IL-10 in Human Newborns : Ontogeny of IL-10 secretion and relation to the secretion of IFN-g, immunoglobulin M, G, and A, and mononuclear cell composition in newborns

The purpose of this work was to elucidate the ontogeny of interleukin-10 (IL-10) secretion from newborn mononuclear cells (MCs), and to examine its relation to the secretion of interferon-g (IFN-g) and immunoglobulins (Igs). The initial hypothesis was that the decreased immunoglobulin (Ig) synthesis of newborn babies was the result of immature cytokine synthesis regulation, which would lead to excessive IL-10 production, leading in turn to suppressed IFN-g secretion. Altogether 57 full-term newborns and 34 adult volunteers were enrolled. Additionally, surface marker compositions of 29 premature babies were included. Enzyme-linked immunoassays were used to determine the amount of secreted IL-10, IFN-g, and Igs, and the surface marker composition of MC were analyzed with a FACScan flow cytometer. The three most important findings were: 1. Cord blood MC, including CD5+ B cells, are able to secrete IL-10. However, when compared with adults, the secretion of IL-10 was decreased. This indicates that reasons other than excessive IL-10 secretion are responsible of reduced IFN-g secretion in newborns. 2. As illustrated by the IL-10 and IFN-g secretion pattern, newborn cytokine profile was skewed towards the Th2 type. However, approximately 25% of newborns had an adult like cytokine profile with both good IL10 and IFN-g secretion, demonstrating that fullterm newborns are not an immunologically homogenous group at the time of birth. 3. There were significant differences in the surface marker composition of MCs between individual neonates. While gestational age correlated with the proportion of some MC types, it is evident that there are many other maternal and fetal factors that influence the maturity and nature of lymphocyte subpopulations in individual neonates. In conclusion, the reduced ability of neonates to secrete Ig and IFN-g is not a consequence of high IL-10 secretion. However, individual newborns differ significantly in their ability to secrete cytokines as well as Igs.

Auger-emitter radiochemotherapy in squamous cell cancers : in vitro and in vivo experiments with In-111-BLMC

Head and neck squamous cell cancer (HNSCC) is the sixth most common cancer worldwide. Despite advances in combined modality therapy (surgery, radiotherapy, chemotherapy) the 5-year survival rate in stage III and IV disease remains at 40% - 60%. Short-range Auger-electron emitters, such as In-111 and In-114m, tagged with a drug, molecule, peptide, protein or nanoparticles brought in close proximity to nuclear DNA represent a fascinating alternative for treating cancer. In this thesis, we studied the usefulness of Indium-111-bleomycin complex (In-111-BLMC) in the diagnostics and potential therapy of HNSCC using in vitro HNSCC cell lines, in vivo nude mice, and in vivo HNSCC patients. In in vitro experiments with HNSCC cell lines, the sensitivity to external beam radiation, BLM, In-111-BLMC, and In-111-Cl3 was studied using the 96-well plate clonogenic assay. The influence of BLM and In-111-BLMC on the cell cycle was measured with flow cytometry. In in vivo nude mice xenograft studies, the activity ratios of In-111-BLMC were obtained in gamma camera images. The effect of In-111-BLMC in HNSCC xenografts was studied. In in vivo patient studies, we determined the tumor uptake of In-111-BLMC with gamma camera and the radioactivity from tumor samples using In-111-BLMC with specific activity of 75, 175, or 375 MBq/mg BLM. The S values, i.e. absorbed dose in a target organ per cumulated activity in a source organ, were simulated for In-111 and In-114m. In vitro studies showed the variation of sensitivity for external beam radiation, BLM, and In-111-BLMC between HNSCC cell lines. IC50 values for BLM were 1.6-, 1.8-, and 2.1-fold higher than In-111-BLMC (40 MBq/mg BLM) in three HNSCC cell lines. Specific In-111 activity of 40 MBq/mgBLM was more effective in killing cells than specific In-111 activity of 195MBq/mgBLM (p=0.0023). In-111-Cl3 alone had no killing effect. The percentage of cells in the G2/M phase increased after exposure to BLM and especially to In-111-BLMC in the three cell lines studied, indicating a G2/M block. The tumor-seeking behavior was shown in the in vivo imaging study of xenografted mice. BLM and In-111-BLMC were more effective than NaCl in reducing xenografted tumor size in HNSCC. The uptake ratios received from gamma images in the in vivo patient study varied from 1.2 to 2.8 in malignant tumors. However, the uptake of In-111-BLMC was unaffected by increasing the injected activity. A positive correlation existed between In-111-BLMC uptake, Ki-67/MIB activity, and number of mitoses. Regarding the S values, In-114m delivered a 4-fold absorbed radiation dose into the tumor compared with In-111, and thus, In-114m-BLMC might be more effective than In-111-BLMC at the DNA level. Auger-electron emitters, such as In-111 and In-114m, might have potential in the treatment of HNSCC. Further studies are needed to develop a radiopharmaceutical agent with appropriate physical properties of the radionuclide and a suitable carrier to bring it to the targeted tissue.

Cyclins in Breast Cancer

Breast cancer is the most common malignancy in women in Western countries. It is a heterogeneous disease with varying biological characteristics and aggressiveness. Family history is one of the strongest predisposing factors for breast cancer. The known susceptibility genes explain only around 25% of all familial breast cancers. At least part of the unknown familial aggregation may be caused by several low-penetrance variants that occur commonly in the general population. Cyclins are cell cycle-regulating proteins. Cyclin expression oscillates during the cell cycle and is under strict control. In cancer cells, cyclin expression often becomes deregulated, leading to uncontrolled cell division and proliferation, one of the hallmarks of cancer. In this study, we investigated the role of cyclins in breast cancer predisposition, pathogenesis, and tumor behavior. Cyclin A immunohistochemistry was evaluated both on traditional large sections and on tissue microarrays (TMA). The concordance of the results was good, indicating that TMA is a reliable method for studying cyclin expression in breast cancer. The expression of cyclins D1, E, and B1 was studied among 1348 invasive breast cancers on TMA. Familial BRCA1/2-mutation negative tumors had significantly more often low cyclin E and high cyclin D1 expression than BRCA1/2 related or sporadic tumors. Unique cyclin E and D1 expression patterns among familial non-BRCA1/2 breast cancers may reflect different predisposition and pathogenesis in these groups and help to differentiate mutation-positive from mutation-negative familial cancers. High cyclin E expression was associated with an aggressive breast cancer phenotype and was an independent marker of poor metastasis-free survival. High cyclin D1 was associated with high grade and high proliferation among estrogen receptor (ER)-positive but with low grade and low proliferation among ER-negative breast cancers. Among ER-positive cancers not treated with chemotherapy, high cyclin D1 showed a trend towards shorter metastasis-free survival. These results suggest that different mechanisms may drive proliferation in ER-negative and -positive breast cancers and that cyclin D1 has a particularly important role in tumorigenesis of hormone receptor-positive breast cancer. High cyclin B1 expression was associated with aggressive breast cancer features and had an independent impact on survival. The results suggest that cyclin B1 immunohistochemistry is a method that could easily be adapted for routine use and is an independent prognostic factor, adding specificity to prognostic evaluation conducted with traditional markers. A commonly occurring cyclin D1 gene polymorphism A870G was associated with increased breast cancer risk in a large material of Finnish and Canadian breast cancer patients. The interaction of the high-activity alleles of cyclin D1 gene and estrogen metabolism gene COMT conferred an even higher risk. These results show that cyclin D1 and COMT act synergistically to contribute to breast cancer progression and that individual risk for breast cancer can be altered by the combined effect of polymorphisms with low-penetrance alleles. By investigating critical cell cycle regulator protein cyclins, we revealed new aspects of breast cancer predisposition, pathogenesis, and clinical course.

New approaches to improve the cord blood unit hematopoietic progenitor cell content

Cord blood is a well-established alternative to bone marrow and peripheral blood stem cell transplantation. To this day, over 400 000 unrelated donor cord blood units have been stored in cord blood banks worldwide. To enable successful cord blood transplantation, recent efforts have been focused on finding ways to increase the hematopoietic progenitor cell content of cord blood units. In this study, factors that may improve the selection and quality of cord blood collections for banking were identified. In 167 consecutive cord blood units collected from healthy full-term neonates and processed at a national cord blood bank, mean platelet volume (MPV) correlated with the numbers of cord blood unit hematopoietic progenitors (CD34+ cells and colony-forming units); this is a novel finding. Mean platelet volume can be thought to represent general hematopoietic activity, as newly formed platelets have been reported to be large. Stress during delivery is hypothesized to lead to the mobilization of hematopoietic progenitor cells through cytokine stimulation. Accordingly, low-normal umbilical arterial pH, thought to be associated with perinatal stress, correlated with high cord blood unit CD34+ cell and colony-forming unit numbers. The associations were closer in vaginal deliveries than in Cesarean sections. Vaginal delivery entails specific physiological changes, which may also affect the hematopoietic system. Thus, different factors may predict cord blood hematopoietic progenitor cell numbers in the two modes of delivery. Theoretical models were created to enable the use of platelet characteristics (mean platelet volume) and perinatal factors (umbilical arterial pH and placental weight) in the selection of cord blood collections with high hematopoietic progenitor cell counts. These observations could thus be implemented as a part of the evaluation of cord blood collections for banking. The quality of cord blood units has been the focus of several recent studies. However, hemostasis activation during cord blood collection is scarcely evaluated in cord blood banks. In this study, hemostasis activation was assessed with prothrombin activation fragment 1+2 (F1+2), a direct indicator of thrombin generation, and platelet factor 4 (PF4), indicating platelet activation. Altogether three sample series were collected during the set-up of the cord blood bank as well as after changes in personnel and collection equipment. The activation decreased from the first to the subsequent series, which were collected with the bank fully in operation and following international standards, and was at a level similar to that previously reported for healthy neonates. As hemostasis activation may have unwanted effects on cord blood cell contents, it should be minimized. The assessment of hemostasis activation could be implemented as a part of process control in cord blood banks. Culture assays provide information about the hematopoietic potential of the cord blood unit. In processed cord blood units prior to freezing, megakaryocytic colony growth was evaluated in semisolid cultures with a novel scoring system. Three investigators analyzed the colony assays, and the scores were highly concordant. With such scoring systems, the growth potential of various cord blood cell lineages can be assessed. In addition, erythroid cells were observed in liquid cultures of cryostored and thawed, unseparated cord blood units without exogenous erythropoietin. This was hypothesized to be due to the erythropoietic effect of thrombopoietin, endogenous erythropoietin production, and diverse cell-cell interactions in the culture. This observation underscores the complex interactions of cytokines and supporting cells in the heterogeneous cell population of the thawed cord blood unit.