Effects of Dietary Fat Oxidation Products and Flavonols on Lipoprotein Oxidation

Various studies suggest that oxidative modifications of low density lipoprotein (LDL), and also other lipoproteins, have an important role in the development of atherosclerosis. In addition to the oxidation products formed endogenously, oxidised triacylglycerols (TAG) and oxysterols in the diet contribute to the oxidised lipoproteins found in circulation. However, studies on both the effect of oxidised dietary lipids on lipoprotein lipid oxidation and the reactions that modify oxidised fat after ingestion have been scarce. Studies on the effects of dietary antioxidants on the lipid oxidation in vivo and the risk of atherosclerosis have been inconclusive. More clinical trials are needed to test the importance of lipoprotein oxidation as a cardiovascular risk factor in humans. In the recent years, various methods have been optimised and applied to the analysis of lipid oxidation products in vivo, and information on the molecular structures of oxidised lipids in plasma, lipoproteins and atherosclerotic plaques has started to accumulate. However, specific structures of oxidised TAG molecules present in these tissues and lipoprotein fractions have not been investigated earlier. In the orginal research in this thesis, an approach based on highperformance liquid chromatographyelectrospray ionisationmass spectrometry (HPLCESIMS) and baseline diene conjugation (BDC) methods was used in order to investigate lipid oxidation level and oxidised TAG molecular structures in pig and human lipoproteins after dietary interventions. The approach was optimised with human LDL samples, which contained various oxidation products of TAG. LDL particles of hyperlipidaemic subjects contained an elevated amount of conjugated dienes. In the pig studies, several oxidised TAG structures with hydroxy, keto, epoxy or aldehydic groups were found in chylomicrons and VLDL after diets rich in sunflower seed oil. Also, the results showed that oxidised sunflower seed oil increased the oxidation of lipoprotein lipids and their TAG molecules. TAG hydroperoxides could be detected neither in the small intestinal mucosa of the pigs fed on the oxidised oil nor in their chylomicrons or VLDL.6 In the clinical studies, dietary flavonol aglycones extracted from sea buckthorn berries did not have an effect on lipoprotein lipid oxidation and other potential risk factors of atherosclerosis, but their absorption was demonstrated. Oil supplementation seemed to increase the bioavailability of the flavonols. Oxidised TAG molecules were detected in LDL particles of the subjects after both flavonol and control diets.

Early Life Intestinal Microbiota in Health and in Atopic Eczema

Decrease in microbial contacts in affluent societies is considered to lie behind the rise in allergic and other chronic inflammatory diseases during the last decades. Indeed, deviations in the intestinal microbiota composition and diversity have been associated with several diseases, such as atopic eczema. However, there is no consensus yet on what would constitute a beneficial or harmful microbiota. The aim of this thesis was to study the microbiota development in healthy infants and to characterize intestinal microbiota signatures associated with disease status and severity in infants with atopic eczema. The methodological aim was to compare and optimize methods for DNA extraction from fecal samples to be used in high-throughput microbiota analyses. It was confirmed that the most critical step in successful microbial DNA extraction from fecal samples is the mechanical cell lysis procedure. Based on this finding, an efficient semi-automated extraction process was developed that can be scaled for use in high-throughput platforms such as phylogenetic microarray used in this series of studies. By analyzing a longitudinal motherchild cohort for 3 years it was observed that the microbiota development is a gradual process, where some bacterial groups reach the degree of adult-type pattern earlier than others. During the breast-feeding period, the microbiota appeared to be relatively simple, while major diversification was found to start during the weaning process. By the age of 3 years, the child’s microbiota composition started to resemble that of an adult, but the bacterial diversity has still not reached the full diversity, indicating that the microbiota maturation extends beyond this age. In addition, at three years of age, the child’s microbiota was more similar to mother’s microbiota than to microbiota of nonrelated women.In infants with atopic eczema, a high total microbiota diversity and abundance of butyrate-producing bacteria was found to correlate with mild symptoms at 6 months. At 18 months, infants with mild eczema had significantly higher microbiota diversity and aberrant microbiota composition when compared to healthy controls at the same age. In conclusion, the comprehensive phylogenetic microarray analysis of early life microbiota shows the synergetic effect of vertical transmission and shared environment on the intestinal microbiota development. By the age of three years, the compositional development of intestinal microbiota is close to adult level, but the microbiota diversification continues beyond this age. In addition, specific microbiota signatures are associated with the existence and severity of atopic eczema and intestinal microbiota seems to have a role in alleviating the symptoms of this disease.

Roles of keratins in intestinal health and disease

Intermediate filament keratins (K) play a pivotal role in protein targeting and epithelialcytoprotection from stress as evidenced by keratin mutations predisposing to human liver and skin diseases and possibly inflammatory bowel disease (IBD). The K8-null (K8-/-) mice exhibit colonic phenotype similar to IBD and marked spontaneous colitis, epithelial hyperproliferation, decreased apoptosis, mistargeting of proteins leading to defective ion transport and diarrhea. The K8-heterozygote (K8+/-) mouse colon appears normal but displays a defective sodium (Na+) and chloride (Cl-) transport similar to, but milder than K8-/-. Characterization of K8+/- colon revealed ~50% less keratins (K7, K8, K19, K20) compared to K8 wild type (K8+/+). A similar ~50% decrease was seen in K8+/- mRNA levels as compared to K8+/+, while the mRNA levels for the other keratins were unaltered. K8+/- keratins were arranged in a normal colonic crypt expression pattern, except K7 which was expressed at the top of crypts in contrast to K8+/+. The K8+/- colon showed mild hyperplasia but no signs of inflammation and no resistance to apoptosis. Experimental colitis induced by using different concentrations of dextran sulphate sodium (DSS) showed that K8+/- mice are slightly more sensitive to induced colitis and showed a delayed recovery compared to K8+/+. Hence, the K8+/- mouse with less keratins and without inflammation, provided a novel model to study direct molecular mechanisms of keratins in intestinal homeostasis and ion transport. Different candidate ion transporters for a possible role in altered ion transport seen in the K8-/- and K8+/- mouse colon were evaluated. Besides normal levels of CFTR, PAT-1 and NHE-3, DRA mRNA levels were decreased 3-4-fold and DRA protein nearly entirely lost in K8-/- caecum, distal and proximal colon compared to K8+/+. In K8+/- mice, DRA mRNA levels were unaltered while decreased DRA protein level and patchy distribution was detected particularly in the proximal colon and as compared to K8+/+. DRA was similarly decreased when K8 was knocked-down in Caco-2 cells, confirming that K8 levels modulate DRA levels in an inflammation-independent manner. The dramatic loss of DRA in colon and caecum of K8-/- mice was responsible for the chloride transport defect. The milder ion transport in K8+/- colon might be related to DRA suggesting a role for K8 in regulation of DRA expression and targeting. The current study demonstrates the importance of keratins in stress protection and cell signaling. Furthermore, we have also successfully developed a novel, simple, fast, cost effective, non-invasive in vivo imaging method for the early diagnosis of murine colitis with specificity for both genetic and experimental colitis. The said modality provides continuous measurements of reactive oxygen and nitrogen species (RONS) and minimizes the use of an increased number of experimental animals by using a luminal derivative chemiluminescent probe, L-012 which provides a cost-effective tool to study the level and longitudinal progression of colitis.

Carriage of herpes simplex virus and human papillomavirus in oral mucosa is rare in young women: a long-term prospective follow-up

Herpes simplex -virus 1 (HSV-1) ja ihmisen papilloomavirus (HPV) infektoivat suun epiteelisoluja. Useimmissa tapauksissa näiden virusten infektio on kliinisesti oireeton. Suun limakalvojen oireeton HPV-infektio on aikuisilla yleinen, ja valtaosa näistä infektioista ovat ohimeneviä ja vain pieni osa johtaa krooniseen HPV-infektioon. Krooninen HPV-infektio lisää riskiä epiteelisolujen transformoitumiseen kohti syöpäsolua. HPV infektio ei ole yksinään riittävä aiheuttamaan syöpäsolun, vaan siihen tarvitaan myös muita riskitekijöitä.. Muut infektiot, kuten esimerkiksi HSV-1-infektio saattaa olla yksi näistä riskitekijöistä. Tämän syventävän opinnäytetyön tutkimuksen tavoitteena oli selvittää HSV-1-infektion esiintymistä naisten suun limakalvonäytteissä kuuden vuoden seurannassa. Toinen tavoitteemme oli selvittää HSV-1 ja HPV -yhteisinfektion esiintymistä suun epiteelisoluissa, erityisesti naisilla joilla havaittiin suun krooninen HPV-infektio. Tämä tutkimus on osa kuusivuotista seurantatutkimusta (Finnish Family HPV -tutkimus), joka suunniteltiin selvittämään HPV-infektioiden dynamiikkaa ja riskitekijöitä 329 suomalaisperheessä. Suun limakalvoilta otettiin harjausnäyte ennen synnytystä ja kuusi kertaa synnytyksen jälkeen kuuden vuoden aikana. Näytteistä eristettyä DNA:ta oli saatavilla riittävästi HSV-1-analyysiä varten yhteensä 304 naiselta (keski-ikä 25.6 vuotta). Kaikkiaan tutkittavia näytteitä oli 1,873, joista HSV-1:n esiintyminen tutkittiin käyttäen kvantitatiivista PCR:ää. Lisäksi epävarmat PCR-tulokset varmistettiin PCR-tuotteen Southern Blot hybridisaatiolla. HSV-1 tuloksia verrattiin aikaisemmin saatuihin HPV-tuloksiin. Suun limakalvonäytteistä 2.2% oli HSV-1-positiivisia ja 19.6% oli HPV-positiivisia. Yhteensä 11.8%:lla äideistä suunäyte oli HSV-1-DNA -positiivinen ainakin kerran seurannan aikana. HSV-1 ja HPV -yhteisinfektio löydettiin vain neljältä äidiltä. Suurin osa äideistä, jotka olivat HSV-1-positiivisia ennen syntymää, pysyivät HSV-1-positiivisina myös synnytyksen jälkeen. Kolmella naisella todettiin persistentti HPV-16 infektio ja kahdella heistä oli samanaikainen HSV-1 infektio. Tuloksemme osoittaa, että HSV-1:n ja HPV:n esiintyminen yhtä aikaa nuorten naisten suun limakalvolla on harvinaista. On suositeltavaa, että yhteisinfektion omaavia äitejä seurataan myös tulevaisuudessa, sillä HSV-1-infektio saattaa olla yksi riskitekijä suun limakalvon malignissa muutoksessa kroonisen HPV-infektion yhteydessä.