Diet and Probiotics during Pregnancy - Endorsing developmental Programming

Maternal obesity has been shown to increase the risk for adverse reproductive health outcomes such as gestational diabetes, hypertension, and preeclampsia. Moreover, several studies have indicated that overnutrition and maternal obesity adversely program the development of offspring by predisposing them to obesity and other chronic diseases later in life. The exact molecular mechanisms leading to developmental programming are not known, but it has recently been suggested that obesity-related low-grade inflammation, gut microbiota and epigenetic gene regulation (in particularly DNA methylation) participate in the developmental programming phenomenon. The aim of this thesis was to evaluate the effect of diet, dietary counseling and probiotic intervention during pregnancy in endorsing favorable developmental programming. The study population consisted of 256 mother-child pairs participating in a prospective, double-blinded dietary counselling and probiotic intervention (Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb12) NAMI (Nutrition, Allergy, Mucosal immunology and Intestinal microbiota) study. Further overweight women were recruited from maternal welfare clinics in the area of Southwest Finland and from the prenatal outpatient clinic at Turku University Hospital. Dietary counseling was aimed to modify women’s dietary intake to comply with the recommended intake for pregnant women. Specifically, counseling aimed to affect the type of fat consumed and to increase the amount of fiber in the women’s diets. Leptin concentration was used as a marker for obesity-related low-grade inflammation, antioxidant vitamin status as an efficiency marker for dietary counselling and epigenetic DNA methylation of obesity related genes as a marker for probiotics influence. Results revealed that dietary intake may modify obesity-associated low-grade inflammation as measured by serum leptin concentration. Specifically, dietary fiber intake may lower leptin concentration in women, whereas the intakes of saturated fatty acids and sucrose have an opposite effect. Neither dietary counselling nor probiotic intervention modified leptin concentration in women, but probiotics tended to increase children’s leptin concentration. Dietary counseling was an efficient tool for improving antioxidant vitamin intake in women, which was reflected in the breast milk vitamin concentration. Probiotic intervention affected DNA methylation of dozens of obesity and weight gain related genes both in women and their children. Altogether these results indicate that dietary components, dietary counseling and probiotic supplementation during pregnancy may modify the intrauterine environment towards favorable developmental programming.

Toll-like receptor 9 in breast cancer

The innate immune system recognizes microbial features leading to the activation of the adaptive immune system. The role of Toll-like receptor 9 (TLR9) is to recognize microbial DNA. In addition to immune cells, TLR9 is widely expressed in breast cancer in addition to other cancers. Breast cancer is the most common cancer in women, affecting approximately one in eight in industrialized countries. In the clinical setting, breast cancer is divided into three clinical subtypes with type-specific treatments. These subtypes are estrogen receptor (ER)-positive, HER2-positive and triple-negative (TNBC) breast cancer. TNBC is the most aggressive subtype that can be further divided into several subtypes. TNBC tumors lack ER, progesterone receptor and HER2 receptor. Therefore, the current clinically used targeted therapies are not suitable for TNBC treatment as TNBC is a collection of diseases rather than one entity. Some TNBC patients are cured with standard chemotherapy, while others rapidly die due to the disease. There are no clinically used iomarkers which would help in predicting which patients respond to chemotherapy. During this thesis project, we discovered a novel good-prognosis TNBC subtype. These tumors have high TLR9 expression levels. Our findings suggest that TLR9 screening in TNBC patient populations might help to identify the patients that are at the highest risk regarding a relapse. To gain better understanding on the role of TLR9 in TNBC, we developed an animal model which mimicks this disease. We discovered that suppression of TLR9 expression in TNBC cells increases their invasive properties in hypoxia. In line with the clinical findings, TNBC cells with low TLR9 expression also formed more aggressive tumors in vivo. TLR9 expression did not, however, affect TNBC tumor responses to doxorubicin. Our results suggest that tumor TLR9 expression may affect chemotherapyrelated immune responses, however, this requires further investigation. Our other findings revealed that DNA released by chemotherapy-killed cells induces TLR9-mediated invasion in living cancer cells. Normally, extracellular self-DNA is degraded by enzymes, but during massive cell death, for example during chemotherapy, the degradation machinery may be exhausted and self-DNA is taken up into living cells activating TLR9. We also discovered that the malaria drug chloroquine, an inhibitor of autophagy and TLR9 signalling does not inhibit TNBC growth in vivo, independently of the TLR9 status. Finally, we found that ERα as well as the sex hormones estrogen and testosterone regulate TLR9 expression and activity in breast cancer cells in vitro. As a conclusion, we suggest that TLR9 is a potential biomarker in TNBC. ------- Sisäsyntyisen immuniteetin tehtävä on tunnistaa mikrobien molekyylirakenteita, mikä saa aikaan adaptiivisen immuunijärjestelmän aktivoitumisen. Tollin kaltainen reseptori 9 (TLR9) on dna:ta tunnistava sisäsyntyisen immuniteetin reseptori, jota ilmennetään myös useissa syövissä, kuten rintasyövässä. Rintasyöpä on naisten yleisin syöpä, johon joka kahdeksas nainen sairastuu elämänsä aikana. Kliinisesti rintasyöpä jaotellaan kolmeen alatyyppiin, joista kolmoisnegatiivinen rintasyöpä on aggressiivisin. Tämän tyypin syövät eivät ilmennä hormonireseptoreja (estrogeeni- ja progesteronireseptori) tai HER2-reseptoria. Tästä johtuen kolmoisnegatiivisten potilaiden hoitoon ei voida käyttää rintasyövän nykyisten hoitosuositusten mukaisia täsmähoitoja. Kolmoisnegatiivinen rintasyöpä ei kuitenkaan ole yksi sairaus, koska molekyylitasolla sen on osoitettu koostuvan lukuisista, biologialtaan erilaisista syöpämuodoista. Tällä hetkellä kliinisessä käytössä ei ole biomarkkeria, jonka avulla kolmoisnegatiivisen rintasyövän alatyypit voisi erottaa toisistaan. Löysimme uuden kolmoisnegatiivisen syövän alatyypin, joka ilmentää vain vähän TLR9-proteiinia. Tällä alatyypillä on erittäin huono ennuste ja tulostemme perusteella TRL9-tason selvittäminen voisi seuloa huonoennusteiset syövät kolmoisnegatiivisten syöpien joukosta. Kehitimme eläinmallin, jolla voidaan tutkia matalan ja korkean TLR9-tason vaikutuksia kolmoisnegatiivisten kasvainten hoitovasteeseen. Toinen löytömme oli, että kemoterapialla tapettujen syöpäsolujen dna saa aikaan elävien syöpäsolujen TLR9-välitteistä invaasiota. Normaalisti entsyymit hajoittavat yksilön oman solunulkoisen dna:n. Erikoistilanteissa, kuten syöpähoitojen yhteydessä, jolloin solukuolema on massiivista, elimistön oma koneisto ei ehdi tuhoamaan solunulkoista dna:ta ja sitä voi kertyä eläviin soluihin, joissa se aktivoi TLR9:n. Kolmanneksi havaitsimme, että malarialääke klorokiini, joka estää TLR9:n toimintaa ja jolla on syövänvastaisia vaikutuksia soluviljelyolosuhteissa, ei estänyt TLR9-positiivisten tai TLR9-negatiivisten kasvainten kasvua käyttämässämme eläinmallissa. Neljänneksi soluviljelykokeittemme tulokset osoittivat, että sukupuolihormonit estrogeeni ja testosteroni sekä estrogeenireseptori osallistuvat TLR9:n ilmentymisen ja aktiivisuuden säätelyyn. Tuloksemme osoittavat, että TLR9 potentiaalinen biomarkkeri kolmoisnegatiivisessa rintasyövässä.

Optimization of immunofluorescence protocols for detection of biomarkers in colorectal and breast cancer tissues

Cancer remains an undetermined question for modern medicine. Every year millions of people ranging from children to adult die since the modern treatment is unable to meet the challenge. Research must continue in the area of new biomarkers for tumors. Molecular biology has evolved during last years; however, this knowledge has not been applied into the medicine. Biological findings should be used to improve diagnostics and treatment modalities. In this thesis, human formalin-fixed paraffin embedded colorectal and breast cancer samples were used to optimize the double immunofluorescence staining protocol. Also, immunohistochemistry was performed in order to visualize expression patterns of each biomarker. Concerning double immunofluorescence, feasibility of primary antibodies raised in different and same host species was also tested. Finally, established methods for simultaneous multicolor immunofluorescence imaging of formalin-fixed paraffin embedded specimens were applied for the detection of pairs of potential biomarkers of colorectal cancer (EGFR, pmTOR, pAKT, Vimentin, Cytokeratin Pan, Ezrin, E-cadherin) and breast cancer (Securin, PTTG1IP, Cleaved caspase 3, ki67).