Long-term effects of fenofibrate on lipoproteins and surrogate markers of macrovascular and microvascular disease in people with type 2 diabetes

Background and aims. Diabetic dyslipidemia is a highly atherogenic triad of increased triglycerides, decreased HDL cholesterol, and small dense LDL. Fibrates have a beneficial effect on diabetic dyslipidemia, and they have reduced cardiovascular events in randomized trials. Fenofibrate has reduced albuminuria and markers of low-grade inflammation and endothelial dysfunction. The present studies were undertaken to characterize the alterations of VLDL and LDL subclasses and to investigate the binding of LDL to arterial wall in type 2 diabetes. Further purpose was to elucidate the effects of fenofibrate on several lipoprotein subclasses, augmentation index (AIx), carotid intima-media thickness (IMT), and renal function. Subjects. 239 type 2 diabetic subjects were recruited among participants of the FIELD (Fenofibrate Intervention and Event Lowering in Diabetes) study at the Helsinki centre. The patients were randomized to fenofibrate (200mg/d) or placebo for 5 years. Additionally, a healthy control group (N = 93) was recruited. Results. VLDL1 triglycerides increased in similar proportion to total triglycerides in type 2 diabetic patients and control subjects. Despite the increase in total apoCIII levels, VLDL apoCIII was decreased in diabetic patients. Enrichment of LDL with apoCIII induced a small increase in binding of LDL to arterial wall proteoglycan. Intrinsic characteristics of diabetic LDL, rather than levels of apoCIII, were responsible for increased proteoglycan binding of diabetic LDL with high apoCIII. Fenofibrate reduced triglycerides, increased LDL size, and shifted HDL subclasses towards smaller particles with no change in levels of HDL cholesterol. High levels of homocysteine were associated with lower increase of HDL cholesterol and apoA-I during fenofibrate treatment. Long-term fenofibrate treatment did not improve IMT, AIx, inflammation, or endothelial function. Fenofibrate decreased creatinine clearance and estimated glomerular filtration rate. No effect on albuminuria was seen with fenofibrate. Instead, Cystatin C was increased during fenofibrate treatment. Conclusions. 1) Elevation of VLDL 1 triglycerides was the major determinant of plasma triglyceride concentration in control subjects and type 2 diabetic patients. 2) LDL with high apoCIII showed multiple atherogenic properties, that were only partially mediated by apoCIII per se in type 2 diabetes 3) Fenofibrate demonstrated no effect on surrogate markers of atherosclerosis. 4) Fenofibrate had no effect on albuminuria and the observed decrease in markers of renal function could complicate the clinical surveillance of the patients. 5) Fenofibrate can be used to treat severe hypertriglyceridemia or in combination therapy with statins, but not to increase HDL levels.

Genetic Variation and Clinical Manifestations in Inflammatory Bowel Disease

Crohn s disease (CD) and ulcerative colitis (UC), collectively known as inflammatory bowel disease (IBD), are characterised by chronic inflammation of the gastrointestinal tract. IBD prevalence in Finland is approximately 3-4 per 1000 inhabitants with a peak incidence in adolescence. The symptoms of IBD include diarrhoea, abdominal pain, fever, and weight loss. The precise aetiology of IBD is unknown but interplay of environmental risk factors and immunologic changes trigger the disease in a genetically susceptible individual. Twin and family studies have provided strong evidence for genetic factors in IBD susceptibility, and genetic factors may be more prominent in CD than UC. The first CD susceptibility gene was identified in 2001. Three common mutations R702W, G908R, and 1007fs of the CARD15/NOD2 gene are shown to associate independently with CD but the magnitude of association varies between different populations. The present study aimed at identifying mutations and genetic variations in IBD susceptibility and candidate genes. In addition, correlation to phenotype was also assessed. One of the main objectives of this study was to evaluate the role of CARD15 in a Finnish CD cohort. 271 CD patients were studied for the three common mutations and the results showed a lower mutation frequency than in other Caucasian populations. Only 16% of the patients carried one of the three mutations. Ileal location as well as stricturing and penetrating behaviour of the disease were associated with occurrence of the mutations. The whole protein coding region of CARD15 was screened for possible Finnish founder mutations. In addition to several sequence variants, five novel mutations (R38M, W355X, P727L, W907R, and R1019X) were identified in five patients. Functional consequences of these novel variants were studied in vitro, and these studies demonstrated a profound impairment of MDP response. Investigation of CARD15 mutation frequency in healthy people across three continents showed a large geographic fluctuation. No simple correlation between mutation frequency and disease incidence was seen in populations studied. The occurrence of double mutant carriers in healthy controls suggested that the penetrance of risk alleles is low. Other main objectives aimed at identifying other genetic variations that are involved in the susceptibility to IBD. We investigated the most plausible IBD candidate genes including TRAF6, SLC22A4, SLC22A5, DLG5, TLR4, TNFRSF1A, ABCB1/MDR1, IL23R, and ATG16L1. The marker for a chromosome 5 risk haplotype and the rare HLA-DRB1*0103 allele were also studied. The study cohort consisted of 699 IBD patients (240 CD and 459 UC), of which 23% had a first-degree relative with IBD. Of the several candidate genes studied, IL23R was associated with CD susceptibility, and TNFRSF1A as well as the HLA-DRB1*0103 allele with UC susceptibility. IL23R variants also showed association with the stricturing phenotype and longer disease duration in CD patients. In addition, TNFRSF1A variants were more common among familial UC and ileocolonic CD. In conclusion, the common CARD15 mutations were shown to account for 16% of CD cases in Finland. Novel CARD15 variants identified in the present study are most likely disease-causing mutations, as judged by the results of in vitro studies. The present study also confirms the IL23R association with CD susceptibility and, in addition, TNFRSF1A and HLA-DRB1*0103 allele association with UC of specific clinical phenotypes.

Noninvasive techniques in assessing coronary artery disease

Conventional invasive coronary angiography is the clinical gold standard for detecting of coronary artery stenoses. Noninvasive multidetector computed tomography (MDCT) in combination with retrospective ECG gating has recently been shown to permit visualization of the coronary artery lumen and detection of coronary artery stenoses. Single photon emission tomography (SPECT) perfusion imaging has been considered the reference method for evaluation of nonviable myocardium, but magnetic resonance imaging (MRI) can accurately depict structure, function, effusion, and myocardial viability, with an overall capacity unmatched by any other single imaging modality. Magnetocardiography (MCG) provides noninvasively information about myocardial excitation propagation and repolarization without the use of electrodes. This evolving technique may be considered the magnetic equivalent to electrocardiography. The aim of the present series of studies was to evaluate changes in the myocardium assessed with SPECT and MRI caused by coronary artery disease, examine the capability of multidetector computed tomography coronary angiography (MDCT-CA) to detect significant stenoses in the coronary arteries, and MCG to assess remote myocardial infarctions. Our study showed that in severe, progressing coronary artery disease laser treatment does not improve global left ventricular function or myocardial perfusion, but it does preserve systolic wall thickening in fixed defects (scar). It also prevents changes from ischemic myocardial regions to scar. The MCG repolarization variables are informative in remote myocardial infarction, and may perform as well as the conventional QRS criteria in detection of healed myocardial infarction. These STT abnormalities are more pronounced in patients with Q-wave infarction than in patients with non-Q-wave infarctions. MDCT-CA had a sensitivity of 82%, a specificity of 94%, a positive predictive value of 79%, and a negative predictive value of 95% for stenoses over 50% in the main coronary arteries as compared with conventional coronary angiography in patients with known coronary artery disease. Left ventricular wall dysfunction, perfusion defects, and infarctions were detected in 50-78% of sectors assigned to calcifications or stenoses, but also in sectors supplied by normally perfused coronary arteries. Our study showed a low sensitivity (sensitivity 63%) in detecting obstructive coronary artery disease assessed by MDCT in patients with severe aortic stenosis. Massive calcifications complicated correct assessment of the lumen of coronary arteries.

Affected sibling pairs with juvenile idiopathic arthritis : An immunogenetic study of the disease in multicase families

Genetic susceptibility to juvenile idiopathic arthritis (JIA) was studied in the genetically homogeneous Finnish population by collecting families with two or three patients affected by this disease from cases seen in the Rheumatism Foundation Hospital. The number of families ranged in different studies from 37 to 45 and the total number of patients with JIA, from among whom these cases were derived, was 2 000 to 2 300. Characteristics of the disease in affected siblings in Finland were compared with a population-based series and with a sibling series from the United States. A thorough clinical and ophthalmological examination was made of all affected patients belonging to sibpair series. Information on the occurrence of chronic rheumatic diseases in parents was collected by questionnaire and diagnoses were confirmed from hospital records. All patients, their parents and most of the healthy sibs were typed for human leukocyte antigen (HLA) alleles in loci A, C, B, DR and DQ. The HLA allele distribution of the cases was compared with corresponding data from Finnish bone marrow donors. The genetic component in JIA was found to be more significant than previously believed. A concordance rate of 25% for a disease with a population prevalence of 1 per 1000 implied a relative risk of 250 for a monozygotic (MZ) twin. An estimate for the sibling risk of an affected individual was about 15- to 20-fold. The disease was basically similar in familial and sporadic cases; the mean age at disease onset was however lower in familial cases, (4.8 years vs 7.4 years). Three sibpairs (3.4 expected) were concordant for the presence of asymptomatic uveitis. Uveitis would thus not appear to have any genetic component of its own, separate from the genetic basis of JIA. Four of the parents had JIA (0.2 cases expected), four had a type of rheumatoid factor-negative arthritis similar to that seen in juvenile patients but commencing in adulthood, and one had spondyloarthropathy (SPA). These findings provide additional support for the conception of a genetic predisposition to JIA and suggest the existence of a new disease entity, JIA of adult onset. Both the linkage analysis of the affected sibpairs and the association analysis of nuclear families provided overwhelming evidence of a major contribution of HLA to the genetic susceptibility to JIA. The association analysis in the Finnish population confirmed that the most significant associations prevailed for DRB1*0801, DQB1*0402, as expected from previous observations, and indicated the independent role of Cw*0401.

Myoblast transplantation and adenoviral VEGF-C transfer in porcine model of coronary artery disease

Heart failure is a common and highly challenging medical disorder. The progressive increase of elderly population is expected to further reflect in heart failure incidence. Recent progress in cell transplantation therapy has provided a conceptual alternative for treatment of heart failure. Despite improved medical treatment and operative possibilities, end-stage coronary artery disease present a great medical challenge. It has been estimated that therapeutic angiogenesis would be the next major advance in the treatment of ischaemic heart disease. Gene transfer to augment neovascularization could be beneficial for such patients. We employed a porcine model to evaluate the angiogenic effect of vascular endothelial growth factor (VEGF)-C gene transfer. Ameroid-generated myocardial ischemia was produced and adenovirus encoding (ad)VEGF-C or β-galactosidase (LacZ) gene therapy was given intramyocardially during progressive coronary stenosis. Angiography, positron emission tomography (PET), single photon emission computed tomography (SPECT) and histology evidenced beneficial affects of the adVEGF-C gene transfer compared to adLacZ. The myocardial deterioration during progressive coronary stenosis seen in the control group was restrained in the treatment group. We observed an uneven occlusion rate of the coronary vessels with Ameroid constrictor. We developed a simple methodological improvement of Ameroid model by ligating of the Ameroid–stenosed coronary vessel. Improvement of the model was seen by a more reliable occlusion rate of the vessel concerned and a formation of a rather constant myocardial infarction. We assessed the spontaneous healing of the left ventricle (LV) in this new model by SPECT, PET, MRI, and angiography. Significant spontaneous improvement of myocardial perfusion and function was seen as well as diminishment of scar volume. Histologically more microvessels were seen in the border area of the lesion. Double staining of the myocytes in mitosis indicated more cardiomyocyte regeneration at the remote area of the lesion. The potential of autologous myoblast transplantation after ischaemia and infarction of porcine heart was evaluated. After ligation of stenosed coronary artery, autologous myoblast transplantation or control medium was directly injected into the myocardium at the lesion area. Assessed by MRI, improvement of diastolic function was seen in the myoblast-transplanted animals, but not in the control animals. Systolic function remained unchanged in both groups.

Mitochondrial helicase Twinkle in mtDNA copy number regulation and a late-onset disease model

Mitochondrial diseases are caused by disturbances of the energy metabolism. The disorders range from severe childhood neurological diseases to muscle diseases of adults. Recently, mitochondrial dysfunction has also been found in Parkinson s disease, diabetes, certain types of cancer and premature aging. Mitochondria are the power plants of the cell but they also participate in the regulation of cell growth, signaling and cell death. Mitochondria have their own genetic material, mtDNA, which contains the genetic instructions for cellular respiration. Single cell may host thousands of mitochondria and several mtDNA molecules may reside inside single mitochondrion. All proteins needed for mtDNA maintenance are, however, encoded by the nuclear genome, and therefore, mutations of the corresponding genes can also cause mitochondrial disease. We have here studied the function of mitochondrial helicase Twinkle. Our research group has previously identified nuclear Twinkle gene mutations underlying an inherited adult-onset disorder, progressive external ophthalmoplegia (PEO). Characteristic for the PEO disease is the accumulation of multiple mtDNA deletions in tissues such as the muscle and brain. In this study, we have shown that Twinkle helicase is essential for mtDNA maintenance and that it is capable of regulating mtDNA copy number. Our results support the role of Twinkle as the mtDNA replication helicase. No cure is available for mitochondrial disease. Good disease models are needed for studies of the cause of disease and its progression and for treatment trials. Such disease model, which replicates the key features of the PEO disease, has been generated in this study. The model allows for careful inspection of how Twinkle mutations lead to mtDNA deletions and further causes the PEO disease. This model will be utilized in a range of studies addressing the delay of the disease onset and progression and in subsequent treatment trials. In conclusion, in this thesis fundamental knowledge of the function of the mitochondrial helicase Twinkle was gained. In addition, the first model for adult-onset mitochondrial disease was generated.

Circulating Glucocorticoid Bioactivity and Serum Glucocorticoid-Responsive Biomarkers during Steroid Therapy in Children and Adolescents with Inflammatory Bowel Disease

Objective: Glucocorticoid therapy is used worldwide to treat various inflammatory and immune conditions, including inflammatory bowel disease (IBD). In IBD, 80% of the patients obtain a positive response to the therapy; however the development of glucocorticoid-related side-effects is common. Our aim was therefore to study the possibility of optimizing glucocorticoid therapy in children and adolescents with IBD by measuring circulating glucocorticoid bioactivity (GBA) and serum glucocorticoid-responsive biomarkers in patients receiving steroid treatment for active disease. Methods: A total of sixty-nine paediatric IBD patients from the Paediatric Outpatient Clinics of the University Hospitals of Helsinki and Tampere participated in the studies. Control patients included 101 non-IBD patients and 41 disease controls in remission. In patients with active disease, blood samples were withdrawn before the glucocorticoid therapy was started, at 2-4 weeks after the initiation of the steroid and at 1-month intervals thereafter. Clinical response to glucocorticoid treatment and the development of steroid adverse events was carefully registered. GBA was analyzed with a COS-1 cell bioassay. The measured glucocorticoid therapy-responsive biomarkers included adipocyte-derived adiponectin and leptin, bone turnover-related collagen markers amino-terminal type I procollagen propeptide (PINP) and carboxyterminal telopeptide of type I collagen (ICTP) as well as insulin-like growth factor 1 (IGF-1) and sex hormone-binding globulin (SHBG), and inflammatory marker high-sensitivity C-reactive protein (hs-CRP). Results: The most promising marker for glucocorticoid sensitivity was serum adiponectin that associated with steroid therapy–related adverse events. Serum leptin indicated a similar trend. In contrast, circulating GBA rose in all subjects receiving glucocorticoid treatment but did not associate with the clinical response to steroids or with glucocorticoid therapy-related side-effects. Of notice, young patients (<10 years) showed similar GBA levels than older patients, despite receiving higher weight-adjusted doses of glucocorticoid. Markers of bone formation were lower in children with active IBD than in the control patients, probably reflecting the suppressive effect of the active inflammation. The onset of the glucocorticoid therapy further suppressed bone turnover. Inflammatory marker hs-CRP decreased readily after the initiation of the steroid, however the decrease did not associate with the clinical response to glucocorticoids. Conclusions: This is the first study to show that adipocyte-derived adiponectin associates with steroid therapy-induced side-effects. Further studies are needed, but it is possible that the adiponectin measurement could aid the recognition of glucocorticoid-sensitive patients in the future. GBA and the other markers reflecting glucocorticoid activity in different tissues changed during the treatment, however their change did not correlate with the therapeutic response to steroids or with the development of glucocorticoid-related side effects and therefore cannot guide the therapy in these patients. Studies such as as the present one that combine clinical data with newly developed biomolecular technology are needed to step-by-step build a general picture of the glucocorticoid actions in different tissues.

Species-specific and Indication-based Use of Antimicrobials in Dogs, Cats, Cattle and Horses in Finland : Data collected using three different methods

Increasing antimicrobial resistance in bacteria has led to the need for better understanding of antimicrobial usage patterns. In 1999, the World Organisation for Animal Health (OIE) recommended that an international ad hoc group should be established to address human and animal health risks related to antimicrobial resistance and the contribution of antimicrobial usage in veterinary medicine. In European countries the need for continuous recording of the usage of veterinary antimicrobials as well as for animal species-specific and indication-based data on usage has been acknowledged. Finland has been among the first countries to develop prudent use guidelines in veterinary medicine, as the Ministry of Agriculture and Forestry issued the first animal species-specific indication-based recommendations for antimicrobial use in animals in 1996. These guidelines have been revised in 2003 and 2009. However, surveillance on the species-specific use of antimicrobials in animals has not been performed in Finland. This thesis provides animal species-specific information on indication-based antimicrobial usage. Different methods for data collection have been utilized. Information on antimicrobial usage in animals has been gathered in four studies (studies A-D). Material from studies A, B and C have been used in an overlapping manner in the original publications I-IV. Study A (original publications I & IV) presents a retrospective cross-sectional survey on prescriptions for small animals at the Veterinary Teaching Hospital of the University of Helsinki. Prescriptions for antimicrobial agents (n = 2281) were collected and usage patterns, such as the indication and length of treatment, were reviewed. Most of the prescriptions were for dogs (78%), and primarily for the treatment of skin and ear infections most of which were treated with cephalexin for a median period of 14 days. Prescriptions for cats (18%) were most often for the treatment of urinary tract infections with amoxicillin for a median length of 10 days. Study B (original publication II) was a retrospective cross-sectional survey where prescriptions for animals were collected from 17 University Pharmacies nationwide. Antimicrobial prescriptions (n = 1038) for mainly dogs (65%) and cats (19%) were investigated. In this study, cephalexin and amoxicillin were also the most frequently used drugs for dogs and cats, respectively. In study C (original publications III & IV), the indication-based usage of antimicrobials of practicing veterinarians was analyzed by using a prospective questionnaire. Randomly selected practicing veterinarians in Finland (n = 262) recorded all their antimicrobial usage during a 7-day study period. Cattle (46%) with mastitis were the most common patients receiving antimicrobial treatment, generally intramuscular penicillin G or intramammary treatment with ampicillin and cloxacillin. The median length of treatment was four days, regardless of the route of administration. Antimicrobial use in horses was evaluated in study D, the results of which are previously unpublished. Firstly, data collected with the prospective questionnaire from the practicing veterinarians showed that horses (n = 89) were frequently treated for skin or wound infections by using penicillin G or trimethoprim-sulfadiazine. The mean duration of treatment was five to seven days. Secondly, according to retrospective data collected from patient records, horses (n = 74) that underwent colic surgery at the Veterinary Teaching Hospital of the University of Helsinki were generally treated according to national and hospital recommendations; penicillin G and gentamicin was administered preoperatively and treatment was continued for a median of three days postoperatively. In conclusion, Finnish veterinarians followed well the national prudent use guidelines. Narrow-spectrum antimicrobials were preferred and, for instance, fluoroquinolones were used sparingly. Prescription studies seemed to give good information on antimicrobials usage, especially when combined with complementary information from patient records. A prospective questionnaire study provided a fair amount of valuable data on several animal species. Electronic surveys are worthwhile exploiting in the future.