up-converting phosphor based on the Yb3+ (sensitizer) and Er3+ (activator) combination. The aim of this work was to prepare nanocrystalline materials with high red (and green) up-conversion luminescence efficiency for use in quantitative whole-blood immunoassays. For coupling to biological compounds, nanometer-sized (crystallite size below 50 nm) up-converting phosphor particles are required. The nanocrystalline ZrO2:Yb3+,Er3+, Y2O2S:Yb3+,Er3+, NaYF4:Yb3+,Er3+ and NaRF4-NaR’F4 (R: Y, Yb, Er) materials, prepared with the combustion, sol-gel, flux, co-precipitation and solvothermal synthesis, were studied using the thermal analysis, FT-IR spectroscopy, transmission electron microscopy, EDX spectroscopy, XANES/EXAFS measurements, absorption spectroscopy, X-ray powder diffraction, as well as up-conversion and thermoluminescence spectroscopies. The effect of the impurities of the phosphors, crystallite size, as well as the crystal structure on the up-conversion luminescence intensity was analyzed. Finally, a new phenomenon, persistent up-conversion luminescence was introduced and discussed. For efficient use in bioassays, more work is needed to yield nanomaterials with smaller and more uniform crystallite sizes. Surface modifications need to be studied to improve the dispersion in water. On the other hand, further work must be carried out to optimize the persistent up-conversion luminescence of the nanomaterials to allow for their use as efficient immunoassay nanomaterials combining the advantages of both up-conversion and persistent luminescence.