Tartrate-Resistant Acid Phosphatase 5b: a Serum Marker of Bone Resorption

Bone is a physiologically dynamic tissue being constantly regenerated throughout life as a consequence of bone turnover by bone-resorbing osteoclasts and bone-forming osteoblasts. In certain bone diseases, such as osteoporosis, the imbalance in bone turnover leads to bone loss and increased fracture risk. Measurement of bone mineral density (BMD) predicts the risk of fracture, but also biochemical markers of bone metabolism have been suggested to be suitable for prediction of fractures and monitoring the efficacy of antiresorptive treatment. Tartrate-resistant acid phosphatase 5b (TRACP 5b) is an enzyme released from osteoclasts into the circulation, from where it can be detected kinetically or immunologically. Conventional assays for serum total TRACP were spectrophotometric and suffered from interference by other acid phosphatases and non-osteoclastic TRACP 5a isoform. Our aim was to develop novel immunoassays for osteoclastic TRACP 5b. Serum TRACP 5b levels were elevated in individuals with high bone turnover, such as children, postmenopausal women, patients with osteoporosis, Paget’s disease and breast cancer patients with bone metastases. As expected, hormone replacement therapy (HRT) in postmenopausal women decreased the levels of serum TRACP 5b. Surprisingly, the highest TRACP 5b levels were observed in individuals with rare autosomal dominant osteopetrosis type II (ADO2), which is characterized by high BMD and fracture risk with simultaneously elevated levels of deficient osteoclasts. In ADO2 patients, elevated levels of serum TRACP 5b were associated with high fracture frequency. It is likely that serum TRACP 5b reflects the number of inactive osteoclasts in ADO2. Similar results supporting the hypothesis that TRACP 5b would reflect the number of osteoclasts instead of their activity were observed with cultured osteoclasts and in animal models. Novel TRACP 5b immunoassays may prove to be of value either as independent or combinatory tools with other bone metabolic markers and BMD measurements in clinical practice and bone research.

Treatment-related renal side-effects in pediatric cancer patients

Background: The long-term side-effects of cancer treatments are of growing importance, since the number of pediatric cancer survivors has considerably increased. Renal side-effects should be noted early to prevent further deterioration. Renal dysfunction may also develop long after cancer treatment. Easy and reliable methods for assessing renal function are needed. Aims: The aims were to find the mechanisms behind methotrexate-induced renal damage by studying renal tubular cells (LLC-PK1cells), and to evaluate the usefulness of laboratory tests in assessing glomerular function in pediatric cancer patients by comparing an isotope clearance method with alternative methods. The aim was also to study the long-term effects of bone marrow transplantation (BMT) and high-dose methotrexate (HD-MTX) treatment in renal function. Results: Methotrexate induced time-dependent renal tubular cell swelling and cell death. In patients treated with HD-MTX a significant decrease in GFR was noted after a follow-up time of one to ten years. One year after BMTthe GFR was reduced, especially in patients treated with total body irradiation (TBI). GFR recovered slightly but remained stable thereafter. In glomerular function assessment the serum cystatin C (cysC) concentration showed a significant association with GFR measured by the isotope method. Conclusions: Methotrexate induced acute damage in renal tubular cells. In assessing GFR the isotope method still remains the method of choice, but the assay of cystatin C was the most reliable of other alternatives. Long-term follow-up of renal function is needed in BMT patients and patients treated with HD-MTX.

Prognostic Markers and Prolonged Interferon-alpha Therapy in Renal Cell Carcinoma

Molekyylimarkkerit ja pitkäaikainen alfainterferonihoito munuaissyövässä Munuaissyöpäpotilaiden viiden vuoden elossaololuku on noin 50 %. Aikaisempien tutkimuksien mukaan viiden vuoden elossaololuku metastasoituneessa munuaissyövässä on 3-16 %, kun käytettiin alfainterferonia sisältävää hoitoa. Tyypillisesti alfainterferonia on käytetty vähemmäin kuin 6 kuukautta. Avoimia kysymyksiä ovat alfainterferonin optimaalinen hoitoannos ja hoidon kesto yksin tai yhdessä uusien täsmähoitojen kanssa. Tärkeimmät tavoitteet olivat tutkia 1) jaksotetun pitkäaikaisen alfainterferonihoidon tehoa ja siedettävyyttä metastasoituneessa munuaissyövässä ja 2) p53-, Ki-67- ja COX-2-proteiinituotannon ennusteellista merkitystä munuaissyövässä. Tutkimuksessa 117 metastasoituneelle munuaissyöpää sairastaneelle potilaalle etsittiin yksilöllinen hänen sietämänsä maksimaalinen hoitoannos rekombinanttia alfa2a-interferonia (Roferon-ATM). Hoitoa pyrittiin jatkamaan 24 kuukauden ajan. Kolmen hoitoviikon jälkeen pidettiin yhden viikon tauko. Hoito lopetettiin, jos ilmaantui vakavia haittavaikutuksia tai tauti eteni. Toisessa tutkimuksessa proteiinituotanto analysoitiin immunohistokemiallisesti munuaissyöpäpotilaiden kasvainnäytteistä, joita oli säilytetty parafiinissa. Kasvainnäytteet oli otettu talteen munuaisen poistoleikkauksen yhteydessä. Nämä potilaat jaettiin kolmeen eri ryhmään: metastasointi primaarivaiheessa (n=29), metastasointi myöhemmin (n=37) ja ei metastasointia (n=51). Keskimääräinen alfainterferonihoidon kesto oli 11 kuukautta (kk) [0,5 – 32 kk]. Objektiivinen hoitovaste todettiin 17 %:lla, tautitilanne pysyi ennallaan 42 %:lla ja myöhäinen vaste (yli 12 kk:tta hoidon aloittamisesta) todettiin 3 %:lla. Aika vasteen saavuttamisesta taudin etenemiseen oli keskimäärin 8 kk ja elinaika 19,1 kk. Viiden vuoden elossaololuku oli 16 %. Jos metastasoituneella munuaissyöpäpotilaalla oli keuhkometastasointi, hän selvisi todennäköisemmin viisi vuotta kuin muut potilaat. Henkeä uhkaavia sivuvaikutuksia ei todettu. Yli 12 kk:n ajan kestävä alfainterferonihoito on hyödyllistä niille potilaille, jotka ovat saaneet objektiivisen hoitovasteen tai tautitilanne on pysynyt ennallaan. Positiivinen p53- ja Ki-67-ekspressio yhdessä viittaavat suureen metastasoinnin todennäköisyyteen. Positiivinen COX-2-ekspressio viittaa viivästyneeseen metastaasien ilmaantumiseen. Metastasoituneilla potilailla positiiviset p53- ja Ki-67-ekspressiot viittaavat huonoon ennusteeseen, mutta positiivinen COX-2 ekspressio viittaa suotuisaan ennusteeseen. Positiivinen COX-2- ja negatiivinen Ki-67-ekspressio yhdessä viittaavat parantuneeseen ennusteeseen metastasoituneessa munuaissyövässä.

Vascular Function in Chronic Kidney Disease and in Renovascular Disease

Cardiovascular mortality is 15 to 30 times higher in patients with chronic kidney disease than in the age-adjusted general population. Even minor renal dysfunction predicts cardiovascular events and death in the general population. In patients with atherosclerotic renovascular disease the annual cardiovascular event and death rate is even higher. The abnormalities in coronary and peripheral artery function in the different stages of chronic kidney disease and in renovascular disease are still poorly understood, nor have the cardiac effects of renal artery revascularization been well characterized, although considered to be beneficial. This study was conducted to characterize myocardial perfusion and peripheral endothelial function in patients with chronic kidney disease and in patients with atherosclerotic renovascular disease. Myocardial perfusion was measured with positron emission tomography (PET) and peripheral endothelial function with brachial artery flow-mediated dilatation. It has been suggested that the poor renal outcomes after the renal artery revascularization could be due to damage in the stenotic kidney parenchyma; especially the reduction in the microvascular density, changes mainly evident at the cortical level which controls almost 80% of the total renal blood flow. This study was also performed to measure the effect of renal artery stenosis revascularization on renal perfusion in patients with renovascular disease. In order to do that a PET-based method for quantification of renal perfusion was developed. The coronary flow reserve of patients with chronic kidney disease was similar to the coronary flow reserve of healthy controls. In renovascular disease the coronary flow reserve was, however, markedly reduced. Flow-mediated dilatation of brachial artery was decreased in patients with chronic kidney disease compared to healthy controls, and even more so in patients with renovascular disease. After renal artery stenosis revascularization, coronary vascular function and renal perfusion did not improve in patients with atherosclerotic renovascular disease, but in patients with bilateral renal artery stenosis, flow-mediated dilatation improved. Chronic kidney disease does not significantly affect coronary vascular function. On the contrary, coronary vascular function was severely deteriorated in patients with atherosclerotic renovascular disease, possibly because of diffuse coronary artery disease and/or diffuse microvascular disease. The peripheral endothelial function was disturbed in patients with chronic kidney disease and even more so in patient with atherosclerotic renovascular disease. Renal artery stenosis dilatation does not seem to offer any benefits over medical treatment in patients with renovascular disease, since revascularization does not improve coronary vascular function or renal perfusion.

First-Line Chemotherapy with Anthracycline and Taxane Combination in Metastatic Breast Cancer. Detection of bone metastases with TRACP 5b

Background: In Finland, breast cancer (BC) is the most common cancer among women, and prostate cancer (PC) that among men. At the metastatic stage both cancers remain essentially incurable. The goals of therapy include palliation of symptoms, improvement or maintenance of quality of life (QoL), delay of disease progression, and prolongation of survival. Balancing between efficacy and toxicity is the major challenge. With increasing costs of new treatments, appropriate use of resources is paramount. When new treatment regimes are introduced into clinical practice a comprehensive assessment of clinical benefit, adverse effects and cost is necessary. Both BC and PC show a predilection to metastasize to bone. Bone metastases cause significant morbidity impairing the patients´ QoL. Diagnosis of bone metastases relies mainly on radiological methods, which however lack optimal sensitivity and specificity. New tools are needed for detection and follow-up of bone metastases. Aims: Anthracyclines and taxanes are effective chemotherapeutic agents in the treatment of metastatic breast cancer (MBC) with different mechanisms of action. Therefore, evaluation of the combination of anthracyclines with taxanes was a justifiable approach in the treatment of MBC patients. We assessed the efficacy, toxicity, cost of treatment and QoL of BC patients treated with first-line chemotherapy for metastatic disease with the combination epirubicin and docetaxel. We also evaluated the diagnostic potential of tartrate-resistant acid phosphatase 5b (TRACP 5b) and carboxyterminal telopeptides of type I collagen (ICTP) in the diagnosis of bone metastases in BC and TRACP 5b in PC patients. Results: The combination of epirubicin and docetaxel was effective in this phase II study, but required individual dose adjustment to avoid neutropenic infections, and the use of growth factors to maintain a feasible dose level. The response rate was 54 % (95 % CI 37-71) and the median overall survival (OS) was 26 months. Of the patients, 87 % were treated for infections. The treatment of adverse events required additional use of health resources mainly due to neutropenic infections, thereby raising direct treatment costs by 20 %. Despite adverse events, the global QoL was not significantly compromised during the treatment. Clinically evident acute cardiac toxicity was not observed. The combination of serum TRACP 5b and ICTP was at least equally sensitive and specific in detection of of bone metastases as commonly used total alkaline phosphatise (tALP) in BC patients. In contrast, TRACP 5b was less specific and sensitive than tALP as a marker of skeletal changes in PC patients. Conclusions: Treatment with epirubicin and docetaxel showed high efficacy in first-line chemotherapy of MBC. The relatively high incidence of neutropenic infections requiring hospitalization increased the treatment costs. Despite adverse events, the global QoL of the patients was not significantly compromised. The combination of TRACP 5b and ICTP showed similar activity as tALP in detecting bone metastases in MBC. In contrast, TRACP 5b was less specific and sensitive than tALP as a marker of skeletal changes in PC.

Utilization of Meat and bone meal for energy production

At the end of the 1990s the stock breeding in the Europe was suffering from the animal disease epidemics such as Bovine spongiform encephalopathy (BSE) and foot –and mouth disease. The European Union (EU) tackled to this problem by tightening the legislation of animal by-products. At this point, rendering and fat producing industries faces new challenges, which they have to cope with in a way of trying to find alternatives to their products (animal fats and meat and bone meal). One of the most promising alternatives to utilize these products was to use them in energy production purposes. The purpose of the Thesis was to examine the utilization possibilities of Meat and bone meal (MBM) for energy production. The first part of the Thesis consists of theory part. The theory part includes evaluation of basic properties of MBM as a fertilizer and as a fuel, legislative evaluation and evaluation of different burning techniques. The second part of the Thesis consists of burning tests in Energy laboratory of LUT with different mixtures of peat and MBM. The purpose of the burning tests was to identify co-firing possibilities of peat and MBM and emission- and ash properties for peat and MBM.

Dissecting the molecular mechanisms of breast cancer bone metastasis for therapeutic targeting

Breast cancer that has metastasized to bone is currently an incurable disease, causing significant morbidity and mortality. The aim of this thesis work was to elucidate molecular mechanisms of bone metastasis and thereby gain insights into novel therapeutic approaches. First, we found that L‐serine biosynthesis genes, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1) and phosphoserine phosphatase (PSPH), were up‐regulated in highly bone metastatic MDA‐MB‐231(SA) cells as compared with the parental breast cancer cell line. Knockdown of serine biosynthesis inhibited proliferation of MDA‐MB‐231(SA) cells, and L‐serine was essential for the formation of bone resorbing osteoclasts. Clinical data demonstrated that high expression of PHGDH and PSAT1 was associated with decreased relapse‐free and overall survival and with features typical of poor outcome in breast cancer. Second, RNA interference screening pointed out heparan sulfate 6‐O‐sulfotransferase 2 (HS6ST2) as a critical gene for transforming growth factor β (TGF‐β)‐induced interleukin 11 (IL‐11) production in MDA‐MB‐231(SA) cells. Exogenous heparan sulfate glycosaminoglycans heparin and K5‐NSOS also inhibited TGF‐β‐induced IL‐11 production in MDA‐MB‐231(SA) cells. Furthermore, K5‐NSOS decreased osteolytic lesion area and tumor burden in bone in mice. Third, we discovered that the microRNAs miR‐204, ‐211 and ‐379 inhibited IL‐11 expression in MDA‐MB‐231(SA) cells through direct targeting of the IL‐11 mRNA. MiR‐379 also inhibited Smad‐mediated signaling. Gene expression profiling of miR‐204 and ‐379 transfected cells indicated that these microRNAs down‐regulate several bone metastasis‐relevant genes, including prostaglandin‐endoperoxide synthase 2 (PTGS2). Taken together, this study identified three potential treatment strategies for bone metastatic breast cancer: inhibition of serine biosynthesis, heparan sulfate glycosaminoglycans and restoration of miR‐204/‐211/‐379.

PET Imaging of Osteomyelitis. Feasibility of 18F-FDG, 68Ga-Chloride and 68Ga-DOTAVAP-P1 Tracers in Staphylococcal Bone Infections

Due to technical restrictions of the database system the title of the thesis does not show corretly on this page. Numbers in the title are in superscript. Please see the PDF-file for correct title. ---- Osteomyelitis is a progressive inflammatory disease of bone and bone marrow that results in bone destruction due to an infective microorganism, most frequently Staphylococcus aureus. Orthopaedic concern relates to the need for reconstructive and trauma-related surgical procedures in the fast grow¬ing population of fragile, aged patients, who have an increased susceptibility to surgical site infections. Depending on the type of osteomyelitis, infection may be acute or a slowly progressing, low-grade infection. Peri-implant infections lead to implant loosening. The emerging antibiotic resistance of com¬mon pathogens further complicates the situation. With current imaging methods, significant limitations exist in the diagnosing of osteomyelitis and implant-related infections. Positron emission tomography (PET) with a glucose analogue, 18F-fluoro¬deoxyglucose (18F-FDG), seems to facilitate a more accurate diagnosis of chronic osteomyelitis. The method is based on the increased glucose consumption of activated inflammatory cells. Unfortunately, 18F-FDG accumulates also in sterile inflammation regions and causes false-positive findings, for exam¬ple, due to post-operative healing processes. Therefore, there is a clinical need for new, more infection-specific tracers. In addition, it is still unknown why 18F-FDG PET imaging is less accurate in the detec¬tion of periprosthetic joint infections, most frequently due to Staphylococcus epidermidis. This doctoral thesis focused on testing novel PET tracers (68Ga-chloride and 68Ga-DOTAVAP-P1) for early detections of bone infections and evaluated the role of pathogen-related factors in the appli¬cations of 18F-FDG PET in the diagnostics of bone infections. For preclinical models of S. epidermidis and S. aureus bone/implant infections, the significance of the causative pathogen was studied with respect to 18F-FDG uptake. In a retrospective analysis of patients with confirmed bone infections, the significance of the presence or absence of positive bacterial cultures on 18F-FDG uptake was evalu¬ated. 18F-FDG and 68Ga-chloride resulted in a similar uptake in S. aureus osteomyelitic bones. However, 68Ga-chloride did not show uptake in healing bones, and therefore it may be a more-specific tracer in the early post-operative or post-traumatic phase. 68Ga-DOTAVAP-P1, a novel synthetic peptide bind¬ing to vascular adhesion protein 1 (VAP-1), was able to detect the phase of inflammation in healing bones, but the uptake of the tracer was elevated also in osteomyelitis. Low-grade peri-implant infec¬tions due to S. epidermidis were characterized by a low uptake of 18F-FDG, which reflects the virulence of the causative pathogen and the degree of leukocyte infiltration. In the clinical study, no relationship was found between the level of 18F-FDG uptake and the presence of positive or negative bacterial cul¬tures. Thus 18F-FDG PET may help to confirm metabolically active infection process in patients with culture-negative, histologically confirmed, low-grade osteomyelitis.

Determinants of Bone Strength and Predictors of Hip Fracture among Finnish Adults. Results from the Health 2000 Survey and the Mini-Finland Health Survey

The objective of this thesis was to identify the determinants of bone strength and predictors of hip fracture in representative samples of Finnish adults. A secondary objective was to construct a simple multifactorial model for hip fracture prediction over a 10-year follow-up period. The study was based on the Health 2000 Survey conducted during 2000 to 2001 (men and women aged 30 years or over, n=6 035) and the Mini-Finland Health Survey conducted during 1978 to 1980 (women aged 45 years or over, n=2 039). Study subjects participated in health interviews and comprehensive health examination. In the Health 2000 Survey, bone strength was assessed by means of calcaneal quantitative ultrasound (QUS). The follow-up information about hip fractures was drawn from the National Hospital Discharge Register. In this study, age, weight, height, serum 25-hydroxyvitamin D (S-25(OH)D), physical activity, smoking and alcohol consumption as well as menopause and eventual HRT in women were found to be associated with calcaneal broadband ultrasound attenuation (BUA) and speed of sound (SOS). Parity was associated with a decreased risk of hip fracture in postmenopausal women. Age, height, weight or waist circumference, quantitative ultrasound index (QUI), S-25(OH)D and fall-related factors, such as maximal walking speed, Parkinson’s disease, and the number of prescribed CNS active medication were significant independent predictors of hip fracture. At the population level, the incremental value of QUS appeared to be minor in hip fracture prediction when the fall-related risk factors were taken into account. A simple multifactorial model for hip fracture prediction presented in this study was based on readily available factors (age, gender, height, waist circumference, and fallrelated factors). Prospective studies are needed to test this model in patient-based study populations.