Major histocompatibility complex and coronary artery disease: Special emphasis on Chlamydia pneumoniae, and periodontitis

Most of the genes in the MHC region are involveed in adaptive and innate immunity, with essential function in inflammatory reactions and in protection against infections. These genes might serve as a candidate region for infection and inflammation associated diseases. CAD is an inflammatory disease. The present set of studies was performed to assess whether the MHC region harbors genetic markers for CAD, and whether these genetic markers explain the CAD risk factors: e.g., C. pneumoniae, periodontitis, and periodontal pathogens. Study I was performed using two separate patient materials and age- and sex-matched healthy controls, categorizing them into two independent studies: the HTx and ACS studies. Both studies consistently showed the HLA-A3– B35– DR1 (35 ancestral haplotype) haplotype as a susceptible MHC genetic marker for CAD. HLA-DR1 alone was associated not only with CAD, but also with CAD risk factor diseases, e.g., diabetes mellitus, and hyperlipidemia. The ACS study further showed the HLA-B*07 and -DRB1*15 -related haplotype as a protective MHC haplotype for CAD. Study II showed that patients with CAD showed signs of chronic C. pneumoniae infection when compared to age- and sex-matched healthy controls. HLA-B*35 or -related haplotypes associated with the C. pneumoniae infection markers. Among these haplotype carriers, males and smokers associated with elevated C. pneumoniae infection markers. Study III showed that CAD patients with periodontitis had elevated serum markers of P. gingivalis and occurrence of the pathogen in saliva. LTA+496C strongly associated with periodontitis, while HLA-DRB1*01 with periodontitis and with the elevated serum antibodies of P. gingivalis. Study IV showed that the increased level of C3/C4 ratio was a new risk factor and was associated with recurrent cardiovascular end-points. The increased C3 and decreased C4 concentrations in serum explained the increased level of the C3/C4 ratio. Both the higher than cut-off value (4.53) and the highest quartile of the C3/C4 ratio were also associated with worst survival, increased end-points, and C4 null alleles. The presence of C4 null alleles associated with decreased serum C4 concentration, and increased C3/C4 ratio. In conclusion, the present studies show that the CAD susceptibility haplotype (HLA-A3− B35− DR1 -related haplotypes, Study I) partially explains the development of CAD in patients possessing several recognized and novel risk factors: diabetes mellitus, increased LDL, smoking, C4B*Q0, C. pneumnoiae, periodontitis, P. gingivalis, and complement C3/C4 ratio (Study II, III, and IV).

Mulibrey nanism : Clinical characteristics and pathophysiologic features of growth restriction, insulin resistance and tumour development

Background: Mulibrey nanism (MUL; Muscle-liver-brain-eye nanism; OMIM 253250) is an autosomal recessive growth disorder more prevalent in Finland than elsewhere in the world. Clinical characteristics include severe prenatal onset growth restriction, cardiopathy, multiple organ manifestations but no major neurological handicap. MUL is caused by mutations in the TRIM37 gene on chromosome 17q22-23, encoding a peroxisomal protein TRIM37 with ubiquitin E3-ligase activity. Nineteen different mutations have been detected, four of them present in the Finnish patients. Objective: This study aimed to characterize clinical and histopathological features of MUL in the national cohort of Finnish patients. Patients and methods: A total of 92 Finnish patients (age 0.7 to 77 years) participated in the clinical follow-up study. Patients hospital records and growth charts were reviewed. Physical, radiographic and laboratory examinations were performed according to a clinical protocol. Thirty patients (18 females) were treated with recombinant human GH for a median period of 5.7 years. Biopsies and autopsy samples were used for the histopathological and immunohistochemical analyses. Results: MUL patients were born small for gestational age (SGA) with immature craniofacial features after prenatal-onset growth restriction. They experienced a continuous deceleration in both height SDS and weight-for-height (WFH) postnatally. In infancy feeding difficulties and frequent pneumonias were common problems. At the time of diagnosis (median age 2.1 years) characteristic craniofacial, radiological and ocular features were the most constant findings. MUL patients showed a dramatic change in glucose metabolism with increasing age. While the children had low fasting glucose and insulin levels, 90% of the adults were insulin resistant, half had type 2 diabetes and an additional 42% showed impaired glucose tolerance (IGT). Seventy percent fulfilled the National Cholesterol Education Program (NCEP) Adult Treatment Panel III criteria for metabolic syndrome as adults. GH therapy improved pre-pubertal growth but had only minor impact on adult height (+5 cm). Interestingly, treated subjects were slimmer and had less frequent metabolic concerns as young adults. MUL patients displayed histologically a disturbed architecture with ectopic tissues and a high frequency of both benign and malignant tumours present in several internal organs. A total of 232 tumorous lesions were detected in our patient cohort. The majority of the tumours showed strong expression of endothelial cell marker CD34 as well as α-smooth muscle actin (α-SMA). Fifteen of the tumours were malignant and seven of them (five Wilms tumours) occurred in the kidney. Conclusions: MUL patients present a distinct postnatal growth pattern. Short-term response of GH treatment is substantial but the long-term impact remains modest. Although MUL patients form a distinct clinical and diagnostic entity, their clinical findings vary considerably from infancy to adulthood. While failure to thrive dominates early life, MUL adults develop metabolic syndrome and have a tendency for malignancies and vascular lesions in several organs. This speaks for a central role of TRIM37 in regulation of key cellular functions, such as proliferation, migration, angiogenesis and insulin signalling.

Inhibition of CYP1A2-mediated drug metabolism in vitro and in humans : With special emphasis on rofecoxib and other NSAIDs

The cytochrome P450 1A2 (CYP1A2) is one of the major metabolizing enzymes. The muscle relaxant tizanidine is a selective substrate of CYP1A2, and the non-steroidal anti-inflammatory drug (NSAID) rofecoxib was thought to modestly in-hibit it. Cases suggesting an interaction between tizanidine and rofecoxib had been reported, but the mechanism was unknown. Also other NSAIDs are often used in combination with muscle relaxants. The aims of this study were to investigate the effect of rofecoxib, several other NSAIDs and female sex steroids on CYP1A2 ac-tivity in vitro and in vivo, and to evaluate the predictability of in vivo inhibition based on in vitro data. In vitro, the effect of several NSAIDs, female sex steroids and model inhibitors on CYP1A2 activity was studied in human liver microsomes, without and with preincubation. In placebo controlled, cross-over studies healthy volunteers ingested a single dose of tizanidine after a pretreament with the inhibitor (rofecoxib, tolfenamic acid or celecoxib) or placebo. Plasma (and urine) concentrations of tizanidine and its metabolites were measured, and the pharmacodynamic effects were recorded. A caffeine test was also performed. In vitro, fluvoxamine, tolfenamic acid, mefenamic acid and rofecoxib potently in-hibited CYP1A2. Ethinylestradiol, celecoxib, desogestrel and zolmitriptan were moderate, and etodolac, ciprofloxacin, etoricoxib and gestodene were weak inhibi-tors of CYP1A2. At 100 µM, other tested NSAIDs and steroids inhibited CYP1A2 less than 35%. Rofecoxib was found to be a mechanism-based inhibitor of CYP1A2. In vivo, rofecoxib greatly increased the plasma concentrations (over ten-fold) and the pharmacodynamic effects of tizanidine. Also the metabolism of caf-feine was impaired by rofecoxib. Despite the relatively strong in vitro CYP1A2 inhibitory effects, tolfenamic acid and celecoxib did not have a significant effect on tizanidine and caffeine concentrations in humans. Competitive inhibition model and the free plasma concentration of the inhibitor predicted well the effect of fluvoxam-ine and the lack of effect of tolfenamic acid and celecoxib on tizanidine concentra-tions in humans, and mechanism-based inhibition model explained the effects of rofecoxib. However, the effects of ciprofloxacin and oral contraceptives were un-derestimated from the in vitro data. Rofecoxib is a potent mechanism-based inhibitor of CYP1A2 in vitro and in vivo. This mechanism may be involved in the adverse cardiovascular effects of rofecoxib. Tolfenamic acid and celecoxib seem to be safe in combination with tizanidine, but mefenamic acid might have some effect on tizanidine concentrations in vivo. Con-sidering the mechanism of inhibition, and using the free plasma concentration of the inhibitor, many but not all CYP1A2 interactions can be predicted from in vitro data.

Suicidal ideation and attempts among psychiatric patients with major depressive disorder

This study is part of an ongoing collaborative research and development project, the Vantaa Depression Study (VDS), between the National Public Health Institute, Helsinki and the Department of Psychiatry of Helsinki University Hospital (HUCH), Peijas hospital, Vantaa. The VDS is a prospective, naturalistic cohort study of 269 secondary-level care psychiatric out- and inpatients with a new episode of DSM-IV major depressive disorder (MDD). 269 patients (Nmales=72, Nfemales=197) with a current DSM-IV MDD were interviewed with semistructured interviews to assess all other psychiatric diagnoses. At 6- and 18-month follow-up the interviews were repeated. Suicidal behaviour was investigated both at intake and follow-up by using a psychometric scale (Scale for Suicidal Ideation) and interviewer's questions as well as the patient's psychiatric records. Patients, who reported suicidal ideation while entering the study were followed up weekly, and their level of suicidal ideation, hopelessness, anxiety and depression was measured. In this study suicidal ideation was common among psychiatric patients with MDD. Almost 60% of the depressed patients reported suicidal ideation and 15% of patients attempted suicide at the baseline. Patients with suicidal ideation or attempts had a clearly higher level of overall psychopathology than non-suicidal patients. During the 18-month follow-up period 8% of patients attempted suicide. The risk of an attempt was markedly higher (RR=7.54) during an episode of major depression compared with a period of remission. Suicide attempt during the follow-up period was predicted by lack of partner, a history of previous suicide attempts and time spent in depression. Suicidal ideation resolved for most of the suicidal patients during the first 2 to 3 months. The duration of suicidal ideation was longer for patients with an initially higher level of psychopathology. Declines both in depression and hopelessness independently predicted the subsequent decline in suicidal ideation. They both could have a causal role in reversing the suicidal process. Thus effective treatment of depression is a credible measure in suicide prevention. Patients with suicidal behaviour often received more antidepressants and had more frequent appointments with mental health professionals than non-suicidal patients. Suicidal patients had also more favourable attitudes towards antidepressant treatment and comparable adherence to treatment than those not suicidal. This study does not support the conception that patient attitudes or adherence to treatments would be a factor differentiating suicidal patients from non-suicidal. Instead, problems with adherence or attitudes seem to be generic to all psychiatric care.

Major Events and the European Code of Police Ethics

Examines the symbolic significance of major events and their security provision in the historical and contemporary context of the European Code of Police Ethics. Stresses the potential of major events to set new practical policing and security standards of technology and in doing so necessitiate the maintenance of professional ethical standards for policing in Europe.

Expression of lactate transporters MCT1, MCT2, MCT4 and the ancillary protein CD147 in horse muscle and red blood cells

Monocarboxylate transporters (MCTs) transport lactate and protons across cell membranes. During intense exercise, lactate and protons accumulate in the exercising muscle and are transported to the plasma. In the horse, MCTs are responsible for the majority of lactate and proton removal from exercising muscle, and are therefore also the main mechanism to hinder the decline in pH in muscle cells. Two isoforms, MCT1 and MCT4, which need an ancillary protein CD147, are expressed in equine muscle. In the horse, as in other species, MCT1 is predominantly expressed in oxidative fibres, where its likely role is to transport lactate into the fibre to be used as a fuel at rest and during light work, and to remove lactate during intensive exercise when anaerobic energy production is needed. The expression of CD147 follows the fibre type distribution of MCT1. These proteins were detected in both the cytoplasm and sarcolemma of muscle cells in the horse breeds studied: Standardbred and Coldblood trotters. In humans, training increases the expression of both MCT1 and MCT4. In this study, the proportion of oxidative fibres in the muscle of Norwegian-Swedish Coldblood trotters increased with training. Simultaneously, the expression of MCT1 and CD147, measured immunohistochemically, seemed to increase more in the cytoplasm of oxidative fibres than in the fast fibre type IIB. Horse MCT4 antibody failed to work in immunohistochemistry. In the future, a quantitative method should be introduced to examine the effect of training on muscle MCT expression in the horse. Lactate can be taken up from plasma by red blood cells (RBCs). In horses, two isoforms, MCT1 and MCT2, and the ancillary protein CD147 are expressed in RBC membranes. The horse is the only species studied in which RBCs have been found to express MCT2, and the physiological role of this protein in RBCs is unknown. The majority of horses express all three proteins, but 10-20% of horses express little or no MCT1 or CD147. This leads to large interindividual variation in the capacity to transport lactate into RBCs. Here, the expression level of MCT1 and CD147 was bimodally distributed in three studied horse breeds: Finnhorse, Standardbred and Thoroughbred. The level of MCT2 expression was distributed unimodally. The expression level of lactate transporters could not be linked to performance markers in Thoroughbred racehorses. In the future, better performance indexes should be developed to better enable the assessment of whether the level of MCT expression affects athletic performance. In human subjects, several mutations in MCT1 have been shown to cause decreased lactate transport activity in muscle and signs of myopathy. In the horse, two amino acid sequence variations, one of which was novel, were detected in MCT1 (V432I and K457Q). The mutations found in horses were in different areas compared to mutations found in humans. One mutation (M125V) was detected in CD147. The mutations found could not be linked with exercise-induced myopathy. MCT4 cDNA was sequenced for the first time in the horse, but no mutations could be detected in this protein.

Myotonic dystrophy type 2 (DM2) : Diagnostic methods and molecular pathology

Myotonic dystrophies type 1 (DM1) and type 2 (DM2) are the most common forms of muscular dystrophy affecting adults. They are autosomal dominant diseases caused by microsatellite tri- or tetranucleotide repeat expansion mutations in transcribed but not translated gene regions. The mutant RNA accumulates in nuclei disturbing the expression of several genes. The more recently identified DM2 disease is less well known, yet more than 300 patients have been confirmed in Finland thus far, and the true number is believed to be much higher. DM1 and DM2 share some features in general clinical presentation and molecular pathology, yet they show distinctive differences, including disease severity and differential muscle and fiber type involvement. However, the molecular differences underlying DM1 and DM2 muscle pathology are not well understood. Although the primary tissue affected is muscle, both DMs show a multisystemic phenotype due to wide expression of the mutation-carrying genes. DM2 is particularly intriguing, as it shows an incredibly wide spectrum of clinical manifestations. For this reason, it constitutes a real diagnostic challenge. The core symptoms in DM2 include proximal muscle weakness, muscle pain, myotonia, cataracts, cardiac conduction defects and endocrinological disturbations; however, none of these is mandatory for the disease. Myalgic pains may be the most disabling symptom for decades, sometimes leading to incapacity for work. In addition, DM2 may cause major socio-economical consequences for the patient, if not diagnosed, due to misunderstanding and false stigmatization. In this thesis work, we have (I) improved DM2 differential diagnostics based on muscle biopsy, and (II) described abnormalities in mRNA and protein expression in DM1 and DM2 patient skeletal muscles, showing partial differences between the two diseases, which may contribute to muscle pathology in these diseases. This is the first description of histopathological differences between DM1 and DM2, which can be used in differential diagnostics. Two novel high-resolution applications of in situ -hybridization have been described, which can be used for direct visualization of the DM2 mutation in muscle biopsy sections, or mutation size determination on extended DNA-fibers. By measuring protein and mRNA expression in the samples, differential changes in expression patterns affecting contractile proteins, other structural proteins and calcium handling proteins in DM2 compared to DM1 were found. The dysregulation at mRNA level was caused by altered transciption and abnormal splicing. The findings reported here indicate that the extent of aberrant splicing is higher in DM2 compared to DM1. In addition, the described abnormalities to some extent correlate to the differences in fiber type involvement in the two disorders.

Two levels of MCT1 and CD147 expression in the equine red blood cells and muscle

Monocarboxylate transporters (MCTs), especially the isoforms MCT1 - MCT4, cotransport lactate and protons across the cell membranes. They are thus essential for pH regulation and homeostasis in glycolytic cells such as red blood cells (RBCs), and skeletal muscle cells during intense exercise. In 70% of the Standardbred horses the lactate transport activity (TA) in RBCs is high and transport is mediated mainly by MCTs. In the rest 30% of the Standardbreds MCT mediated transport route is not active and the TA is low. MCTs need an ancillary protein for their proper localization and functioning in the plasma membrane. The ancillary protein for MCT1 and MCT4 is a member of immunoglobulin superfamily, CD147. Here we determined the expression of MCT isoforms and CD147 in equine RBCs and gluteal muscle. We sequenced the cDNA of horse MCT1 and CD147 to achieve horse-specific antibodies and to reveal sequence variations that may affect the TA of RBCs. The amount of MCT1 and CD147 mRNA in muscle were also studied. ---- In all, 73 horses representing different breeds were used. Blood samples were drawn from the jugular vein and muscle samples were taken either from gluteal muscle using biopsy needle or during castration from expendable cremaster muscle. The TA of RBCs was studied using radiolabeled lactate and the amount of MCT isoforms and CD147 in the plasma membranes using Western blotting. The level of mRNA in muscle cells was determined using qPCR. Isoforms MCT1 and MCT2 were found in the RBCs and isoforms MCT1 and MCT4 in the muscle cells of horses. The TA of RBCs was dependent on the expression of CD147 and MCT1 in the plasma membrane. Sequence variations were found in the cDNA of both MCT1 and CD147, but they did not explain the inactivity of MCT1 mediated transport route. The single nucleotide polymorphism (SNP) Met125Val in CD147 that existed parallel with an SNP in 3´-untranslated region explained, however, attenuation in CD147 expression in Standardbreds. A single mutation Ile51Val also decreased the expression of CD147 in one Warmblood. The MCT1 and CD147 mRNA concentrations in the gluteal muscle were higher in horses with higher MCT1 and CD147 expression in RBCs and lower in horses with minor expression of CD147 and MCT1. This suggests that the bimodal distribution of TA is due to differences in transcriptional regulation that is functioning in parallel in MCT1 and CD147 gene.

Cyclin dependent protein kinases as drug targets – potential in cardiovascular diseases

Complications of atherosclerosis such as myocardial infarction and stroke are the primary cause of death in Western societies. The development of atherosclerotic lesions is a complex process, including endothelial cell dysfunction, inflammation, extracellular matrix alteration and vascular smooth muscle cell (VSMC) proliferation and migration. Various cell cycle regulatory proteins control VSMC proliferation. Protein kinases called cyclin dependent kinases (CDKs) play a major role in regulation of cell cycle progression. At specific phases of the cell cycle, CDKs pair with cyclins to become catalytically active and phosphorylate numerous substrates contributing to cell cycle progression. CDKs are also regulated by cyclin dependent kinase inhibitors, activating and inhibitory phosphorylation, proteolysis and transcription factors. This tight regulation of cell cycle is essential; thus its deregulation is connected to the development of cancer and other proliferative disorders such as atherosclerosis and restenosis as well as neurodegenerative diseases. Proteins of the cell cycle provide potential and attractive targets for drug development. Consequently, various low molecular weight CDK inhibitors have been identified and are in clinical development. Tylophorine is a phenanthroindolizidine alkaloid, which has been shown to inhibit the growth of several human cancer cell lines. It was used in Ayurvedic medicine to treat inflammatory disorders. The aim of this study was to investigate the effect of tylophorine on human umbilical vein smooth muscle cell (HUVSMC) proliferation, cell cycle progression and the expression of various cell cycle regulatory proteins in order to confirm the findings made with tylophorine in rat cells. We used several methods to determine our hypothesis, including cell proliferation assay, western blot and flow cytometric cell cycle distribution analysis. We demonstrated by cell proliferation assay that tylophorine inhibits HUVSMC proliferation dose-dependently with an IC50 value of 164 nM ± 50. Western blot analysis was used to determine the effect of tylophorine on expression of cell cycle regulatory proteins. Tylophorine downregulates cyclin D1 and p21 expression levels. The results of tylophorine’s effect on phosphorylation sites of p53 were not consistent. More sensitive methods are required in order to completely determine this effect. We used flow cytometric cell cycle analysis to investigate whether tylophorine interferes with cell cycle progression and arrests cells in a specific cell cycle phase. Tylophorine was shown to induce the accumulation of asynchronized HUVSMCs in S phase. Tylophorine has a significant effect on cell cycle, but its role as cell cycle regulator in treatment of vascular proliferative diseases and cancer requires more experiments in vitro and in vivo.