Campylobacter Infections of Domestic Origin in Finland

Campylobacter jejuni and C. coli are the leading causes of human bacterial gastroenteritis in developed countries. Most human Campylobacter infections are sporadic and a seasonal peak in the distribution of infections can be seen in the summer months in several countries, including Finland. A variety of risk factors for Campylobacter infections have been identified; handling and eating poultry, drinking unpasteurized milk, contact with domestic animals, and travelling abroad. However, the relative importance of the different risk factors in sporadic cases of Campylobacter infection remains unknown. In most cases, the infection is self-limiting and no specific treatment is required. Campylobacter enteritis can cause a wide range of complications, including reactive arthritis (ReA) that is reported in 1-5% of the cases. Seven clinical microbiology laboratories serving different geographical areas of Finland, participated in this multi-centre study, conducted during a seasonal peak in 2002. In a matched case-control study, domestically-acquired sporadic Campylobacter infections from three geographical areas were collected. The final study comprised 100 cases and 137 controls. Risk factors for sporadic domestically-acquired Campylobacter infections were identified on the basis of a questionnaire; swimming in natural waters was found to be a novel risk factor for Campylobacter infection. Other independent risk factors were tasting or eating raw or undercooked meat and drinking untreated water from a dug well. The role of bacterial strain and host characteristics are not fully understood in Campylobacter infections. Exposure factors, demographical characteristics, and the serotype of the Campylobacter isolate may affect the severity of the enteritis. This cross-sectional study comprised 114 patients with C. jejuni enteritis, diagnosed in three clinical microbiology laboratories; most of the patients had participated in the previous case-control study. Swimming was associated with age ≤ 5 years and serotype Pen 6,7 was found significantly more often among patients reporting swimming. The geographical distribution among serotypes varied; serotype Pen 4-complex appeared more often in patients from urban areas and serotype Pen 21 among patients from more rural areas. Thus, risk factors and sources of infection for C. jejuni infection may vary among individuals depending on age and geographical location. The in vitro susceptibilities of C. jejuni and C. coli strains isolated from patients infected abroad (85 strains) or domestically (393 strains) revealed that susceptibility to erythromycin is still high, even among isolates of foreign origin. However, the novel antimicrobial agent telithromycin did not offer any advantage over erythromycin; isolates with high minimal inhibitory concentrations (MICs) for erythromycin also showed reduced susceptibility to telithromycin. Reduced susceptibility to fluoroquinolones was detected almost exclusively among isolates of foreign origin and half of these isolates with high MICs for fluoroquinolones also showed elevated MICs for doxycycline. Questionnaires concerning complications associated with C. jejuni enteritis were sent to patients two months after becoming ill; 201 patients from seven different geographical areas were included in the study. Musculoskeletal complications after C. jejuni infection were commonly reported by patients (39%). The incidence of classical ReA was 4% and that of Achilles enthesopathy and/or heel pain 9%. Other C. jejuni-associated reactive joint symptoms were commonly reported, however, due to their milder nature seldom seen and diagnosed by a physician. The severity of the enteritis may predict further complications; stomach ache during enteritis was connected to the development of later joint pain. Early antimicrobial treatment, within two days from the start of symptoms, shortened the duration of diarrhoea by two days but did not prevent later musculoskeletal complications. Campylobacter is an important human enteropathogen and causes a significant burden of illness. As the incidence of Campylobacter infections is high, the importance of the infection and the occurrence of complications will increase. This stresses the importance of understanding the risk factors for acquiring Campylobacter infection and how bacterial strain and host characteristics may affect the risk for infection. The role of antimicrobial treatment for acute Campylobacter enteritis seems to be marginal and should be used restrictively.

Molecular and clinical characteristics of tricarboxylic acid cycle-associated tumors

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a rare, dominantly inherited tumor predisposition syndrome characterized by benign cutaneous and uterine (ULM) leiomyomas, and sometimes renal cell cancer (RCC). A few cases of uterine leiomyosarcoma (ULMS) have also been reported. Mutations in a nuclear gene encoding fumarate hydratase (FH), an enzyme of the mitochondrial tricarboxylic acid cycle (TCA cycle), underlie HLRCC. As a recessive condition, germline mutations in FH predispose to a neurological defect, FH deficiency (FHD). Hereditary paragangliomatosis (HPGL) is a dominant disorder associated with paragangliomas and pheochromocytomas. Inherited mutations in three genes encoding subunits of succinate dehydrogenase (SDH), also a TCA cycle enzyme, predispose to HPGL. Both FH and SDH seem to act as tumor suppressors. One of the consequences of the TCA cycle defect is abnormal activation of HIF1 pathway ( pseudohypoxia ) in the HLRCC and HPGL tumors. HIF1 drives transcription of genes encoding e.g. angiogenetic factors which can facilitate tumor growth. Recently hypoxia/HIF1 has been suggested to be one of the causes of genetic instability as well. One of the aims of this study was to broaden the clinical definers of HLRCC. To determine the cancer risk and to identify possible novel tumor types associated with FH mutations eight Finnish HLRCC/FHD families were extensively evaluated. The extension of the pedigrees and the Finnish Cancer Registry based tumor search yielded genealogical and cancer data of altogether 868 individuals. The standardized incidence ratio-based comparison of HLRCC/FHD family members with general Finnish population revealed 6.5-fold risk for RCC. Moreover, risk for ULMS was highly increased. However, according to the recent and more stringent diagnosis criteria of ULMS many of the HLRCC uterine tumors previously considered malignant are at present diagnosed as atypical or proliferative ULMs (with a low risk of recurrence). Thus, the formation of ULMS (as presently defined) in HLRCC appears to be uncommon. Though increased incidence was not observed, interestingly the genetic analyses suggested possible association of breast and bladder cancer with loss of FH. Moreover, cancer cases were exceptionally detected in an FHD family. Another clinical finding was the conventional (clear cell) type RCC of a young Spanish HLRCC patient. Conventional RCC is distinct from the types previously observed in this syndrome but according to these results, FH mutation may underlie some of young conventional cancer cases. Secondly, the molecular pathway from defective TCA cycle to tumor formation was intended to clarify. Since HLRCC and HPGL tumors display abnormally activated HIF1, the hypothesis on the link between HIF1/hypoxia and genetic instability was of interest to study in HLRCC and HPGL tumor material. HIF1α (a subunit of HIF1) stabilization was confirmed in the majority of the specimens. However, no repression of MSH2, a protein of DNA mismatch repair system, or microsatellite instability (MSI), an indicator of genetic instability, was observed. Accordingly, increased instability seems not to play a role in the tumorigenesis of pseudohypoxic TCA cycle-deficient tumors. Additionally, to study the putative alternative functions of FH, a recently identified alternative FH transcript (FHv) was characterized. FHv was found to contain instead of exon 1, an alternative exon 1b. Differential subcellular distribution, lack of FH enzyme activity, low mRNA expression compared to FH, and induction by cellular stress suggest FHv to have a role distinct from FH, for example in apoptosis or survival. However, the physiological significance of FHv requires further elucidation.

Sindbis virus and Pogosta disease in Finland

Sindbis virus (SINV) (genus Alphavirus, family Togaviridae) is an enveloped virus with a genome of single-stranded, positive-polarity RNA of 11.7 kilobases. SINV is widespread in Eurasia, Africa, and Australia, but clinical infection only occurs in a few geographically restricted areas, mainly in Northern Europe. In Europe, antibodies to SINV were detected from patients with fever, rash, and arthritis for the first time in the early 1980s in Finland. It became evident that the causative agent of this syndrome, named Pogosta disease, was closely related to SINV. The disease is also found in Sweden (Ockelbo disease) and in Russia (Karelian fever). Since 1974, for unknown reason, the disease has occurred as large outbreaks every seven years in Finland. This study is to a large degree based on the material collected during the 2002 Pogosta disease outbreak in Finland. We first developed SINV IgM and IgG enzyme immunoassays (EIA), based on highly purified SINV, to be used in serodiagnostics. The EIAs correlated well with the hemagglutination inhibition (HI) test, and all individuals showed neutralizing antibodies. The sensitivities of the IgM and IgG EIAs were 97.6% and 100%, and specificities 95.2% and 97.6%, respectively. E1 and E2 envelope glycoproteins of SINV were shown to be recognized by IgM and IgG in the immunoblot early in infection. We isolated SINV from five patients with acute Pogosta disease; one virus strain was recovered from whole blood, and four other strains from skin lesions. The etiology of Pogosta disease was confirmed by these first Finnish SINV strains, also representing the first human SINV isolates from Europe. Phylogenetic analysis indicated that the Finnish SINV strains clustered with the strains previously isolated from mosquitoes in Sweden and Russia, and seemed to have a common ancestor with South-African strains. Northern European SINV strains could be maintained locally in disease-endemic regions, but the phylogenetic analysis also suggests that redistribution of SINV tends to occur in a longitudinal direction, possibly with migratory birds. We searched for SINV antibodies in resident grouse (N=621), whose population crashes have previously coincided with human SINV outbreaks, and in migratory birds (N=836). SINV HI antibodies were found for the first time in birds during their spring migration to Northern Europe, from three individuals: red-backed shrike, robin, and song thrush. Of the grouse, 27.4% were seropositive in 2003, one year after a human outbreak, but only 1.4% of the grouse were seropositive in 2004. Thus, grouse might contribute to the human epidemiology of SINV. A total of 86 patients with verified SINV infection were recruited to the study in 2002. SINV RNA detection or virus isolation from blood and/or skin lesions was successful in eight patients. IgM antibodies became detectable within the first eight days of illness, and IgG within 11 days. The acute phase of Pogosta disease was characterized by arthritis, itching rash, fatigue, mild fever, headache, and muscle pain. Half of the patients reported in self-administered questionnaires joint symptoms to last > 12 months. Physical examination in 49 of these patients three years after infection revealed persistent joint manifestations. Arthritis (swelling and tenderness in physical examination) was diagnosed in 4.1% (2/49) of the patients. Tenderness in palpation or in movement of a joint was found in 14.3% of the patients in the rheumatologic examination, and additional 10.2% complained persisting arthralgia at the interview. Thus, 24.5% of the patients had joint manifestations attributable to the infection three years earlier. A positive IgM antibody response persisted in 3/49 of the patients; both two patients with arthritis were in this group. Persistent symptoms of SINV infection might have considerable public health implications in areas with high seroprevalence. The age-standardized seroprevalence of SINV (1999-2003, N=2529) in the human population in Finland was 5.2%. The seroprevalence was high in North Karelia, Kainuu, and Central Ostrobothnia. The incidence was highest in North Karelia. Seroprevalence in men (6.0%) was significantly higher than in women (4.1%), however, the average annualized incidence in the non-epidemic years was higher in women than in men, possibly indicating that infected men are more frequently asymptomatic. The seroprevalence increased with age, reaching 15.4% in persons aged 60-69 years. The incidence was highest in persons aged 50-59 years.

Molecular genetic studies on nemaline myopathy and related disorders

Nemaline myopathy (NM) is a rare muscle disorder characterised by muscle weakness and nemaline bodies in striated muscle tissue. Nemaline bodies are derived from sarcomeric Z discs and may be detected by light microscopy. The disease can be divided into six subclasses varying from very severe, in some cases lethal forms to milder forms. NM is usually the consequence of a gene mutation and the mode of inheritance varies between NM subclasses and different families. Mutations in six genes are known to cause NM; nebulin (NEB), alpha-actin, alpha-tropomyosin (TPM3), troponin T1, beta-tropomyosin (TPM2) and cofilin 2, of which nebulin and -actin are the most common. One of the main interests of my research is NEB. Nebulin is a giant muscle protein (600-900 kDa) expressed mainly in the thin filaments of striated muscle. Mutations in NEB are the main cause of autosomal recessive NM. The gene consists of 183 exons. Thus being gigantic, NEB is very challenging to investigate. NEB was screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC) and sequencing. DNA samples from 44 families were included in this study, and we found and published 45 different mutations in them. To date, we have identified 115 mutations in NEB in a total of 96 families. In addition, we determined the occurrence in a world-wide sample cohort of a 2.5 kb deletion containing NEB exon 55 identified in the Ashkenazi Jewish population. In order to find the seventh putative NM gene a genome-wide linkage study was performed in a series of Turkish families. In two of these families, we identified a homozygous mutation disrupting the termination signal of the TPM3 gene, a previously known NM-causing gene. This mutation is likely a founder mutation in the Turkish population. In addition, we described a novel recessively inherited distal myopathy, named distal nebulin myopathy, caused by two different homozygous missense mutations in NEB in six Finnish patients. Both mutations, when combined in compound heterozygous form with a more disruptive mutation, are known to cause NM. This study consisted of molecular genetic mutation analyses, light and electron microscopic studies of muscle biopsies, muscle imaging and clinical examination of patients. In these patients the distribution of muscle weakness was different from NM. Nemaline bodies were not detectable with routine light microscopy, and they were inconspicuous or absent even using electron microscopy. No genetic cause was known to underlie cap myopathy, a congenital myopathy characterised by cap-like structures in the muscle fibres, until we identified a deletion of one codon of the TPM2 gene, in a 30-year-old cap myopathy patient. This mutation does not change the reading frame of the gene, but a deletion of one amino acid does affect the conformation of the protein produced. In summary, this thesis describes a novel distal myopathy caused by mutations in the nebulin gene, several novel nebulin mutations associated with nemaline myopathy, the first molecular genetic cause of cap myopathy, i.e. a mutation in the beta-tropomyosin gene, and a founder mutation in the alpha-tropomyosin gene underlying autosomal recessive nemaline myopathy in the Turkish population.

New molecular strategies for prevention of fibroproliferative vascular disease : An experimental approach to restenosis

Vascular intimal hyperplasia is a major complication following angioplasty. The hallmark feature of this disorder is accumulation of dedifferentiated smooth muscle cells (SMCs) to the luminal side of the injured artery, cellular proliferation, migration, and synthesis of extracellular matrix. This finally results in intimal hyperplasia, which is currently considered an untreatable condition. According to current knowledge, a major part of neointimal cells derive from circulating precursor cells. This has outdated the traditional in vitro cell culture methods of studying neointimal cell migration and proliferation using cultured medial SMCs. Somatostatin and some of its analogs with different selectivity for the five somatostatin receptors (sst1 through sst5) have been shown to have vasculoprotective properties in animal studies. However, clinical trials using analogs selective for sst2/sst3/sst5 to prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) have failed to show any major benefits. Sirolimus is a cell cycle inhibitor that has been suggested to act synergistically with the protein-tyrosine kinase inhibitor imatinib to inhibit intimal hyperplasia in rat already at well-tolerated submaximal oral doses. The mechanisms behind this synergy and its long-term efficacy are not known. The aim of this study was to set up an ex vivo vascular explant culture model to measure neointimal cell activity without excluding the participation of circulating progenitor cells. Furthermore, two novel potential vasculoprotective treatment strategies were evaluated in detail in rat models of intimal hyperplasia and in the ex vivo explant model: sst1/sst4-selective somatostatin receptor analogs and combination treatment with sirolimus and imatinib. This study shows how whole vessel explants can be used to study the kinetics of neointimal cells and their progenitors, and to evaluate the anti-migratory and anti-proliferative properties of potential vasculoprotective compounds. It also shows how the influx of neointimal progenitor cells occurs already during the first days after vascular injury, how the contribution of cell migration is more important in the injury response than cell proliferation, and how the adventitia actively contribute in vascular repair. The vasculoprotective effect of somatostatin is mediated preferentially through sst4, and through inhibition of cell migration rather than of proliferation, which may explain why sst2/sst3/sst5-selective analogs have failed in clinical trials. Furthermore, a brief early oral treatment with the combination of sirolimus and imatinib at submaximal doses results in long-term synergistic suppression of intimal hyperplasia. The synergy is a result of inhibition of post-operative thrombocytosis and leukocytosis, inhibition of neointimal cell migration to the injury-site, and maintenance of cell integrity by inhibition of apoptosis and SMC dedifferentiation. In conclusion, the influx of progenitor cells already during the first days after injury and the high neointimal cell migratory activity underlines the importance of early therapeutic intervention with anti-migratory compounds to prevent neointimal hyperplasia. Sst4-selective analogs and the combination therapy with sirolimus and imatinib represent potential targets for the development of such vasculoprotective therapies.

Target Cell Topography and Cytoskeletal Reorganization in Natural Killer Cell Activity

The immune system has to recognize and destroy abnormal or infected cells to maintain homeostasis. Natural killer (NK) cells directly recognize and kill transformed or virus-infected cells without prior sensitization. We have studied both virus-infected and tumor cells in order to identify the target structures involved in triggering NK activity. Mouse/human cell hybrids containing various human chromosomes were used as targets. The human chromosome responsible for activating NK cell killing was identified to chromosome number 6. The results suggest that activated NK cells recognize ligands that are encoded on human chromosome 6. We showed that the ligand on the target cell side was intercellular adhesion molecule 2 (ICAM-2). There was no difference in the level of expression of ICAM-2, however, but a drastic difference was seen in the distribution of the molecule: ICAM-2 was evenly distributed on the surface of the NK-resistant cells, but almost totally redistributed to the tip of uropods, bud-like extensions, which were absent from the parental cells. Interestingly, the gene coding for cytoskeletal linker protein ezrin has been localized to human chromosome 6, and there was a colocalization of ezrin and ICAM-2 in the uropods. Furthermore, the transfected human ezrin into NK cell-resistant cells induced uropod formation, ICAM-2 and ezrin redistribution to newly formed uropods, and sensitized target cells to NK cell killing. These data reveal a novel form of NK cell recognition: target structures are already present on normal cells; they become detectable only after abnormal redistribution into hot spots on the target cell membrane. NK cells are central players in the defence against virus infections. They inhibit the spread of infection, allowing time for specific immune responses to develop. The virus-proteins that directly activate human NK cell killing are largely unknown. We studied the sensitivity of virus-specific early proteins of Semliki Forest virus (SFV) to NK killing. The viral non-structural proteins (nsP1-4) translated early in the virus cycle were transfected in NK-resistant cells. Viral early gene nsP1 alone efficiently sensitized target cells to NK activity, and the tight membrane association of nsP1 seems to be critical in the triggering of NK killing. NsP1 protein colocalized with (redistributed) ezrin in filopodia-like structures to which the NK cells were bound. The results suggest that also in viral infections NK cells react to rapid changes in membrane topography. Based on the results of this thesis, a new model of target cell recognition of NK cells can be suggested: reorganization of the cytoskeleton induces alterations in cell surface topography, and this new pattern of surface molecules is recognized as "altered-self".

Novel prognostic factors in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a malignant clonal blood disease that originates from a pluripotent hematopoietic stem cell. The cytogenetic hallmark of CML, the Philadelphia chromosome (Ph), is formed as a result of reciprocal translocation between chromosomes 9 and 22, which leads to a formation of a chimeric BCR-ABL fusion gene. The BCR-ABL protein is a constitutively active tyrosine kinase that changes the adhesion properties of cells, constitutively activates mitogenic signaling, enhances cell proliferation and reduces apoptosis. This results in leukemic growth and the clinical disease, CML. With the advent of targeted therapies against the BCR-ABL fusion protein, the treatment of CML has changed considerably during the recent decade. In this thesis, the clinical significance of different diagnostic methods and new prognostic factors in CML have been assessed. First, the association between two different methods for measuring CML disease burden (the RQ-PCR and the high mitotic index metaphase FISH) was assessed in bone marrow and peripheral blood samples. The correlation between positive RQ-PCR and metaphase FISH samples was high. However, RQ-PCR was more sensitive and yielded measurable transcripts in 40% of the samples that were negative by metaphase FISH. The study established a laboratory-specific conversion factor for setting up the International Scale when standardizing RQ-PCR measurements. Secondly, the amount of minimal residual disease (MRD) after allogeneic hematopoietic stem cell transplantation (alloHSCT) was determined. For this, metaphase FISH was done for the bone marrow samples of 102 CML patients. Most (68%), had no residual cells during the entire follow-up time. Some (12 %) patients had minor (<1%) MRD which decreased even further with time, whereas 19% had a progressive rise in MRD that exceeded 1% or had more than 1% residual cells when first detected. Residual cells did not become eradicated spontaneously if the frequency of Ph+ cells exceeded 1% during follow-up. Next, the impact of deletions in the derivative chromosome 9, was examined. Deletions were observed in 15% of the CML patients who later received alloHSCT. After alloHSCT, there was no difference in the total relapse rate in patients with or without deletions. Nor did the estimates of overall survival, transplant-related mortality, leukemia-free survival and relapse-free time show any difference between these groups. When conventional treatment regimens are used, the der(9) status could be an important criterion, in conjunction with other prognostic factors, when allogeneic transplantation is considered. The significance of der(9) deletions for patients treated with tyrosine kinase inhibitors is not clear and requires further investigation. In addition to the der(9) status of the patient, the significance of bone marrow lymphocytosis as a prognostic factor in CML was assessed. Bone marrow lymphocytosis during imatinib therapy was a positive predictive factor and heralded optimal response. When combined with major cytogenetic response at three months of treatment, bone marrow lymphocytosis predicted a prognostically important major molecular response at 18 months of imatinib treatment. Although the validation of these findings is warranted, the determination of the bone marrow lymphocyte count could be included in the evaluation of early response to imatinib treatment already now. Finally, BCR-ABL kinase domain mutations were studied in CML patients resistant against imatinib treatment. Point mutations detected in the kinase domain were the same as previously reported, but other sequence variants, e.g. deletions or exon splicing, were also found. The clinical significance of the other variations remains to be determined.

Vascular growth factors and progenitor cells in cardiac allograft arteriosclerosis

Heart transplantation is the only therapeutic modality for many end-stage heart diseases but poor long-term survival remains a challenging problem. This is mainly due to the development of cardiac allograft arteriosclerosis (TxCAD) that is an accelerated form of coronary artery disease. Both traditional cardiovascular and transplantation-related risk factors for TxCAD have been identified but options for therapy are limited. TxCAD involves dysfunction of cardiac allograft vascular cells. Activated endothelial cells (EC) regulate allograft inflammation and secrete smooth muscle cell (SMC) growth factors. In turn, SMC and their progenitors invade the intima of the injured vessels and occlude the affected coronary arteries. Different vascular growth factors have to be delicately regulated in normal vascular development. In the present study, experimental heterotopic transplantation models were used to study the role of angiogenic and pro-inflammatory vascular endothelial growth factor (VEGF), EC growth factor angiopoietin (Ang), and SMC mitogen platelet-derived growth factor (PDGF) in the development of TxCAD. Pharmacological and gene transfer approaches were used to target these growth factors and to assess their therapeutic potential. This study shows that alloimmune response in heart transplants upregulates VEGF expression, and induces allograft angiogenesis that involves donor-derived primitive EC. Intracoronary adenoviral VEGF gene transfer increased macrophage infiltration, intimal angiogenesis and TxCAD. VEGF inhibition with PTK787 decreased allograft inflammation and TxCAD, and simultaneous PDGF inhibition with imatinib further decreased TxCAD. Specific inhibition of two VEGF-receptors (VEGFR) decreased allograft inflammation and TxCAD, and VEGFR-2 inhibition normalized the density of primitive and mature capillaries in the allografts. Adenovirus-mediated transient Ang1 expression in the allograft had anti-inflammatory and anti-arteriosclerotic effects. Adeno-associated virus (AAV)-mediated prolonged Ang1 or Ang2 expression had similar anti-inflammatory effects. However, AAV-Ang1 activated allograft SMC whereas AAV-Ang2 had no effects on SMC activation and decreased the development of TxCAD. These studies indicate an interplay of inflammation, angiogenesis and arteriosclerosis in cardiac allografts, and show that vascular growth factors are important regulators in the process. Also, VEGF inhibition, PDGF inhibition and angiopoietin therapy with clinically-relevant pharmacological agents or novel gene therapy approaches may counteract vascular dysfunction in cardiac allografts, and have beneficial effects on the survival of heart transplant patients in the future.

Antizyme Inhibitor 2 : A Novel Regulator of Cellular Polyamine Homeostasis

Polyamines are organic polycations that participate in various physiological functions, including cell proliferation, differentiation and apoptosis. Cellular polyamines originate from endogenous biosynthesis and exogenous sources. Their subcellular pool is under strict control, achieved by regulating their uptake and metabolism. Polyamine-induced proteins called antizymes (AZ) act as key regulators of intracellular polyamine concentration. They regulate both the transport of polyamines and the activity and degradation of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. AZs themselves are negatively regulated by antizyme inhibitor (AZIN). AZIN functions as a positive regulator of cellular polyamine homeostasis, which by binding to AZs reactivates ODC and induces the uptake of polyamines. In various pathological conditions, including cancer, polyamine levels are misregulated. Polyamine homeostasis has therefore become an attractive target for therapeutic interventions and it is thus crucial to characterize the molecular basis underlying the homeostatic regulation. A novel human AZIN-resembling protein was previously identified in our group. The purpose of this study was to elucidate the function and distribution of this protein, termed as an antizyme inhibitor 2 (AZIN2). According to my results, AZIN2 functions as a novel regulator of polyamine homeostasis. It shows no enzymatic activity, but instead it binds AZs and negates their activity, which subsequently leads to reactivation of ODC and inhibition of its degradation. Expression of AZIN2 is restricted to terminally differentiated cells, such as mast cells (MC) and neurosecretory cells. In these actively secreting cell types, AZIN2 localizes to subcellular vesicles or granules where its function is important for the vesicle-mediated secretion. In MCs, AZIN2 localizes to the serotonin-containing subset of MC granules, and its expression is coupled to MC activation. The functional role of polyamines as potential mediators of MC activity was also investigated, and it was observed that the secretion of serotonin is selectively dependent on activation of ODC. In neurosecretory cells, AZIN2-positive vesicles localize mainly to the trans-Golgi network (TGN). Depletion of AZIN2 or cellular polyamines causes selective fragmentation of the TGN and retards secretion of proteins. Since addition of exogenous polyamines reverses these effects, the data indicate that AZIN2 and its downstream effectors, polyamines, are functionally implicated in the regulation of secretory vesicle transport. My studies therefore reveal a novel function for polyamines as modulators of both constitutive and regulated secretion. Based on the results, I propose that the role of AZIN2 is to act as a local in situ activator of polyamine biosynthesis.

The role of AIP and CDKN1B/p27Kip1 in endocrine neoplasia

Identification of genes predisposing to tumor syndromes has raised general awareness of tumorigenesis. Genetic testing of tumor susceptibility genes aids the recognition of individuals at increased risk of tumors. Identification of novel predisposing genes enables further studies concerning the classification of potential associated tumors and the definition of target patient group. Pituitary adenomas are common, benign neoplasms accounting for approximately 15% of all intracranial tumors. Accurate incidence estimation is challenging since a great portion of these adenomas are small and asymptomatic. Clinically relevant adenomas, that cause symptoms due to the expansion of the cell mass or the over-secretion of normally produced hormones, occur in approximately one of 1 000 individuals. Although the majority of pituitary adenomas are sporadic, a minority occur as components of familial syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 syndrome is caused by germ-line mutations in the MEN1 gene, whereas most of the CNC patients carry the mutated protein kinase A (PKA) regulatory subunit-1-α (PRKAR1A) gene. Recently, other conditions predisposing to endocrine tumors have been identified: Pituitary Adenoma Predisposition (PAP) and MEN type 4 (MEN4). PAP was originally identified in a genetically homogeneous Finnish population. In a population based cohort from Northern Finland, aryl hydrocarbon receptor-interacting protein (AIP) gene mutations were found in 16% of all patients diagnosed with growth hormone (GH) producing pituitary adenoma, and in 40% of the subset of patients who were diagnosed under the age of 35 years. Since AIP mutations were originally described in a defined, homogeneous population from Northern Finland, it was relevant to study whether mutations also occur in more heterogeneous populations. In patient cohorts with different ethnic origins and variable clinical phenotypes, germ-line AIP mutations were detectable at low frequencies (range 0.8-7.4%). AIP mutation-positive patients were often diagnosed with a GH-producing adenoma at a young age, and usually had no family history of endocrine tumors. The low frequency of AIP mutations in randomly selected patients, and the lack of any family history of pituitary adenomas create a challenge for the identification of PAP patients. Our preliminary study suggests that AIP immunohistochemistry may serve as a pre-screening tool to distinguish between the AIP mutation-negative and the mutation-positive tumors. Tumors of various endocrine glands are components of MEN1 and CNC syndromes. Somatic MEN1 and PRKAR1A mutations in sporadic pituitary adenomas are rare, but occur in some of the other tumors related to these syndromes. The role of AIP mutations in endocrine neoplasia was studied and our results indicated that somatic AIP mutations are rare or non-existent in sporadic tumors of endocrine glands (0 of 111). Furthermore, germ-line AIP mutations in prolactin producing adenomas (2 of 9) confirmed the role of this pituitary tumor type in the PAP phenotype. Thyroid disorders are common in the general population, and the majority of them are sporadic. Interestingly, it has been suggested that thyroid disorders might be more common in PAP families. For this reason we studied germ-line AIP mutations in 93 index cases from familial non-medullary thyroid cancer (NMTC) families. The underlying gene or genes for familial NMTC have not been identified yet. None of the patients had any potentially pathogenic AIP mutation. This suggests that AIP is unlikely to play a role in familial NMTCs. A novel multiple endocrine syndrome was originally described in rats with phenotypic features of human MEN type 1 and 2. Germ-line mutations of cyclin-dependent kinase inhibitor 1B (CDKN1B also known as p27Kip1) gene were reported later in these rats and a germ-line mutation was also identified in one human family with MEN1-like phenotype (later named MEN4). To confirm the importance of this gene’s mutations in humans, we performed a mutation screening in MEN-like patients and in patients with pituitary adenoma. Our results indicate that CDKN1B/p27Kip1 mutations appear in a small portion of MEN1-like patients (one of 36), and that such mutations are rare or non-existent in both familial (0 of 19) and sporadic pituitary adenoma patients (0 of 50). In conclusion, this work strengthens the tumor susceptibility role of AIP and CDKN1B/p27Kip1 in endocrine neoplasia. Clarifying the PAP phenotype facilitates the identification of potential AIP mutation carriers. Genetic counseling can be offered to the relatives and follow-up of the mutation carriers can be organized, hence an earlier diagnosis is feasible.

Novel insights on functions of myotilin/palladin family members

Poikkijuovaisen luuranko- ja sydänlihaksen supistumisyksikkö, sarkomeeri, koostuu tarkoin järjestyneistä aktiini- ja myosiinisäikeistä. Rakenne eroaa muista solutyypeistä, joissa aktiinisäikeistö muovautuu jatkuvasti ja sen järjestyminen säätelee solun muotoa, solujakautumista, soluliikettä ja solunsisäisten organellien kuljetusta. Myotilin, palladin ja myopalladin kuuluvat proteiiniperheeseen, jonka yhteispiirteenä ovat immunoglobuliinin kaltaiset (Igl) domeenit. Proteiinit liittyvät aktiinitukirankaan ja niiden arvellaan toimivan solutukirangan rakenne-elementteinä ja säätelijöinä. Myotilinia ja myopalladinia ilmennetään poikkijuovaisessa lihaksessa. Sen sijaan palladinin eri silmukointimuotoja tavataan monissa kudostyypeissä kuten hermostossa, ja eri muodoilla saattaa olla solutyypistä riippuvia tehtäviä. Poikkijuovaisessa lihaksessa kaikki perheen jäsenet sijaitsevat aktiinisäikeitä yhdistävässä Z-levyssä ja ne sitovat Z-levyn rakenneproteiinia, -aktiniinia. Myotilingeenin pistemutaatiot johtavat periytyviin lihastauteihin, kun taas palladinin mutaatioiden on kuvattu liittyvän periytyvään haimasyöpään ja lisääntyneeseen sydäninfarktin riskiin. Tässä tutkimuksessa selvitettin myotilinin ja pallainin toimintaa. Kokeissa löydettiin uusia palladinin 90-92kDa alatyyppiin sitoutuvia proteiineja. Yksi niistä on aktiinidynamiikkaa säätelevä profilin. Profilinilla on kahdenlaisia tehtäviä; se edesauttaa aktiinisäikeiden muodostumista, mutta se voi myös eristää yksittäisiä aktiinimolekyylejä ja edistää säikeiden hajoamista. Solutasolla palladinin ja profilinin sijainti on yhtenevä runsaasti aktiinia sisältävillä solujen reuna-alueilla. Palladinin ja profilinin sidos on heikko ja hyvin dynaaminen, joka sopii palladinin tehtävään aktiinisäideiden muodostumisen koordinoijana. Toinen palladinin sitoutumiskumppani on aktiinisäikeitä yhteensitova -aktiniini. -Aktiniini liittää solutukirangan solukalvon proteiineihin ja ankkuroi solunsisäisiä viestintämolekyylejä. Sitoutumista välittävä alue on hyvin samankaltainen palladinissa ja myotilinissa. Luurankolihaksen liiallinen toistuva venytys muuttaa Z-levyjen rakennetta ja muotoa. Prosessin aikana syntyy uusia aktiinifilamenttejä sisältäviä tiivistymiä ja lopulta uusia sarkomeereja. Löydöstemme perusteella myotilinin uudelleenjärjestyminen noudattaa aktiinin muutoksia. Tämä viittaa siihen, että myotilin liittää yhteen uudismuodostuvia aktiinisäikeitä ja vakauttaa niitä. Myotilin saattaa myös ankkuroida viesti- tai rakennemolekyylejä, joiden tehtävänä on edesauttaa Z-levyjen uudismuodostusta. Tulostemme perusteella arvelemme, että myotilin toimii Z-levyjen rakenteen vakaajana ja aktiinisäikeiden säätelijänä. Palladinin puute johtaa sikiöaikaiseen kuolemaan hiirillä, mutta myotilinin puutoksella ei ole samanlaisia vaikutuksia. Tuotettujen myotilin poistogeenisten hiirten todetiin syntyvän ja kehittyvän normaalisti eikä niillä esiintynyt rakenteellisia tai toiminnallisia häiriöitä. Toisaalta aiemmissa kokeissa, joissa hiirille on siirretty ihmisen lihastautia aikaansaava myotilingeeni, nähdään samankaltaisia kuin sairailla ihmisillä. Näin ollen muuntunut myotilin näyttä olevan lihaksen toiminnalle haitallisempi kuin myotilinin puute. Myotilinin ja palladinin yhteisvaikutusta selvittääksemme risteytimme myotilin poistegeenisen hiiren ja hiirilinjan, joka ilmentää puutteellisesti palladinin 200 kDa muotoa. Puutteellisesti 200 kDa palladinia ilmentävien hiirten sydänlihaksessa todettiin vähäisiä hienorakenteen muutoksia, mutta risteytetyillä hiirillä tavattiin rakenteellisia ja toiminnallisia muutoksia myös luurankolihaksessa. Tulosten perusteella voidaan todeta, että palladinin 200 kDa muoto säätelee sydänlihassolujen rakennetta. Luurankolihaksessa sen sijaan myotilinilla ja palladinilla näyttäisi olevan päällekkäisiä tehtäviä.

Amyloid diseases at old age : a pathological, epidemiological, and genetic study

Along with the increased life span of individuals, the burden of old age-associated diseases has inevitably increased. Alzheimer s disease (AD), probably the most well known geriatric disease, belongs to the old age-associated amyloid diseases. The purpose of this study was to investigate the frequency, genetic and health-associated risk factors, mutual association, and amyloid proteins in two old age-associated amyloid disorders senile systemic amyloidosis (SSA) and cerebral amyloid angiopathy (CAA) as part of the prospective population-based Vantaa 85+ autopsy study on a Finnish population aged 85 years or more (Studies I-III), completed with a case report on a patient with advanced AGel amyloidosis (Study IV). The numbers of patients investigated in the studies (I-III) were 256, 74, and 63, respectively. The diagnosis and grading of amyloid were based upon histological examination of tissue samples obtained post mortem and stained with Congo red. The amyloid fibril and associated proteins were characterized by immunohistochemical staining methods. The genotype frequencies of 20 polymorphisms in 9 genes and information on health-associated risk factors in subjects with and without SSA and CAA were compared. In a Finnish population ≥ 95 years of age, SSA and CAA occurred in 36% and 49% of the subjects, respectively. In total, two-thirds of these very elderly individuals had SSA, CAA, or both. However, in only 14% of the population these two conditions co-occurred. In subjects 85 years or older, the prevalence of SSA was 25%. In this population, SSA was associated with age at the time of death (p=0.002), myocardial infarctions (MIs; p=0.004), the G/G (Val/Val) genotype of the exon 24 polymorphism in the alpha2-macroglobulin (α2M) gene (p=0.042) and with the H2 haplotype of the tau gene (p=0.016). In contrast, the presence of CAA was strongly associated with APOE e4 (p=0.0003), with histopathological AD (p=0.0005), and with clinical dementia (p=0.01) in both e4+ (p=0.02) and e4- (p=0.06) individuals. Apart from demonstrating the amyloid fibril proteins, complement proteins 3d (C3d) and 9 (C9) were detected in the amyloid deposits of CAA and AGel amyloidosis, and α2M protein was found in fibrous scar tissue close to SSA. In conclusion, this first population based study on SSA shows that both SSA and CAA are common in very elderly individuals. Old age, MIs, the exon 24 polymorphism of the α2M gene, and H1/H2 polymorphism of the tau gene associate with SSA while clinical dementia and APOE ε4 genotype associate with CAA. The high prevalence of CAA, combined with its association with clinical dementia independent of APOE genotype, neuropathological AD, or SSA, also highlights its clinical significance in the very aged, among which the serious end stage complications of CAA, namely multiple infarctions and hemorrhages, are rare. The report on a patient having advanced AGel amyloidosis added knowledge on the disease and showed that this generally benign condition occasionally may lead to death. Further studies are warranted to confirm the findings in other populations. Also, the role of α2M and tau in the pathogenesis of SSA and the involvement of complement in the process of amyloid beta (Aβ) protein elimination from the brain remain to be clarified. Finally, the high prevalence of SSA in the elderly raises the need for prospective clinical studies to define its clinical significance.

Nuorten männiköiden kehitys - taimikonhoito ja ensiharvennus

The aim of this study was to investigate the effects of location, site type, regeneration method and precommercial thinning on the characteristics and development of young, even-aged, pure Scots pine stands. In addition, the effects of timing and intensity of first commercial thinning on the yield and profitability during the rotation period were also studied. The stand characteristics and external quality of young Scots pine stands and stand-level growth models were based on extensive inventory data of the Finnish Forest Research Institute for young Scots pine stands (3 measurement times, 192 stands). The effect of precommercial thinning on stand development was examined on the basis of long-term experiments (13 stands, 169 plots). The effect of timing and intensity of the first commercial thinning on yield and profitability were based on measurements made in first commercial thinnings (27 stands of Metsähallitus), and the further stand development was modeled using the MOTTI simulator. The thesis was based on four articles and a summary. Stand level growth models were developed for young, even-aged Scots pine stands. The models reliably predicted the development up until the first commercial thinning stage. The stand density of young Scots pine stands in Finland was moderately low compared to the target values. In addition, the external quality of pines was low on average. The low stand density and poor external quality will result in the need for quality tree selection in thinnings, if high quality sawn timber is required. In Northern Finland, only 20% of the dominant trees were classified as normal. This will lead to the situation where external quality will remain relatively poor up until the end of rotation. Early and light precommercial thinning (Hdom 3 m, to a density of 3000 trees per hectare) increased the thinning removal by 40% compared to late and more intensive precommercial thinning (at 7 meters to a density of 2000 trees per hectare). A model for the effect of precommercial thinning on merchantable thinning removal at the first commercial thinning was developed for forest management planning purposes. When the recommended time of first commercial thinning was delayed from a dominant height of 12 m to 16 m, or by ten years, the yield of merchantable wood was doubled. Simultaneously, the current value of the stumpage revenues (with 4% interest rate) was increased on the average by 65% (330 € per hectare). Variation in stumpage prices or interest rates did not have any effect on the final results. Without exception, delaying the first commercial thinning by ten years seemed to be the most profitable method. This presupposes that precommercial thinning has been carried out at the right time and that tree quality aspects do not be specially considered. Furthermore, the wood yield and economic outcome from the entire rotation were similar regardless of whether the first thinning was performed at the time currently recommended or ten years later.

Community forestry and environmental literacy in northern Thailand : Towards collaborative natural resource management and conservation

Conservation and sustainable management of tropical forests needs a holistic approach: in addition to ecological concerns, socio-economic issues including cultural aspects must be taken into consideration. An ability to adapt practices is a key to successful collaborative natural resource management. Achieving this requires local participation and understanding of local conceptions of the environment. This study examined these issues in the context of northern Thailand. Northern uplands are the home of much of the remaining natural forest in Thailand and several ethnic minority groups commonly referred to as hill tribes. The overall purpose of this study was to grasp a regional view of an ethnically diverse forested area and to elicit prospects to develop community forestry for conservation purposes and for securing people s livelihood. Conservation was a central goal of management as the forests in the area were largely designated as protected. The aim was to study local perceptions, objectives, values and practices of forest management, under the umbrella of the concept environmental literacy, as well as the effects of forest policy on community management goals and activities. Environmental literacy refers to holistic understanding of the environment. It was used as a tool to examine people s views, interests, knowledge and motivation associated to forests. The material for this study was gathered in six villages in Chiang Mai Province. Three minority groups were included in the study, the Karen, Hmong and Lawa, and also the Thai. Household and focus group interviews were conducted in the villages. In addition, officials at district, regional and national levels, workers of non-governmental organisations, and academics were interviewed, and some data were gathered from the students of a local school. The results showed that motivation for protecting the forests existed among each ethnic group studied. This was a result of culture and traditions evolved in the forest environment but also of a need to adapt to a changed situation and environment and to outside pressures. The consequences of deforestation were widely agreed on in the villages, and the impact of socio-economic changes on the forests and livelihood was also recognised. The forest was regarded as a source of livelihood providing land, products and services essential to the people inhabiting rural uplands. Traditions, fire control, cooperation, reforestation, separation of protected and utilisable areas, and rules were viewed as central for conservation. For the villagers, however, conservation meant sustainable use, whereas the government has tended to prefer strict restrictions on forest resource use. Thus, conflicts had arisen. Between communities, cooperation was more dominant than conflict. The results indicated that the heterogeneity of forest dwellers, although it has to be recognised, should not be overemphasised: ethnic diversity can be considered as no major obstacle for successful community forestry. Collaborative management is particularly important in protected areas in order to meet the conservation goals while providing opportunities for livelihood. Forest management needs more positive incentives and increased dialogue.

Is prosopis a curse or a blessing? : An ecological economic analysis of an invasive alien tree species in Sudan.

The issue of the usefulness of different prosopis species versus their status as weeds is a matter of hot debate around the world. The tree Prosopis juliflora had until 2000 been proclaimed weedy in its native range in South America and elsewhere in the dry tropics. P. juliflora or mesquite has a 90-year history in Sudan. During the early 1990s a popular opinion in central Sudan and the Sudanese Government had begun to consider prosopis a noxious weed and a problematic tree species due to its aggressive ability to invade farmlands and pastures, especially in and around irrigated agricultural lands. As a consequence prosopis was officially declared an invasive alien species also in Sudan, and in 1995 a presidential decree for its eradication was issued. Using a total economic valuation (TEV) approach, this study analysed the impacts of prosopis on the local livelihoods in two contrasting irrigated agricultural schemes. Primarily a problem-based approach was used in which the derivation of non-market values was captured using ecological economic tools. In the New Halfa Irrigation Scheme in Kassala State, four separate household surveys were conducted due to diversity between the respective population groups. The main aim was here to study the magnitude of environmental economic benefits and costs derived from the invasion of prosopis in a large agricultural irrigation scheme on clay soil. Another study site, the Gandato Irrigation Scheme in River Nile State represented impacts from prosopis that an irrigation scheme was confronted with on sandy soil in the arid and semi-arid ecozones along the main River Nile. The two cases showed distinctly different effects of prosopis but both indicated the benefits to exceed the costs. The valuation on clay soil in New Halfa identified a benefit/cost ratio of 2.1, while this indicator equalled 46 on the sandy soils of Gandato. The valuation results were site-specific and based on local market prices. The most important beneficial impacts of prosopis on local livelihoods were derived from free-grazing forage for livestock, environmental conservation of the native vegetation, wood and non-wood forest products, as well as shelterbelt effects. The main social costs from prosopis were derived from weeding and clearing it from farm lands and from canalsides, from thorn injuries to humans and livestock, as well as from repair expenses vehicle tyre punctures. Of the population groups, the tenants faced most of the detrimental impacts, while the landless population groups (originating from western and eastern Sudan) as well as the nomads were highly dependent on this tree resource. For the Gandato site the monetized benefit-cost ratio of 46 still excluded several additional beneficial impacts of prosopis in the area that were difficult to quantify and monetize credibly. In River Nile State the beneficial impact could thus be seen as completely outweighing the costs of prosopis. The results can contributed to the formulation of national and local forest and agricultural policies related to prosopis in Sudan and also be used in other countries faced with similar impacts caused by this tree.