Prognostic significance of cyclooxygenase-2 and associated molecules in gastric cancer

Background and aims: Low stage and curative surgery are established factors for improved survival in gastric cancer. However, not all low-stage patients have a good prognosis. Cyclooxygenase-2 (COX-2) is known to associate with reduced survival in several cancers, and has been shown to play an important role in gastric carcinogenesis. Since new and better prognostic markers are needed for gastric cancer, we studied the prognostic significance of COX-2 and of markers that associate with COX-2 expression. We also studied markers reflecting proliferation and apoptosis, and evaluated their association with COX-2. Our purpose was to construct an accurate prognostic model by combining tissue markers and clinicopathogical factors. Materials and methods: Of 342 consecutive patients who underwent surgery for gastric cancer at Meilahti Hospital, Helsinki University Central Hospital, 337 were included in this study. Low stages I to II were represented by 141 (42%) patients, and high stages III to IV by 196 (58%). Curative surgery was performed on 176 (52%) patients. Survival data were obtained from the national registers. Slides from archive tissue blocks were prepared for immunohistochemistry by use of COX-2, human antigen R (HuR), cyclin A, matrix metalloproteinases 2 and 9 (MMP-2, MMP-9), and Ki-67 antibodies. Immunostainings were scored by microscopy, and scores were entered into a database. Associations of tumor markers with clinicopathological factors were calculated, as well as associations with p53, p21, and results of flow cytometry from earlier studies. Survival analysis was performed by the Kaplan-Meier method, and Cox multivariate models were reconstructed. Cell culture experiments were performed to explore the effect of small interfering (si)RNA of HuR on COX-2 expression in a TMK-1 gastric cancer cell line. Results: Overall 5-year survival was 35.1%. Study I showed that COX-2 was an independent prognostic factor, and that the prognostic impact of COX-2 was more pronounced in low-stage patients. Cytoplasmic HuR expression also associated with reduced survival in gastric cancer patients in a non-independent manner. Cell culture experiments showed that HuR can regulate COX-2 expression in TMK-1 cells in vitro, with an association also between COX-2 and HuR tissue expression in a clinical material. In Study II, cyclin A was an independent prognostic factor and was associated with HuR expression in the gastric cancer material. The results of Study III showed that epithelial MMP-2 associated with survival in univariate, but not in multivariate analysis. However, MMP-9 showed no prognostic value. MMP-2 expression was associated with COX-2 expression. In Study IV, the prognostic power of COX-2 was compared with that of all tested markers associated with survival in Studies I to III, as well as with p21, p53, and flow cytometry results. COX-2 and p53 were independent prognostic factors, and COX-2 expression was associated with that of p53 and Ki-67 and also with aneuploidy. Conclusions: COX-2 is an independent prognostic factor in gastric cancer, and its prognostic power emerges especially in low stage cancer. COX-2 is regulated by HuR, and is associated with factors reflecting invasion, proliferation, and apoptosis. In an extended multivariate model, COX-2 retained its position as an independent prognosticator. COX-2 can be considered a promising new prognostic marker in gastric cancer.

Cystatin C, a measure of renal function, as prognostic risk marker in acute heart failure : Studies on the cardiorenal syndrome

Acute heart failure (AHF) is a complex syndrome associated with exceptionally high mortality. Still, characteristics and prognostic factors of contemporary AHF patients have been inadequately studied. Kidney function has emerged as a very powerful prognostic risk factor in cardiovascular disease. This is believed to be the consequence of an interaction between the heart and kidneys, also termed the cardiorenal syndrome, the mechanisms of which are not fully understood. Renal insufficiency is common in heart failure and of particular interest for predicting outcome in AHF. Cystatin C (CysC) is a marker of glomerular filtration rate with properties making it a prospective alternative to the currently used measure creatinine for assessment of renal function. The aim of this thesis is to characterize a representative cohort of patients hospitalized for AHF and to identify risk factors for poor outcome in AHF. In particular, the role of CysC as a marker of renal function is evaluated, including examination of the value of CysC as a predictor of mortality in AHF. The FINN-AKVA (Finnish Acute Heart Failure) study is a national prospective multicenter study conducted to investigate the clinical presentation, aetiology and treatment of, as well as concomitant diseases and outcome in, AHF. Patients hospitalized for AHF were enrolled in the FINN-AKVA study, and mortality was followed for 12 months. The mean age of patients with AHF is 75 years and they frequently have both cardiovascular and non-cardiovascular co-morbidities. The mortality after hospitalization for AHF is high, rising to 27% by 12 months. The present study shows that renal dysfunction is very common in AHF. CysC detects impaired renal function in forty percent of patients. Renal function, measured by CysC, is one of the strongest predictors of mortality independently of other prognostic risk markers, such as age, gender, co-morbidities and systolic blood pressure on admission. Moreover, in patients with normal creatinine values, elevated CysC is associated with a marked increase in mortality. Acute kidney injury, defined as an increase in CysC within 48 hours of hospital admission, occurs in a significant proportion of patients and is associated with increased short- and mid-term mortality. The results suggest that CysC can be used for risk stratification in AHF. Markers of inflammation are elevated both in heart failure and in chronic kidney disease, and inflammation is one of the mechanisms thought to mediate heart-kidney interactions in the cardiorenal syndrome. Inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) correlate very differently to markers of cardiac stress and renal function. In particular, TNF-α showed a robust correlation to CysC, but was not associated with levels of NT-proBNP, a marker of hemodynamic cardiac stress. Compared to CysC, the inflammatory markers were not strongly related to mortality in AHF. In conclusion, patients with AHF are elderly with multiple co-morbidities, and renal dysfunction is very common. CysC demonstrates good diagnostic properties both in identifying impaired renal function and acute kidney injury in patients with AHF. CysC, as a measure of renal function, is also a powerful prognostic marker in AHF. CysC shows promise as a marker for assessment of kidney function and risk stratification in patients hospitalized for AHF.

Prognostic importance of the tumour microenvironment in follicular lymphoma patients treated with immunochemotherapy

Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma. It is an indolent and clinically heterogeneous disease, which is generally considered incurable. Currently, immunochemotherapy has significantly improved the outcome of FL patients. This is based on the combination of rituximab, a monoclonal anti-CD20 antibody, with chemotherapy, and is used at present as a standard first-line therapy in FL. Thus far, however, patients have been selected for treatment based on clinical risk factors and indices that were developed before the rituximab era. Therefore, there is a growing need to understand the molecular mechanisms underlying the disease, which would not only provide information to predict survival in the rituximab era, but also enable the design of more targeted therapeutic strategies. In this study, our aim was to identify genes predicting the outcome in FL patients treated with immunochemotherapy. Thus, we performed a cDNA microarray with 24 FL patients. When gene expression differences from diagnostic tumour samples were related to the clinical outcome, we identified novel genes with a prognostic impact on survival. The expression of selected genes was further characterized with quantitative PCR and immunohistochemistry (IHC). Interestingly, the prognostic influence of these genes was often associated with their expression in non-malignant cells instead of tumour cells. Based on the observed gene expression patterns, we analyzed the abundance and prognostic value of non-malignant immune cells in 95-98 FL patients treated with immunochemotherapy. We observed that a high content of tumour-associated macrophages was a marker of a favourable prognosis. In contrast, the accumulation of mast cells correlated with a poor outcome and was further associated with tumour vascularity. Increased microvessel density also correlated with an inferior outcome. In addition, we used the same microarray data with a systems biology approach to identify signalling pathways or groups of genes capable of separating patients with favourable or adverse outcomes. Among the transcripts, there were many genes associated with signal transducers and activators of the transcription (STAT5a) pathway. When IHC was used as validation, STAT5a expression was mostly observed in T-cells and follicular dendritic cells, and expression was found to predict a favourable outcome. In cell cultures, rituximab was observed to induce the expression of STAT5a-associated interleukins in human lymphoma cell lines, which might provide a possible link for the cross-talk between rituximab-induced FL cells and their microenvironment. In conclusion, we have demonstrated that the microenvironment has a prognostic role in FL patients treated with immunochemotherapy. The results also address the importance of re-evaluating the prognostic markers in the rituximab era of lymphoma therapies.

Consequences of Vertebral Fractures

Vertebral fractures occur due to forces applied to spinal structures. When the bone tissue is weakened, vertebral fractures can result from a minor trauma. Adult vertebral fractures are commonly considered to be an indication for osteoporosis. In children osteoporosis is a rare condition, and pediatric vertebral fractures are usually clearly trauma-related. The aims of this dissertation are to produce knowledge of the epidemiology of osteoporotic vertebral fractures and to analyse their association with total and cause-specific mortality, to find indicators with which to identify individuals who are at great risk of subsequent fractures, to study the incidence of pediatric vertebral fractures and need for their operative treatment and hospital care. The Mobile-Clinic and Mini-Finland Health surveys of the adult population were used as materials in this research. Record linkages to the Finnish Hospital Discharge Register and the Official Cause of Death register were used to study mortality and hospitalization in the same population group. These registers were also used to evaluate epidemiology, mortality, hospitalization and the need for operative management of pediatric vertebral fracture patients. The main findings and conclusions of the present dissertation are: 1. The presence of a thoracic vertebral fracture in adults is a significant predictor of cancer and respiratory mortality. In women, but not in men, vertebral fractures strongly predict mortality due to injuries. Most of these deaths in the study group were hip fracture related. 2. Severe thoracic vertebral fracture in adults was a strong predictor of a subsequent hip fracture, whereas mild or moderate fractures and the number of compressed vertebrae were much weaker predictors, 3. Pediatric spinal fractures were rare: The incidence was 66 per one million children per year. In younger children cervical spine was most often affected, whereas in older children fractures of the thoracic and lumbar spine were more common. Maturation of spinal structures seems to play a major role in the typical injury patterns in children. Thirty per cent of pediatric spinal fractures required surgical treatment. The current study focuses on consequences of vertebral fractures in general, without evaluating further the causation of the studied phenomena. Further studies are needed to clarify the mechanisms of association between vertebral fractures and specific causes of mortality. A severe vertebral fracture appears to indicate a substantial risk of a subsequent hip fracture. If such a fracture is identified from a chest radiograph, urgent clinical evaluation, treatment of osteoporosis and protective measures against falls are recommended.

Painehaavapotilaille tehtyjen hoitointerventioiden määrä ja tulokset HYKS:n plastiikkakirurgian klinikassa

Keräsimme ICD-10 koodilla L89 HYKS:n plastiikkakirurgian klinikassa hoidetuista 401:stä potilaasta hoitotiedot vuosilta 2000-2008. Plastiikkakirurgiseen leikkaukseen ja hoidon arvioon päätyneiden potilaiden keski-ikä oli 60,3 (18-97v.). Painehaavan riskitekijät selvitettiin. Yksittäisistä riskitekijöistä yleisimpiä olivat paraplegia ( n=77; 17% ) ja diabetes/ASO tauti ( n=53; 12 % ). Yhteensä painehaavaleikkauksia tehtiin 347 , joista revisioleikkauksia 108 ja varsinaisia sulkuleikkauksia oli 239. Keskimäärin potilaat tarvitsivat 1.7 operaatiota. Leikatuista painehaavoista gradus III- haavoja oli 35,1% ; n= 84 ja gradus IV 25,5% ; n= 61, pinta-ala haavoilla oli keskimäärin 30,5 cm2. Istuinkyhmyjen ja sacrumin painehaavat olivat potilasaineistossamme yleisiä (20% ja 27% operoiduista painehaavoista). Gradus III haavoja leikattiin keskimäärin 1,6 kertaa ja gradus IV haavoja keskimäärin 2,0 kertaa. Postoperatiivisia poliklinikkakäyntejä tarvittiin keskimäärin 2,3, gradus III painehaavapotilailla 2,0 ja gradus IV potilailla 2,6 käyntiä. Leikkausmenetelmistä eri kielekerekonstruktiot olivat selkeästi eniten käytetty leikkausmenetelmä painehaavaleikkauksissa: 58 % rekonstruktioleikkauksista suoritettiin käyttäen jonkinlaista iho- lihaskielekettä. (134 sanaa)

Sex steroids in Sjögren's syndrome

Sjögren s syndrome (SS) is a strongly female dominant autoimmune disease. SS targets mainly salivary and lacrimal glands and leads to loss of the secreting acinar cells of these glands. Accordingly, secretion of the affected glands is diminished and the main symptoms of SS, dryness of mouth and eyes, follow. In addition to these sicca symptoms, SS patients suffer from severe fatigue and can have various extraglandular symptoms. To date, the etiology of SS still remains unknown. Female dominance and the late onset of the disease simultaneously with remarkable hormonal changes in the body (menopause, adrenopause) encouraged us to hypothesize that sex steroids, especially androgens, are involved in the onset and progression of SS. We confirmed our hypothesis and showed that patients with SS suffer from androgen depletion both systemically and locally in the target tissue of SS, salivary glands. We especially focused on the local androgen environment in salivary glands and demonstrated that healthy salivary glands contain a complete enzymatic machinery for local synthesis of androgens and estrogens from pro-hormone dehydroepiandrosterone (DHEA). However, in SS salivary glands the enzymes catalyzing the local androgen synthesis are defective and, in a subgroup of patients, practically non-functional. Probably due to this local defect in DHEA processing, therapy with DHEA was found unbeneficial for SS patients in the treatment of fatigue. We also studied the effect of the local androgen depletion on salivary glands. We found that in salivary gland cells and healthy labial salivary glands androgens upregulate integrin subunits α1 and α2, which are important for the communication, differentiation and function of the acinar cells. On the contrary, in SS salivary glands DHEA failed to upregulate these signaling molecules, again probably due to defective processing of DHEA into active androgens. Our finding highlights the importance of the local androgen environment and local DHEA processing for the function and welfare of salivary glands. In conclusion, this study showed that patients with SS are androgen depleted both systemically and locally in salivary glands. SS patients also have a defective local sex steroid synthesizing enzymatic machinery further impairing the local androgen depletion. We also showed that the local androgen defect leads to decreased expression of acinar cell specific integrin molecules, which impairs the signaling between the acinar cells and basement membrane and might thus explain the acinar cell loss seen in SS salivary glands. By showing the importance of the local sex steroid imbalance in SS we have clarified some etiopathogenetic mechanisms of SS, which have thus far remained unknown.

Henkilökunnan päihdeongelmat leikkaus- ja teho-osastoilla Suomessa

Anestesiahenkilökunnalla on useissa kansainvälisissä tutkimuksissa todettu esiintyvän enemmän päihteiden väärinkäyttöä kuin muilla erikoisaloilla. Tämän on arveltu johtuvan lääkkeiden helposta saatavuudesta, työn stressaavuudesta tai tahattomasta altistumisesta leikkaussalissa höyrystyneille lääkeaineille. Suomalaisia tutkimuksia aiheesta ei ole julkaistu. Tutkielman tarkoituksena on selvittää sähköpostikyselyiden ja asiantuntijahaastatteluiden avulla henkilökunnan päihdeongelmien yleisyyttä Suomen leikkaus- ja teho-osastoilla. Tavoitteena on myös saada tietoa muun muassa väärinkäytetyistä aineista, ovatko väärinkäyttäjät saaneet hoitoa ongelmaansa ja ovatko he myöhemmin pystyneet palaamaan entiseen työhönsä. Saatujen tulosten mukaan Suomen leikkaus- ja teho-osastojen henkilökunnalla ei näyttäisi esiintyvän sen enempää päihdeongelmia kuin väestössä keskimäärin. Alkoholi on selvästi yleisimmin väärinkäytetty aine, mutta myös opioidien ja bentsodiatsepiinien väärinkäyttöä esiintyy. Huolestuttavaa on, että vaikka suurin osa väärinkäyttäjistä sai hoitoa ongelmaansa, heistä vain kolmasosa pystyi palaamaan entiseen työhönsä ongelmitta. Tutkielmaa käytetään hyväksi Huume- ja päihdetyöryhmän työskentelyssä ja loppuraportissa. Alkuvuodesta 2011 ilmestyvässä loppuraportissa on toimenpide-ehdotuksia, joiden avulla leikkaus- ja teho-osastoilla voidaan ennaltaehkäistä henkilökunnan päihdeongelmia sekä lääkeainevarkauksia.

Depression and Cognition in Type 2 Diabetes from a Life Course Perspective

Background: Type 2 diabetes is linked to several complications which add to both physical and mental distress. Depression is a common co-morbidity of diabetes which can occur both as a cause and a consequence of type 2 diabetes. Depression has been shown to correlate with glucose regulation and treating depression might prove beneficial for glucose regulation as well as for mental well being. Another complication which might affect diabetes management is cognitive decline. Several risk factors and complications of diabetes might modify the risk for developing cognitive impairment, which is increased 1.5 times among subjects with type 2 diabetes. Type 2 diabetes, depression and impaired cognitive performance have all been linked to low birth weight. This thesis aimed to explore the effects and interactions of birth weight, depression and cognitive ability in relation to type 2 diabetes from a life course perspective. Subjects and methods: Studies I, II and V were part of the Helsinki Birth Cohort Study. 2003 subjects participated in an extensive clinical examination at an average age of 61 years. A standard glucose tolerance test (OGTT) was performed and depressive symptoms were assessed using the Beck Depression Inventory (BDI). In addition data was obtained from child welfare clinics and national registers. A subset of the cohort (n=1247) also performed a test on cognitive performance (CogState ®) at the average age of 64. Studies III and IV were randomised clinical trials where mildly depressed diabetic subjects were treated with paroxetine or placebo and the effect on metabolic parameters and quality of life was assessed. The first trial included 14 women and lasted 10 weeks, while the second trial included 43 subjects, both men and women, and lasted 6 months. Results: Type 2 diabetes was positively associated with the occurrence of depressive symptoms. Among diabetic subjects 23.6% had depressive symptoms, compared to 16.7% of subjects with normal glucose tolerance (OR = 1.77, p<0.001). Formal mediation analysis revealed that cardiovascular disease (CVD) is likely to act as a mediator in the association. Furthermore, low birth weight was found to modify the association between type 2 diabetes, CVD and depression. The association between BDI score and having type 2 diabetes or CVD was twice as strong in the subgroup with low birth weight (≤ 2500g) compared with the group with birth weight > 2500g (p for interaction 0.058). In the six months long randomised clinical trial (study IV) paroxetine had a transient beneficial effect on glycosylated haemoglobin A1c (GHbA1c) and quality of life when compared to placebo after three months of treatment. In study V we found that subjects with known diabetes had a consistently poorer level of cognitive performance than subjects with normal glucose tolerance in most of the tested cognitive domains. This effect was further amplified among those born with a small birth weight (p for interaction 0.002). Conclusions: Type 2 diabetes is associated with a higher occurrence of depressive symptoms compared to subjects with normal glucose tolerance. This association is especially strong among subjects with CVD and those born with a low birth weight. Treating depressed diabetic subjects with paroxetine has no long term effect on glucose regulation. Physicians should be aware of depression as an important co-morbidity of type 2 diabetes. Both depression and the cognitive decline often seen among diabetic subjects are increased if the subject is born with a low birth weight. Physicians should recognise low birth weight as an additional risk factor and modifier of diabetic complications.

Breathing, swallowing and voice in laryngeal disorsers

The aim of this work was to examine how breathing, swallowing and voicing are affected in different laryngeal disorders. For this purpose, we examined four different patient groups: patients who had undergone total laryngectomy, anterior cervical decompression (ACD), or injection laryngoplasty with autologous fascia (ILAF), and patients with dyspnea during exercise. We studied the problems and benefits related to the automatic speech valve used for the rehabilitation of speech in laryngectomized patients. The device was given to 14 total laryngectomized patients who used the traditional valve especially well. The usefulness of voice and intelligibility of speech were assessed by speech pathologists. The results demonstrated better performance with the traditional valve in both dimensions. Most of the patients considered the automatic valve a helpful additional device but because of heavier breathing and the greater work needed for speech production, it was not suitable as a sole device in speech rehabilitation. Dysphonia and dysphagia are known complications of ACD. These symptoms are caused due to the stretching of tissue needed during the surgery, but the extent and the recovery from them was not well known before our study. We studied two patient groups, an early group with 50 patients who were examined immediately before and after the surgery and a late group with 64 patients who were examined 3 9 months postoperatively. Altogether, 60% reported dysphonia and 69% dysphagia immediately after the operation. Even though dysphagia and dysphonia often appeared after surgery, permanent problems seldom occurred. Six (12 %) cases of transient and two (3 %) permanent vocal cord paresis were detected. In our third study, the long-term results of ILAF in 43 patients with unilateral vocal cord paralysis were examined. The mean follow-up was 5.8 years (range 3 10). Perceptual evaluation demonstrated improved results for voice quality, and videostroboscopy revealed complete or partial glottal closure in 83% of the patients. Fascia showed to be a stable injection material with good vocal results. In our final study we developed a new diagnostic method for exertional laryngeal dyspnea by combining a cardiovascular exercise test with simultaneous fiberoptic observation of the larynx. With this method, it is possible to visualize paradoxal closure of the vocal cords during inspiration, which is a diagnostic criterion for vocal cord dysfunction (VCD). We examined 30 patients referred to our hospital because of suspicion of exercise-induced vocal cord dysfunction (EIVCD). Twenty seven out of thirty patients were able to perform the test. Dyspnea was induced in 15 patients, and of them five had EIVCD and four high suspicion of EIVCD. With our test it is possible to set an accurate diagnosis for exertional laryngeal dyspnea. Moreover, the often seen unnecessary use of asthma drugs among these patients can be avoided.

Health-related quality of life, symptoms and comorbidity in inflammatory bowel disease

Health-related quality of life (HRQoL) measurement has become an important outcome in treatment trials and in health policy decisions. HRQoL can be measured by using generic or disease-specific tools. Generic instruments can be used for comparing health status among patients in different health states and conditions but they do not focus specifically on the issues relevant in a particular disease. Disease-specific tools may be more responsive to changes within a specific condition. In earlier studies, impairment of HRQoL has been evident in patients with inflammatory bowel disease (IBD), especially when the disease is active. Data about the impact of comorbidity or demographic characteristics of the patients on HRQoL are partly controversial. This study, which comprised 2913 adult IBD patients, examined HRQoL using the disease-specific IBDQ and the general 15D instruments. The 15D scores of IBD patients were compared with scores of a gender and age matched general population sample. Frequency of IBD symptoms and arrangement of therapy were studied and compared with those of IBD patients in an earlier European study. Furthermore, data of other chronic diseases of the patients were obtained from the Social Insurance Institution s reimbursement register and comorbidity of IBD patients was compared with that of age and gender matched controls. --- Of the respondents, 37% reported that they suffered from disturbing IBD symptoms weekly. In 17% of the patients, the symptoms greatly affected the ability to enjoy leisure activities, and 14% stated that these symptoms greatly affected their capacity to work. Despite that, the great majority (93%) of patients expressed satisfaction with their current treatment, which exceeded the rate observed in the other European patients. The mean IBDQ score was 163, as the possible range is 32-224, and disease activity was strongly correlated with HRQoL. Older age, comorbid diseases, and female gender were also related to impairment of HRQoL. Lower HRQoL scores were seen also in newly-diagnosed patients and in those with a history of surgery, especially after stoma or ileal pouch-anal anastomosis (IPAA) operation. The range of 15D scores was 0.30-1.00, with mean of 0.87. As with the IBDQ, disease activity, older age and history of surgery were correlated with the score. Both the newly-diagnosed patients and patients with a long-lasting disease had lower scores than average even after adjusting for age. The 15D scores of IBD patients were significantly lower than those of the control group. A strong correlation was seen between the 15D and the IBDQ scores. Comorbidity with other chronic diseases was observed in 29% of IBD patients. Connective tissue diseases, chronic obstructive pulmonary diseases, pernicious anaemia, and coronary heart disease (CHD) were significantly increased in patients with IBD. Especially female IBD patients appeared to be at increased risk for CHD, and patients who reported weekly IBD symptoms had a higher risk for having other chronic diseases in addition to IBD. Comorbidity impaired HRQoL, as measured with both generic and disease-specific tools. In conclusion, HRQoL is impaired in IBD patients. An understanding of predictors of HRQoL will help to recognise patients who will need special support.

Estramustine and its Derivatives in Potentiating Radiotherapy of Prostate Cancer

The purpose of this study was to deepen our knowledge of the combined use of estramustine and radiotherapy in the treatment of prostate cancer. Prostate cancer is a common disease, with a high variability between subjects in its malignant potential. In many cases, the disease is an incidental finding with little or no clinical significance. In other cases, however, prostate cancer may be an aggressive malignant disease, which, if the initial treatment fails, lacks an effective cure and may lead to severe symptoms, metastasis, and death despite all treatment. In many cases, the methods of treatment available at the moment provide cure or significant regression of symptoms, but often at the cost of considerable side effects. Estramustine, a cytostatic drug used for treating advanced cancer of the prostate, has been shown to inhibit prostate cancer progression and also to increase the sensitivity of cancer cells to radiotherapy. The goals of this study were, first, to find out whether it is possible to use either estramustine or an antibody against estramustine binding protein as carrier molecules for bringing therapeutic radioisotopes into prostate cancer cells, and, secondly, to gain more understanding of the mechanisms behind the known radiosensitising effect of estramustine. Estramustine and estramustine binding protein antibody were labelled with iodine-125 to study the biodistribution of these substances in mice. In the first experiment, both of the substances accumulated in the prostate, but radioiodinated estramustine also showed affinity to the liver and the lungs. Since the radiolabelled antibody was found out to accumulate more selectively to the prostate, we studied its biodistribution in nude mice with DU-145 human prostate cancer implants. In this experiment, the prostate and the tumour accumulated more radioactivity than other organs, but we concluded that the difference in the dose of radiation compared to other organs was not sufficient for the radioiodinated antibody to be advocated as a carrier molecule for treating prostate cancer. Mice with similar DU-145 prostate cancer implants were then treated with estramustine and external beam irradiation, with and without neoadjuvant estramustine treatment. The tumours responded to the treatment as expected, showing the radiation potentiating effect of estramustine. In the third experiment, this effect was found without an increase in the amount of apoptosis in the tumour cells, despite previous suggestions to the contrary. In the fourth experiment, we gave a similar treatment to the mice with DU-145 tumours. A reduction in proliferation was found in the groups treated with radiotherapy, and an increased amount of tumour hypoxia and tumour necrosis in the group treated with both neoadjuvant estramustine and radiation. This finding is contradictory to the suggestion that the radiation sensitising effect of estramustine could be attributed to its angiogenic activity.

Developmental-like responses in injured mature central neurons

Traumatic insults to the central nervous system are frequently followed by profound and irreversible neuronal loss as well as the inability of the damaged neurons to regenerate. One of the major therapeutic challenges is to increase the amount of surviving neurons after trauma. Thus it is crucial to understand how injury affects neuronal responses and which conditions are optimal for survival to prevent neuronal loss. During development neuronal survival is thought to be dependent on the competition for the availability of survival-promoting molecules called neurotrophic factors. Much less is known on the survival mechanisms of mature neurons under traumatic conditions. Increasing amount of evidence points towards the possibility that after injury neuronal responses might aquire some developmental characteristics. One of the important examples is the change in the responses to the neurotransmitter GABA: it is inhibitory in the intact mature neurons, but can induce excitation during development and after trauma. An important step in the maturation of GABAergic transmission in the CNS is the developmental shift in the action of GABAA receptor from depolarization in immature neurons to hyperpolarization in mature neurons. GABAA-mediated responses are tightly linked to the homeostasis of the chloride anion (Cl-), which in neurons is mainly regulated by Na+-K+-2Cl- cotransporter NKCC1 and K+-Cl- cotransporter KCC2. Trauma-induced functional downregulation of KCC2 promotes a shift from hyperpolarizing GABAA-mediated responses to depolarizing. Other important consequences of neuronal trauma are the emergence of dependency of central neurons on brain-derived neuro¬trophic factor (BDNF) for survival, as well as the upregulation of neurotrophin receptor p75NTR. Our aim was to answer the question whether these post-traumatic events are interrelated, and whether the regulation of BDNF and KCC2 expression is different under traumatic conditions and in intact neurons. To study responses of injured mature central neurons, we used an in vitro and in vivo axotomy models. For in vitro studies, we lesioned organotypic hippocampal slices between CA3 and CA1 regions, which resulted in selective axotomy of the CA3 neurons and denervation of the CA1 neurons. Some experiments were repeated in vivo by lesioning the neurons of the corticospinal tract at the internal capsule level, or by lesioning spinal motoneurons at the ventral root. We show that intact mature neurons do not require BDNF for survival, whereas in axotomized neurons apoptosis is induced upon BDNF deprivation. We further show that post-traumatic dependency on BDNF is mediated by injury-induced upregulation of p75NTR. Post-traumatic increase in p75NTR is induced by GABAA-mediated depolarization, consequent opening of voltage-gated Ca2+ channels, and the activation of Rho kinase ROCK. Thus, post-traumatic KCC2 downregulation leads to the dependency on BDNF through the induction of p75NTR upregulation. Neurons that survive after axotomy over longer period of time lose BDNF dependency and regain normal KCC2 levels. This phenomenon is promoted by BDNF itself, since after axotomy contrary to normal conditions KCC2 is upregulated by BDNF. The developmentally important thyroid hormone thyroxin regulates BDNF expression during development. We show that in mature intact neurons thyroxin downregulates BDNF, whereas after axotomy thyroxin upregulates BDNF. The elevation of BDNF expression by thyroxin promoted survival of injured neurons. In addition, thyroxin also enhanced axonal regeneration and promoted the regaining of normal levels of KCC2. Thus we show that this hormone acts at several levels on the axotomy-initiated chain of events described in the present work, and could be a potential therapeutic agent for the injured neurons. We have also characterized a previously unknown downregulatory interaction between thyroxin and KCC2 in intact neurons. In conclusion, we identified several important interactions at the neurotrophin-protein and hormone-neurotrophin level that acquire immature-like characteristics after axotomy and elucidated an important part of the mechanism by which axotomy leads to the requirement of BDNF trophic support. Based on these findings, we propose a new potential therapeutic strategy where developmentally crucial agents could be used to enhance survival and regeneration of axotomized mature central neurons.

Simplified Treatment of Childhood Acute Bone and Joint Infections

Acute childhood osteomyelitis (OM), septic arthritis (SA), and their combination osteomyelitis with adjacent septic arthritis (OM+SA), are treated with long courses of antimicrobials and immediate surgery. We conducted a prospective multi-center randomized trial among Finnish children at age 3 months to 15 years in 1983-2005. According to the two-by-two factorial study design, children with OM or OM+SA received 20 or 30 days of antimicrobials, whereas those with SA were treated for 10 or 30 days. In addition, the whole series was randomized to be treated with clindamycin or a first-generation cephalosporin. Cases were included only if the causative agent was isolated. The treatment was instituted intravenously, but only for the first 2-4 days. Percutaneous aspiration was done to obtain a representative sample for bacteriology, but all other surgical intervention was kept at a minimum. A total of 265 patients fulfilled our strict inclusion criteria and were analyzed; 106 children had OM, 134 SA, and 25 OM+SA. In the OM group, one child in the long and one child in the short-term treatment group developed sequelae. One child with SA twice developed a late re-infection of the same joint, but the causative agents differed. Regarding surgery, diagnostic arthrocentesis or corticotomy was the only surgical procedure performed in most cases. Routine arthrotomy was not required even in hip arthritis. Serum C-reactive protein (CRP) proved to be a reliable laboratory index in the diagnosis and monitoring of osteoarticular infections. The recovery rate was similar regardless of whether clindamycin or a first-generation cephalosporin was used. We conclude that a course of 20 days of these well-absorbing antimicrobials is sufficient for OM or OM+SA, and 10 days for SA in most cases beyond the neonatal age. A short intravenous phase of only 2-5 days often suffices. CRP gives valuable information in monitoring the course of illness. Besides diagnostic aspiration, surgery should be reserved for selected cases.

Crosstalk between the sympathetic nervous system, inflammation and coagulation in gestational diabetes; a therapeutic approach in postmenopausal hypertension

The occurrence of gestational diabetes (GDM) during pregnancy is a powerful sign of a risk of later type 2 diabetes (T2D) and cardiovascular diseases (CVDs). The physiological basis for this disease progression is not yet fully understood, but increasing evidence exists on interplay of insulin resistance, subclinical inflammation, and more recently, on unbalance of the autonomic nervous system. Since the delay in development of T2D and CVD after GDM ranges from years to decades, better understanding of the pathophysiology of GDM could give us new tools for primary prevention. The present study was aimed at investigating the role of the sympathetic nervous system (SNS) in GDM and its associations with insulin and a variety of inflammatory cytokines and coagulation and fibrinolysis markers. This thesis covers two separate study lines. Firstly, we investigated 41 women with GDM and 22 healthy pregnant and 14 non-pregnant controls during the night in hospital. Blood samples were drawn at 24:00, 4:00 and 7:00 h to determine the concentrations of plasma glucose, insulin, noradrenaline (NA) and adrenomedullin, markers of subclinical inflammation, coagulation and fibrinolysis variables and platelet function. Overnight holter ECG recording was performed for analysis of heart rate variability (HRV). Secondly, we studied 87 overweight hypertensive women with natural menopause. They were randomised to use a central sympatholytic agent, moxonidine (0.3mg twice daily), the β-blocking agent atenolol (50 mg once daily+blacebo once daily) for 8 weeks. Inflammatory markers and adiponectin were analysed at the beginning and after 8 weeks. Activation of the SNS (increase in NA, decreased HRV) was seen in pregnant vs. non-pregnant women, but no difference existed between GDM and normal pregnancy. However, modulation (internal rhythm) of HRV was attenuated in GDM. Insulin and inflammatory cytokine levels were comparable in all pregnant women but nocturnal variation of concentrations of C-reactive protein, serum amyloid A and insulin were reduced in GDM. Levels of coagulation factor VIII were lower in GDM compared with normal pregnancy, whereas no other differences were seen in coagulation and fibrinolysis markers. No significant associations were seen between NA and the studied parameters. In the study of postmenopausal women, moxonidine treatment was associated with favourable changes in the inflammatory profile, seen as a decrease in TNFα concentrations (increase in atenolol group) and preservation of adiponectin levels (decrease in atenolol group). In conclusion, our results did not support our hypotheses of increased SNS activity in GDM or a marked association between NA and inflammatory and coagulation markers. Reduced biological variation of HRV, insulin and inflammatory cytokines suggests disturbance of autonomic and hormonal regulatory mechanisms in GDM. This is a novel finding. Further understanding of the regulatory mechanisms could allow earlier detection of risk women and the possibility of prevention. In addition, our results support consideration of the SNS as one of the therapeutic targets in the battle against metabolic diseases, including T2D and CVD.

Interrelations between Dry Eye Syndrome and Tear Fluid Phospholipid Transfer Protein

The simplified model of human tear fluid (TF) is a three-layered structure composed of a homogenous gel-like layer of hydrated mucins, an aqueous phase, and a lipid-rich outermost layer found in the tear-air interface. It is assumed that amphiphilic phospholipids are found adjacent to the aqueous-mucin layer and externally to this a layer composed of non-polar lipids face the tear-air interface. The lipid layer prevents evaporation of the TF and protects the eye, but excess accumulation of lipids may lead to drying of the corneal epithelium. Thus the lipid layer must be controlled and maintained by some molecular mechanisms. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. The aim of this thesis was to investigate the presence and molecular mechanisms of lipid transfer proteins in human TF. The purpose was also to study the role of these proteins in the development of dry eye syndrome (DES). The presence of TF PLTP and CETP was studied by western blotting and mass spectrometry. The concentration of these proteins was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. To study the molecular mechanisms involved in PLTP mediated lipid transfer Langmuir monolayers and asymmetrical flow field-flow fractionation (AsFlFFF) was used. Ocular tissue samples were stained with monoclonal antibodies against PLTP to study the secretion route of PLTP. Heparin-Sepharose affinity chromatography was used for PLTP pull-down experiments and co-eluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. To study whether PLTP plays any functional role in TF PLTP-deficient mice were examined. The activity of PLTP was also studied in dry eye patients. PLTP is a component of normal human TF, whereas CETP is not. TF PLTP concentration was about 2-fold higher than that in human plasma. Inactivation of PLTP by heat treatment or immunoinhibition abolished the phospholipid transfer activity in tear fluid. PLTP was found to be secreted from lacrimal glands. PLTP seems to be surface active and is capable of accepting lipid molecules without the presence of lipid-protein complexes. The active movement of radioactively labeled lipids and high activity form of PLTP to acceptor particles suggested a shuttle model of PLTP-mediated lipid transfer. In this model, PLTP physically transports lipids between the donor and acceptor. Protein-protein interaction assays revealed ocular mucins as PLTP interaction partners in TF. In mice with a full deficiency of functional PLTP enhanced corneal epithelial damage, increased corneal permeability to carboxyfluorescein, and decreased corneal epithelial occludin expression was demonstrated. Increased tear fluid PLTP activity was observed among human DES patients. These results together suggest a scavenger property of TF PLTP: if the corneal epithelium is contaminated by hydrophobic material, PLTP could remove them and transport them to the superficial layer of the TF or, alternatively, transport them through the naso-lacrimal duct. Thus, PLTP might play an integral role in tear lipid trafficking and in the protection of the corneal epithelium. The increased PLTP activity in human DES patients suggests an ocular surface protective role for this lipid transfer protein.