Cholesterol Disturbances in Inherited Neurodegenerative Diseases : Studies on Npc1, Ppt1 and Ctsd deficient mice

The central nervous system (CNS) is the most cholesterol-rich organ in the body. Cholesterol is essential to CNS functions such as synaptogenesis and formation of myelin. Significant differences exist in cholesterol metabolism between the CNS and the peripheral organs. However, the regulation of cholesterol metabolism in the CNS is poorly understood compared to our knowledge of the regulation of cholesterol homeostasis in organs reached by cholesterol-carrying lipoprotein particles in the circulation. Defects in CNS cholesterol homeostasis have been linked to a variety of neurodegenerative diseases, including common diseases with complex pathogenetic mechanisms such as Alzheimer s disease. In spite of intense effort, the mechanisms which link disturbed cholesterol homeostasis to these diseases remain elusive. We used three inherited recessive neurodegenerative disorders as models in the studies included in this thesis: Niemann-Pick type C (NPC), infantile neuronal ceroid lipofuscinosis and cathepsin D deficiency. Of these three, NPC has previously been linked to disturbed intracellular cholesterol metabolism. Elucidating the mechanisms with which disturbances of cholesterol homeostasis link to neurodegeneration in recessive inherited disorders with known genetic lesions should shed light on how cholesterol is handled in the healthy CNS and help to understand how these and more complex diseases develop. In the first study we analyzed the synthesis of sterols and the assembly and secretion of lipoprotein particles in Npc1 deficient primary astrocytes. We found that both wild type and Npc1 deficient astrocytes retain significant amounts of desmosterol and other cholesterol precursor sterols as membrane constituents. No difference was observed in the synthesis of sterols and the secretion of newly synthesized sterols between Npc1 wild type, heterozygote or knockout astrocytes. We found that the incorporation of newly synthesized sterols into secreted lipoprotein particles was not inhibited by Npc1 mutation, and the lipoprotein particles were similar to those excreted by wild type astrocytes in shape and size. The bulk of cholesterol was found to be secreted independently of secreted NPC2. These observations demonstrate the ability of Npc1 deficient astrocytes to handle de novo sterols, and highlight the unique sterol composition in the developing brain. Infantile neuronal ceroid lipofuscinosis is caused by the deficiency of a functional Ppt1 enzyme in the cells. In the second study, global gene expression studies of approximately 14000 mouse genes showed significant changes in the expression of 135 genes in Ppt1 deficient neurons compared to wild type. Several genes encoding for enzymes of the mevalonate pathway of cholesterol biosynthesis showed increased expression. As predicted by the expression data, sterol biosynthesis was found to be upregulated in the knockout neurons. These data link Ppt1 deficiency to disturbed cholesterol metabolism in CNS neurons. In the third study we investigated the effect of cathepsin D deficiency on the structure of myelin and lipid homeostasis in the brain. Our proteomics data, immunohistochemistry and western blotting data showed altered levels of the myelin protein components myelin basic protein, proteolipid protein and 2 , 3 -cyclic nucleotide 3 phosphodiesterase in the brains of cathepsin D deficient mice. Electron microscopy revealed altered myelin structure in cathepsin D deficient brains. Additionally, plasmalogen-derived alkenyl chains and 20- and 24-carbon saturated and monounsaturated fatty acids typical for glycosphingolipids were found to be significantly reduced, but polyunsaturated species were significantly increased in the knockout brains, pointing to a decrease in white matter. The levels of ApoE and ABCA1 proteins linked to cholesterol efflux in the CNS were found to be altered in the brains of cathepsin D deficient mice, along with an accumulation of cholesteryl esters and a decrease in triglycerols. Together these data demonstrate altered myelin architecture in cathepsin D deficient mice and link cathepsin D deficiency to aberrant cholesterol metabolism and trafficking. Basic research into rare monogenic diseases sheds light on the underlying biological processes which are perturbed in these conditions and contributes to our understanding of the physiological function of healthy cells. Eventually, understanding gained from the study of disease models may contribute towards establishing treatment for these disorders and further our understanding of the pathogenesis of other, more complex and common diseases.

Periodontitis and peri-implantitis biomarkers in human oral fluids and the null-allele mouse model

Tissue destruction associated with the periodontal disease progression is caused by a cascade of host and microbial factors and proteolytic enzymes. Aberrant laminin-332 (Ln-332), human beta defensin (hBD), and matrix metalloproteinase (MMP) functions have been found in oral inflammatory diseases. The null-allele mouse model appears as the next step in oral disease research. The MMP-8 knock-out mouse model allowed us to clarify the involvement of MMP-8 in vivo in oral and related inflammatory diseases where MMP-8 is suggested to play a key role in tissue destruction. The cleaved Ln-332 γ2-chain species has been implicated in the apical migration of sulcular epithelial cells during the formation of periodontal pockets. We demonstrated that increased Ln-332 fragment levels in gingival crevicular fluid (GCF) are strongly associated with the severity of inflammation in periodontitis. Porphyromonas gingivalis trypsin-like proteinase can cleave an intact Ln-332 γ2-chain into smaller fragments and eventually promote the formation of periodontal pockets. hBDs are components of an innate mucosal defense against pathogenic microbes. Our results suggest that P. gingivalis trypsin-like proteinase can degrade hBD and thus reduce the innate immune response. Elevated levels and the increased activity of MMPs have been detected in several pathological tissue-destructive conditions where MMPs are shown to cleave extracellular matrix (ECM) and basement membrane (BM) molecules and to facilitate tissue destruction. Elevated levels of MMP-8 have been reported in many inflammatory diseases. In periodontitis, MMP-8 levels in gingival crevicular fluid (GCF) and in peri-implant sulcular fluid (PISF) are elevated at sites of active inflammation, and the increased levels of MMP-8 are mainly responsible for collagenase activity, which leads to tissue destruction. MMP-25, expressed by neutrophils, is involved in inflammatory diseases and in ECM turnover. MMP-26 can degrade ECM components and serve as an activator of other MMP enzymes. We further confirmed that increased levels and activation of MMP-8, -25, and -26 in GCF, PISF, and inflamed gingival tissue are associated with the severity of periodontal/peri-implant inflammation. We evaluated the role of MMP-8 in P. gingivalis-induced periodontitis by comparing MMP-8 knock-out (MMP8-/-) and wild-type mice. Surprisingly, MMP-8 significantly attenuated P. gingivalis-induced site-specific alveolar bone loss. We also evaluated systemic changes in serum immunoglobulin and lipoprotein profiles among these mouse groups. P. gingivalis infection increased HDL/VLDL particle size in the MMP-8-/- mice, which is an indicator of lipoprotein responses during systemic inflammation. Serum total LPS and IgG antibody levels were enhanced in both mice groups. P. gingivalis-induced periodontitis, especially in MMP-8-/- mice, is associated with severe alveolar bone loss and with systemic inflammatory and lipoprotein changes that are likely to be involved in early atherosclerosis.

Inflammation-induced atherogenesis, liver alterations, and cardiovascular outcome

Cardiovascular diseases, which presently are considered inflammatory diseases, affect millions of people worldwide. Chronic infections may contribute to the systemic inflammation suggested to increase the risk for cardiovascular diseases. Such chronic infections are periodontitis and Chlamydia pneumoniae infection. They are highly prevalent as approximately 10% of adult population and 30% of people over 50 years old are affected by severe periodontitis and 70-80% of elderly people are seropositive for C. pneumoniae. Our general aim was to investigate the role of infection and inflammation in atherosclerosis both in animal and human studies. We aimed to determine how the two pathogens alter the atherosclerosis-associated parameters, and how they affect the liver inflammation and lipid composition. Furthermore, we evaluated the association between matrix metalloproteinase-8 (MMP-8), a proteinase playing a major role in inflammation, and the future cardiovascular diseases (CVD) events in a population-based cohort. For the animal experiments, we used atherosclerosis-susceptible apolipoprotein E deficient (apoE-/-) mice. They were kept in germ free conditions and fed with a normal chow diet. The bacteria were administered either intravenously (A. actinomycetemcomitans) or intranasally (C. pneumoniae). Several factors were determined from serum as well as from aortic and hepatic tissues. We also determined how cholesterol efflux, a major event in the removal of excess cholesterol from the tissues, and endothelial function were affected by these pathogens. In the human study, serum MMP-8 and its tissue inhibitor (TIMP-1) concentrations were measured and their associations during the follow-up time of 10 years with CVD events were determined. An infection with A. actinomycetemcomitans increased concentrations of inflammatory mediators, MMP production, and cholesterol deposit in macrophages, decreased lipoprotein particle size, and induced liver inflammation. C. pneumoniae infection also elicited an inflammatory response and endothelial dysfunction, as well as induced liver inflammation, microvesicular appearance and altered fatty acid profile. In the population-based cohort, men with increased serum MMP-8 concentration together with subclinical atherosclerosis (carotid artery intima media thickness > 1mm) had a three-fold increased risk for CVD death during the follow-up. The results show that infections with A. actinomycetemcomitans and C. pneumoniae induce proatherogenic changes, as well as affect the liver. These data therefore support the concept that common infections have systemic effects and could be considered as cardiovascular risk factors. Furthermore, our data indicate that, as an independent predictor of fatal CVD event, serum MMP-8 could have a clinical significance in diagnosing cardiovascular diseases.

Latent TGF-β binding proteins -3 and -4 : transcriptional control and extracellular matrix targeting

Extracellular matrix (ECM) is a complex network of various proteins and proteoglycans which provides tissues with structural strength and resilience. By harvesting signaling molecules like growth factors ECM has the capacity to control cellular functions including proliferation, differentiation and cell survival. Latent transforming growth factor β (TGF-β) binding proteins (LTBPs) associate fibrillar structures of the ECM and mediate the efficient secretion and ECM deposition of latent TGF-β. The current work was conducted to determine the regulatory regions of LTBP-3 and -4 genes to gain insight into their tissue-specific expression which also has impact on TGF-β biology. Furthermore, the current research aimed at defining the ECM targeting of the N-terminal variants of LTBP-4 (LTBP-4S and -4L), which is required to understand their functions in tissues and to gain insight into conditions in which TGF-β is activated. To characterize the regulatory regions of LTBP-3 and -4 genes in silico and functional promoter analysis techniques were employed. It was found that the expression of LTBP-4S and -4L are under control of two independent promoters. This finding was in accordance with the observed expression patterns of LTBP-4S and -4L in human tissues. All promoter regions characterized in this study were TATAless, GC-rich and highly conserved between human and mouse species. Putative binding sites for Sp1 and GATA family of transcription factors were recognized in all of these regulatory regions. It is possible that these transcription factors control the basal expression of LTBP-3 and -4 genes. Smad binding element was found within the LTBP-3 and -4S promoter regions, but it was not present in LTBP-4L promoter. Although this element important for TGF-β signaling was present in LTBP-4S promoter, TGF-β did not induce its transcriptional activity. LTBP-3 promoter activity and mRNA expression instead were stimulated by TGF-β1 in osteosarcoma cells. It was found that the stimulatory effect of TGF-β was mediated by Smad and Erk MAPK signaling pathways. The current work explored the ECM targeting of LTBP-4S and identified binding partners of this protein. It was found that the N-terminal end of LTBP-4S possesses fibronectin (FN) binding sites which are critical for its ECM targeting. FN deficient fibroblasts incorporated LTBP-4S into their ECM only after addition of exogenous FN. Furthermore, LTBP-4S was found to have heparin binding regions, of which the C-terminal binding site mediated fibroblast adhesion. Soluble heparin prevented the ECM association of LTBP-4S in fibroblast cultures. In the current work it was observed that there are significant differences in the secretion, processing and ECM targeting of LTBP-4S and -4L. Interestingly, it was observed that most of the secreted LTBP-4L was associated with latent TGF-β1, whereas LTBP-4S was mainly secreted as a free form from CHO cells. This thesis provides information on transcriptional regulation of LTBP-3 and -4 genes, which is required for the deeper understanding of their tissue-specific functions. Further, the current work elucidates the structural variability of LTBPs, which appears to have impact on secretion and ECM targeting of TGF-β. These findings may advance understanding the abnormal activation of TGF-β which is associated with connective tissue disorders and cancer.

Impact of the Human Bacterial Environment on Mycobacteriosis and Allergy

My work describes two sectors of the human bacterial environment: 1. The sources of exposure to infectious non-tuberculous mycobacteria. 2. Bacteria in dust, reflecting the airborne bacterial exposure in environments protecting from or predisposing to allergic disorders. Non-tuberculous mycobacteria (NTM) transmit to humans and animals from the environment. Infection by NTM in Finland has increased during the past decade beyond that by Mycobacterium tuberculosis. Among the farm animals, porcine mycobacteriosis is the predominant NTM disease in Finland. Symptoms of mycobacteriosis are found in 0.34 % of slaughtered pigs. Soil and drinking water are suspected as sources for humans and bedding materials for pigs. To achieve quantitative data on the sources of human and porcine NTM exposure, methods for quantitation of environmental NTM are needed. We developed a quantitative real-time PCR method, utilizing primers targeted at the 16S rRNA gene of the genus of Mycobacterium. With this method, I found in Finnish sphagnum peat, sandy soils and mud high contents of mycobacterial DNA, 106 to 107 genome equivalents per gram. A similar result was obtained by a method based on the Mycobacterium-specific hybridization of 16S rRNA. Since rRNA is found mainly in live cells, this result shows that the DNA detected by qPCR mainly represented live mycobacteria. Next, I investigated the occurrence of environmental mycobacteria in the bedding materials obtained from 5 pig farms with high prevalence (>4 %) of mycobacteriosis. When I used for quantification the same qPCR methods as for the soils, I found that piggery samples contained non-mycobacterial DNA that was amplified in spite of several mismatches with the primers. I therefore improved the qPCR assay by designing Mycobacterium-specific detection probes. Using the probe qPCR assay, I found 105 to 107 genome equivalents of mycobacterial DNA in unused bedding materials and up to 1000 fold more in the bedding collected after use in the piggery. This result shows that there was a source of mycobacteria in the bedding materials purchased by the piggery and that mycobacteria increased in the bedding materials during use in the piggery. Allergic diseases have reached epidemic proportions in urbanized countries. At the same time, childhood in rural environment or simple living conditions appears to protect against allergic disorders. Exposure to immunoreactive microbial components in rural environments seems to prevent allergies. I searched for differences in the bacterial communities of two indoor dusts, an urban house dust shown to possess immunoreactivity of the TH2-type and a farm barn dust with TH1-activity. The immunoreactivities of the dusts were revealed by my collaborators, in vitro in human dendritic cells and in vivo in mouse. The dusts accumulated >10 years in the respiratory zone (>1.5 m above floor), thus reflecting the long-term content of airborne bacteria at the two sites. I investigated these dusts by cloning and sequencing of bacterial 16S rRNA genes from dust contained DNA. From the TH2-active urban house dust, I isolated 139 16S rRNA gene clones. The most prevalent genera among the clones were Corynebacterium (5 species, 34 clones), Streptococcus (8 species, 33 clones), Staphylococcus (5 species, 9 clones) and Finegoldia (1 species, 9 clones). Almost all of these species are known as colonizers of the human skin and oral cavity. Species of Corynebacterium and Streptococcus have been reported to contain anti-inflammatory lipoarabinomannans and immunmoreactive beta-glucans respectively. Streptococcus mitis, found in the urban house dust is known as an inducer of TH2 polarized immunity, characteristic of allergic disorders. I isolated 152 DNA clones from the TH1-active farm barn dust and found species quite different from those found from the urban house dust. Among others, I found DNA clones representing Bacillus licheniformis, Acinetobacter lwoffii and Lactobacillus each of which was recently reported to possess anti-allergy immunoreactivity. Moreover, the farm barn dust contained dramatically higher bacterial diversity than the urban house dust. Exposure to this dust thus stimulated the human dendritic cells by multiple microbial components. Such stimulation was reported to promote TH1 immunity. The biodiversity in dust may thus be connected to its immunoreactivity. Furthermore, the bacterial biomass in the farm barn dust consisted of live intact bacteria mainly. In the urban house dust only ~1 % of the biomass appeared as intact bacteria, as judged by microscoping. Fragmented microbes may possess bioactivity different from that of intact cells. This was recently shown for moulds. If this is also valid for bacteria, the different immunoreactivities of the two dusts may be explained by the intactness of dustborne bacteria. Based on these results, we offer three factors potentially contributing to the polarized immunoreactivities of the two dusts: (i) the species-composition, (ii) the biodiversity and (iii) the intactness of the dustborne bacterial biomass. The risk of childhood atopic diseases is 4-fold lower in the Russian compared with the Finnish Karelia. This difference across the country border is not explainable by different geo-climatic factors or genetic susceptibilities of the two populations. Instead, the explanation must be lifestyle-related. It has already been reported that the microbiological quality of drinking water differs on the two sides of the borders. In collaboration with allergists, I investigated dusts collected from homes in the Russian Karelia and in the Finnish Karelia. I found that bacterial 16S rRNA genes cloned from the Russian Karelian dusts (10 homes, 234 clones) predominantly represented Gram-positive taxa (the phyla Actinobacteria and Firmicutes, 67%). The Russian Karelian dusts contained nine-fold more of muramic acid (60 to 70 ng mg-1) than the Finnish Karelian dusts (3 to 11 ng mg-1). Among the DNA clones isolated from the Finnish side (n=231), Gram-negative taxa (40%) outnumbered the Gram-positives (34%). Out of the 465 DNA clones isolated from the Karelian dusts, 242 were assigned to cultured validly described bacterial species. In Russian Karelia, animal-associated species e.g. Staphylococcus and Macrococcus were numerous (27 clones, 14 unique species). This finding may connect to the difference in the prevalence of allergy, as childhood contacts with pets and farm animals have been connected with low allergy risk. Plant-associated bacteria and plant-borne 16S rRNA genes (chloroplast) were frequent among the DNA clones isolated from the Finnish Karelia, indicating components originating from plants. In conclusion, my work revealed three major differences between the bacterial communtites in the Russian and in the Finnish Karelian homes: (i) the high prevalence of Gram-positive bacteria on the Russian side and of Gram-negative bacteria on the Finnish side and (ii) the rich presence of animal-associated bacteria on the Russian side whereas (iii) plant-associated bacteria prevailed on the Finnish side. One or several of these factors may connect to the differences in the prevalence of allergy.

Acidic pH and acidic enzymes in atherosclerosis

Atherosclerosis is an inflammatory disease characterized by accumulation of lipids and fibrous connective tissue in the arterial wall. Recently, it has been suggested that decrease in the pH of extracellular fluid of the arterial intima may enhance LDL accumulation by increasing binding of the LDL to matrix proteoglycans and also by making the plaque more favorable for acidic enzymes to be active. Many lysosomal acidic enzymes have been found in atherosclerotic plaques. In this thesis, we were able to induce secretion of lysosomal acidic cathepsin F from human monocyte-derived macrophages by stimulation with angiotensin II. We also showed that LDL pre-proteolyzed with cathepsin S was more prone to subsequent hydrolytic modifications by lipases. Especially acidic secretory sphingomyelinase was able to hydrolyze pre-proteolyzed LDL even at neutral pH. We also showed that the proteolyzed and lipolyzed LDL particles were able to bind more efficiently to human aortic proteoglycans. In addition, the role of extracellular acidic pH on the ability of macrophages to internalize LDL was studied. At acidic pH, the production of cell surface proteoglycans in macrophages was increased as well as the binding of native and modified LDL to cell surface proteoglycans. Furthermore, macrophages cultured at acidic pH showed increased internalization of modified and native LDL leading to foam cell formation. This thesis revealed various mechanisms by which acidic pH can increase LDL retention and accumulation in the arterial intima and has the potential to increase the progression of atherosclerosis.

Nephrin-associated molecules in human glomerular diseases

The glomerular epithelial cells and their intercellular junctions, termed slit diaphragms, are essential components of the filtration barrier in the kidney glomerulus. Nephrin is a transmembrane adhesion protein of the slit diaphragm and a signalling molecule regulating podocyte physiology. In congenital nephrotic syndrome of the Finnish type, mutation of nephrin leads to disruption of the permeability barrier and leakage of plasma proteins into the urine. This doctoral thesis hypothesises that novel nephrin-associated molecules are involved in the function of the filtration barrier in health and disease. Bioinformatics tools were utilized to identify novel nephrin-like molecules in genomic databases, and their distribution in the kidney and other tissues was investigated. Filtrin, a novel nephrin homologue, is expressed in the glomerular podocytes and, according to immunoelectron microscopy, localizes at the slit diaphragm. Interestingly, the nephrin and filtrin genes, NPHS1 and KIRREL2, locate in a head-to-head orientation on chromosome 19q13.12. Another nephrin-like molecule, Nphs1as was cloned in mouse, however, no expression was detected in the kidney but instead in the brain and lymphoid tissue. Notably, Nphs1as is transcribed from the nephrin locus in an antisense orientation. The glomerular mRNA and protein levels of filtrin were measured in kidney biopsies of patients with proteinuric diseases, and marked reduction of filtrin mRNA levels was detected in the proteinuric samples as compared to controls. In addition, altered distribution of filtrin in injured glomeruli was observed, with the most prominent decrease of the expression in focal segmental glomerulosclerosis. The role of the slit diaphragm-associated genes for the development of diabetic nephropathy was investigated by analysing single nucleotide polymorphisms. The genes encoding filtrin, densin-180, NEPH1, podocin, and alpha-actinin-4 were analysed, and polymorphisms at the alpha-actinin-4 gene were associated with diabetic nephropathy in a gender-dependent manner. Filtrin is a novel podocyte-expressed protein with localization at the slit diaphragm, and the downregulation of filtrin seems to be characteristic for human proteinuric diseases. In the context of the crucial role of nephrin for the glomerular filter, filtrin appears to be a potential candidate molecule for proteinuria. Although not expressed in the kidney, the nephrin antisense Nphs1as may regulate the expression of nephrin in extrarenal tissues. The genetic association analysis suggested that the alpha-actinin-4 gene, encoding an actin-filament cross-linking protein of the podocytes, may contribute to susceptibility for diabetic nephropathy.

Applications of coralline hydroxyapatite with bioabsorbable containment and reinforcement as bone graft substitute : An experimental study

The aim of the present experimental study was to find out if the applications of coralline hydroxyapatite (HA) can be improved by using bioabsorbable containment or binding substance with particulate HA in mandibular contour augmentation and by using bioabsorbable fibre-reinforced HA blocks in filling bone defects and in anterior lumbar interbody fusion. The use of a separate curved polyglycolide (PGA) containment alone or together with a fast resorbing polyglycolide/polylactide (PGA/PLA) binding substance were compared to the conventional non-contained method in ridge augmentation in sheep. The contained methods decreased HA migration, but the augmentations did not differ significantly. The use of the containment caused a risk for wound dehiscence and infection. Histologically there was a rapid connective tissue ingrowth into the HA graft and it was more abundant with the PGA containment compared to the non-contained augmentation and even additionally rich when the HA particles were bound with PGA/PLA copolymer. However, the bone ingrowth was best in the non-contained augmentation exceeding 10-12 % of the total graft area at 24 weeks. Negligible or no bone ingrowth was seen in the cases where the polymer composite was added to the HA particles and, related to that, foreign-body type cells were seen at the interface between the HA and host bone. The PGA and poly-dl/l-lactide (PDLLA) fibre-reinforced coralline HA blocks were studied in the metaphyseal and in the diaphyseal defects in rabbits. A rapid bone ingrowth was seen inside the both types of implants. Both PGA and PDLLA fibres induced an inflammatory fibrous reaction around themselves but it did not hinder the bone ingrowth. The bone ingrowth pattern was directed according to the loading conditions so that the load-carrying cortical ends of the implants as well as the implants sited in the diaphyseal defects were the most ossified. The fibre-reinforced coralline HA implants were further studied as stand-alone grafts in the lumbar anterior interbody implantation in pigs. The strength of the HA implants proved not to be adequate, the implants fractured in six weeks and the disc space was gradually lost similarly to that of the discectomized spaces. Histologically, small quantities of bone ingrowth was seen in some of the PGA and PDLLA reinforced coralline implants while no bone formation was identified in any of the PDLLA reinforced synthetic porous HA implants. While fragmented, the inner structure of the implants was lost, the bone ingrowth was minimal, and the disc was replaced by the fibrous connective tissue. When evaluated radiologically the grade of ossification was assessed as better than histologically, and, when related to the histologic findings, CT was more dependable than the plain films to show ossification of the implanted disc space. Local kyphosis was a frequent finding along with anterior bone bridging and ligament ossification as a consequence of instability of the implanted segment.

Evaluation of the Biocompatibility of Poly (ortho ester), Copolymer of ε-caprolactone/D,L-lactide and the Composite of Copolymer of ε-caprolactone/D,L-lactide and Tricalciumphosphate as Bone Filling Material

The purpose of this series of studies was to evaluate the biocompatibility of poly (ortho) ester (POE), copolymer of ε-caprolactone and D,L-lactide [P (ε-CL/DL-LA)] and the composite of P(ε-CL/DL-LA) and tricalciumphosphate (TCP) as bone filling material in bone defects. Tissue reactions and resorption times of two solid POE-implants (POE 140 and POE 46) with different methods of sterilization (gamma- and ethylene oxide sterilization), P(ε-CL/DL-LA)(40/60 w/w) in paste form and 50/50 w/w composite of 40/60 w/w P(ε-CL/DL-LA) and TCP and 27/73 w/w composite of 60/40 w/w P(ε-CL/DL-LA) and TCP were examined in experimental animals. The follow-up times were from one week to 52 weeks. The bone samples were evaluated histologically and the soft tissue samples histologically, immunohistochemically and electronmicroscopically. The results showed that the resorption time of gamma sterilized POE 140 was eight weeks and ethylene oxide sterilized POE 140 13 weeks in bone. The resorption time of POE 46 was more than 24 weeks. The gamma sterilized rods started to erode from the surface faster than ethylene oxide sterilized rods for both POEs. Inflammation in bone was from slight to moderate with POE 140 and moderate with POE 46. No highly fluorescent layer of tenascin or fibronectin was found in the soft tissue. Bone healing at the sites of implantation was slower than at control sites with the copolymer in small bone defects. The resorption time for the copolymer was over one year. Inflammation in bone was mostly moderate. Bone healing at the sites of implantation was also slower than at the control sites with the composite in small and large mandibular bone defects. Bone formation had ceased at both sites by the end of follow-up in large mandibular bone defects. The ultrastructure of the connective tissue was normal during the period of observation. It can be concluded that the method of sterilization influenced the resorption time of both POEs. Gamma sterilized POE 140 could have been suitable material for filling small bone defects, whereas the degradation times of solid EO-sterilized POE 140 and POE 46 were too slow to be considered as bone filling material. Solid material is difficult to contour, which can be considered as a disadvantage. The composites were excellent to handle, but the degradation time of the polymer and the composites were too slow. Therefore, the copolymer and the composite can not be recommended as bone filling material.

Adipose tissue inflammation, liver fat and insulin resistance in humans

BACKGROUND: Obesity is closely associated with insulin resistance, which is a pathophysiologic condition contributing to the important co-morbidities of obesity, such as the metabolic syndrome and type 2 diabetes mellitus. In obese subjects, adipose tissue is characterized by inflammation (macrophage infiltration, increased expression insulin resistance genes and decreased expression of insulin sensitivity genes). Increased liver fat, without excessive alcohol consumption, is defined as non-alcoholic fatty liver disease (NAFLD) and also associated with obesity and insulin resistance. It is unknown whether and how insulin resistance is associated with altered expression of adipocytokines (adipose tissue-derived signaling molecules), and whether adipose tissue inflammation and NAFLD coexist independent of obesity. Genetic factors could explain variation in liver fat independent of obesity but the heritability of NAFLD is unknown. AIMS: To determine whether acute regulation of adipocytokine expression by insulin in adipose tissue is altered in obesity. To investigate the relationship between adipose tissue inflammation and liver fat content independent of obesity. To assess the heritability of serum alanine aminotransferase (ALT) activity, a surrogate marker of liver fat. METHODS: 55 healthy normal-weight and obese volunteers were recruited. Subcutaneous adipose tissue biopsies were obtained for measurement of gene expression before and during 6 hours of euglycemic hyperinsulinemia. Liver fat content was measured by proton magnetic resonance spectroscopy, and adipose tissue inflammation was assessed by gene expression, immunohistochemistry and lipidomics analysis. Genetic factors contributing to serum ALT activity were determined in 313 twins by statistical heritability modeling. RESULTS: During insulin infusion the expression of insulin sensitivity genes remains unchanged, while the expression of insulin resistance genes increases in obese/insulin-resistant subjects compared to insulin-sensitive subjects. Adipose tissue inflammation is associated with liver fat content independent of obesity. Adipose tissue of subjects with high liver fat content is characterized infiltrated macrophages and increased expression of inflammatory genes, as well as by increased concentrations of ceramides compared to equally obese subjects with normal liver fat. A significant heritability for serum ALT activity was verified. CONCLUSIONS: Effects of insulin infusion on adipose tissue gene expression in obese/insulin-resistant subjects are not only characterized by hyporesponse of insulin sensitivity genes but also by hyperresponse of insulin resistance and inflammatory genes. This suggests that in obesity, the impaired insulin action contributes or self-perpetuates alterations in adipocytokine expression in adipose tissue. Adipose tissue inflammation is increased in subjects with high liver fat compared to equally obese subjects with normal liver fat content. Concentrations of ceramides, the putative mediators of insulin resistance, are increased in adipose tissue in subjects with high liver fat. Genetic factors contribute significantly to variation in serum ALT activity, a surrogate marker of liver fat. These data imply that adipose tissue inflammation and increased liver fat content are closely interrelated, and determine insulin resistance even independent of obesity.

Peripheral facial palsy : Grading, Etiology, and Melkersson-Rosenthal Syndrome

The objective of this study was to assess the utility of two subjective facial grading systems, to evaluate the etiologic role of human herpesviruses in peripheral facial palsy (FP), and to explore characteristics of Melkersson-Rosenthal syndrome (MRS). Intrarater repeatability and interrater agreement were assessed for Sunnybrook (SFGS) and House-Brackmann facial grading systems (H-B FGS). Eight video-recorded FP patients were graded in two sittings by 26 doctors. Repeatability for SFGS was from good to excellent and agreement between doctors from moderate to excellent by intraclass correlation coefficient and coefficient of repeatability. For H-B FGS, repeatability was from fair to good and agreement from poor to fair by agreement percentage and kappa coefficients. Because SFGS was at least as good in repeatability as H-B FGS and showed more reliable results in agreement between doctors, we encourage the use of SFGS over H-B FGS. Etiologic role of human herpesviruses in peripheral FP was studied by searching DNA of herpes simplex virus (HSV) -1 and -2, varicella-zoster virus (VZV), human herpesvirus (HHV) -6A, -6B, and -7, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) by PCR/microarray methods in cerebrospinal fluid (CSF) of 33 peripheral FP patients and 36 controls. Three patients and five controls had HHV-6 or -7 DNA in CSF. No DNA of HSV-1 or -2, VZV, EBV, or CMV was found. Detecting HHV-7 and dual HHV-6A and -6B DNA in CSF of FP patients is intriguing, but does not allow etiologic conclusions as such. These DNA findings in association with FP and the other diseases that they accompanied require further exploration. MRS is classically defined as a triad of recurrent labial or oro-facial edema, recurrent peripheral FP, and plicated tongue. All three signs are present in the minority of patients. Edema-dominated forms are more common in the literature, while MRS with FP has received little attention. The etiology and true incidence of MRS are unknown. Characteristics of MRS were evaluated at the Departments of Otorhinolaryngology and Dermatology focusing on patients with FP. There were 35 MRS patients, 20 with FP and they were mailed a questionnaire (17 answered) and were clinically examined (14 patients). At the Department of Otorhinolaryngology, every MRS patient had FP and half had the triad form of MRS. Two patients, whose tissue biopsies were taken during an acute edema episode, revealed nonnecrotizing granulomatous findings typical for MRS, the other without persisting edema and with symptoms for less than a year. A peripheral blood DNA was searched for gene mutations leading to UNC-93B protein deficiency predisposing to HSV-1 infections; no gene mutations were found. Edema in most MRS FP patients did not dominate the clinical picture, and no progression of the disease was observed, contrary to existing knowledge. At the Department of Dermatology, two patients had triad MRS and 15 had monosymptomatic granulomatous cheilitis with frequent or persistent edema and typical MRS tissue histology. The clinical picture of MRS varied according to the department where the patient was treated. More studies from otorhinolaryngology departments and on patients with FP would clarify the actual incidence and clinical picture of the syndrome.

Bioabsorbable materials for osteosynthesis and small joint arthroplasty in the hand

Fractures and arthritic joint destruction are common in the hand. A reliable and stable fracture fixation can be achieved by metal implants, which however, become unnecessary or even harmful after consolidation. The silicone implant arthroplasty is the current method of choice for reconstruction of metacarpophalangeal joints in rheumatoid patients. However, the outcome tends to worsen with long-term follow-up and implant-related complications become frequent. To address these problems, bioabsorbable implants were designed for the hand area. Aims of the studies were: 1) to evaluate the biomechanical stabilities provided by self- reinforced (SR) bioabsorbable implants in a transverse and an oblique osteotomy of small tubular bones and to compare them with those provided by metal implants; 2) to evaluate the SR poly-L/DL-lactide 70/30 plate for osteosynthesis in a proof-of-principle type of experiment in three cases of hand injuries; and 3) to evaluate the poly-L/D-lactide (PLA) 96/4 joint scaffold, a composite joint implant with a supplementary intramedullary Polyactive® stem and Swanson silicone implant in an experimental small joint arthroplasty model. Methods used were: 1) 112 fresh frozen human cadaver and 160 pig metacarpal bones osteotomised transversally or obliquely, respectively, and tested ex vivo in three point bending and in torsion; 2) three patient cases of complex hand injuries; and 3) the fifth metacarpophalangeal joints reconstructed in 18 skeletally-mature minipigs and studied radiologically and histologically. The initial fixation stabilities provided by bioabsorbable implants in the tubular bones of the hand were comparable with currently-employed metal fixation techniques, and were sufficient for fracture stabilisation in three preliminary cases in the hand. However, in torsion the stabilities provided by bioabsorbable implants were lower than that provided by metal counterparts. The bioabsorbable plate enhanced the bending stability for the bioabsorbable fixation construct. PLA 96/4 joint scaffolds demonstrated good biocompatibility and enabled fibrous tissue in-growth in situ. After scaffold degradation, a functional, stable pseudarthrosis with dense fibrous connective tissue was formed. However, the supplementary Polyactive® stem caused a deleterious tissue reaction and therefore the stem can not be applied to the composite joint implant. The bioabsorbable implants have potential for use in clinical hand surgery, but have to await validation in clinical patient series and controlled trials.

X-ray scattering and diffraction enhanced imaging studies of in vitro breast tissues

This thesis is a study of the x-ray scattering properties of tissues and tumours of the breast. Clinical radiography is based on the absorption of the x-rays when passing right through the human body and gives information about the densities of the tissues. Besides being absorbed, x-rays may change their direction within the tissues due to elastic scattering or even to refraction. The phenomenon of scattering is a nuisance to radiography in general, and to mammography in particular, because it reduces the quality of the images. However, scattered x-rays bear very useful information about the structure of the tissues at the supra-molecular level. Some pathologies, like breast cancer, produce alterations to the structures of the tissues, being especially evident in collagen-rich tissues. On the other hand, the change of direction due to refraction of the x-rays on the tissue boundaries can be mapped. The diffraction enhanced imaging (DEI) technique uses a perfect crystal to convert the angular deviations of the x-rays into intensity variations, which can be recorded as images. This technique is of especial interest in the cases were the densities of the tissues are very similar (like in mammography) and the absorption images do not offer enough contrast. This thesis explores the structural differences existing in healthy and pathological collagen in breast tissue samples by the small-angle x-ray scattering (SAXS) technique and compares these differences with the morphological information found in the DEI images and the histo-pathology of the same samples. Several breast tissue samples were studied by SAXS technique in the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Scattering patterns of the different tissues of the breast were acquired and compared with the histology of the samples. The scattering signals from adipose tissue (fat), connective tissue (collagen) and necrotic tissue were identified. Moreover, a clear distinction could be done between the scattering signals from healthy collagen and from collagen from an invasive tumour. Scattering from collagen is very characteristic. It includes several scattering peaks and scattering features that carry information about the size and the spacing of the collagen fibrils in the tissues. It was found that the collagen fibrils in invaded tumours were thinner and had a d-spacing length 0,7% longer that fibrils from healthy tumours. The scattering signals from the breast tissues were compared with the histology by building colour-coded maps across the samples. They were also imaged with the DEI technique. There was a total agreement between the scattering maps, the morphological features seen in the images and the information of the histo- pathological examination. The thesis demonstrates that the x-ray scattering signal can be used to characterize tissues and that it carries important information about the pathological state of the breast tissues, thus showing the potential of the SAXS technique as a possible diagnostic tool for breast cancer.

Consent Practices and Biomedical Knowledge Production in Tissue Economies

The use of human tissue sample collections has become an important tool in biomedical research. The collection, use and distribution of human tissue samples, which include blood and diagnostic tissue samples, from which DNA can be extracted and analyzed has also become a major bio-political preoccupation, not only in national contexts, but also at the transnational level. The foundation of medical research rests on the relationship between the doctor and the research subject. This relationship is a social one, in that it is based on informed consent, privacy and autonomy, where research subjects are made aware of what they are getting involved in and are then able to make an informed decision as to whether or not to participate. Within the post-genomic era, however, our understanding of what constitutes informed consent, privacy and autonomy is changing in relation to the needs of researchers, but also as a reflection of policy aspirations. This reflects a change in the power relations between the rights of the individual in relation to the interests of science and society. Using the notions of tissue economies and biovalue (Waldby, 2002) this research explores the changing relationship between sources and users of samples in biomedical research by examining the contexts under which human tissue samples and the information that is extracted from them are acquired, circulated and exchanged in Finland. The research examines how individual rights, particularly informed consent, are being configured in relation to the production of scientific knowledge in tissue economies in Finland from the 1990s to the present. The research examines the production of biovalue through the organization of scientific knowledge production by examining the policy context of knowledge production as well as three case studies (Tampere Research Tissue Bank, Hereditary Non-polyposis Colorectal Cancer and the Finnish Genome Information Center) in which tissues are acquired, circulated and exchanged in Finland. The research shows how interpretations of informed consent have become divergent and the elements and processes that have contributed to these differences. This inquiry shows how the relationship between the interests of individuals is re-configured in relation to the interests of science and society. It indicates how the boundary between interpretations of informed consent, on the one hand, and social and scientific interests, on the other, are being re-drawn and that this process is underscored, in part, by the economic, commercial and preventive potential that research using tissue samples are believed to produce. This can be said to fundamentally challenge the western notion that the rights of the individual are absolute and inalienable within biomedical legislation.

Merkel cell carcinoma : Epidemiological study with special reference to polyomavirus and vascular factors in pathogenesis

Merkel cell carcinoma (MCC) is a rare cutaneous malignancy that occurs predominantly on sun exposed skin areas. A new polyomavirus (MCPyV) was identified in MCC tumor tissues in 2008 suggesting that a viral infection might be an etiological factor. A typical MCC is a rapidly growing painless purple nodule. In its early stage it can be misjudged by its appearance as a cyst or abscess. Recurrences are common and approximately half of the patients will develop lymph node metastases and third of the patents will have distant metastases. It affects mostly elderly persons at an average age of 70 at the time of diagnosis. MCC was first described in 1972 and the first MCC patient in Finland was identified in 1983. MCC has been poorly recognized, but increased awareness and better diagnostic accuracy has increased the incidence since the early years. In this study, all cases with a notation of MCC during 1979 2008 were obtained from the Finnish Cancer Registry. Based on this data, the incidence is 0.11 for men and 0.12 for women. It is similar than that of other Nordic countries, but lower than in the USA. For clinical series, the files of patients diagnosed with MCC during 1983 2004 were reviewed, and the tissue samples were re-evaluated, if available (n=181). Third of the patients were men, and the most common site of the primary tumor was the head and neck (53%). The majority of the patients (86%) presented with a clinically node-negative (Stage I or II) disease, but the disease recurred in 38% of them. The treatment schemes were heterogeneous. No additional benefit from a wide margin (≥2 cm) was found compared to a margin of 0.1-1.9 cm, but intralesional excision was more often associated with local recurrence. None of the patients with Stage I-II disease who had received postoperative radiotherapy had local recurrence during the follow-up period. The 5-year relative survival ratio for Stage I disease was 68%, for Stage II 67%, for Stage III 16%, and for Stage IV 0%. The relative excess risk of death was significantly lower among women than among men. Some of these tissue samples were further analyzed for vascular invasion (n=126) by immunohistochemistry using vascular endothelial markers CD-31 and D2-40. Vascular invasion was seen in 93% of the samples and it was observed already in very small, <5mm tumors. The tissue samples were also analyzed for the presence of MCPyV by using a polymerase chain reaction (PCR) and quantitative PCR. MCPyV DNA was present in 80% of 114 samples studied. The patients with virus-positive tumors had better overall survival than patients with virus-negative tumors. Immunohistochemical analyses were performed for the expression of VEGFR-2 (n=21) and endostatin (n=19), but they had no prognostic value. Our results support the concept of treating MCC with margin-negative excision and radiotherapy to the tumor bed to reduce local recurrence. The finding of a high frequency of lymphovascular invasion reduces its value as a prognostic factor, but emphasizes the role of sentinel node biopsy even in very small primary MCC.