Balance of growth factors in the human perinatal lung : Implications for physiological lung development and link to bronchopulmonary dysplasia

The aims of this Thesis was to evaluate the role of proangiogenic placental growth factor (PlGF), antiangiogenic endostatin and lymphangiogenic vascular endothelial growth factor (VEGF) -C as well as the receptors vascular endothelial growth factor receptor (VEGFR) -2 and VEGFR-3 during lung development and in development of lung injury in preterm infants. The studied growth factors were selected due to a close relationship with VEGF-A; a proangiogenic growth factor important in normal lung angiogenesis and lung injury in preterm infants. The thesis study consists of three analyses. I: Lung samples from fetuses, preterm and term infants without lung injury, as well as preterm infants with acute and chronic lung injury were stained by immunohistochemistry for PlGF, endostatin, VEGF-C, VEGFR-2 and VEGFR-3. II: Tracheal aspirate fluid (TAF) was collected in the early postnatal period from a patient population consisting of 59 preterm infants, half developing bronchopulmonary dysplasia (BPD) and half without BPD. PlGF, endostatin and VEGF-C concentrations were measured by commercial enzyme-linked immunosorbent assay (ELISA). III: Cord plasma was collected from very low birth weight (VLBW) (n=92) and term (n=48) infants in conjuncture with birth and endostatin concentrations were measured by ELISA. I: All growth factors and receptors studied were consistently stained in immunohistochemistry throughout development. For endostatin in early respiratory distress syndrome (RDS), no alveolar epithelial or macrophage staining was seen, whereas in late RDS and BPD groups, both alveolar epithelium and macrophages stained positively in approximately half of the samples. VEGFR-2 staining was fairly consistent, except for the fact that capillary endothelial staining in the BPD group was significantly decreased. II: During the first postnatal week in TAF mean PlGF concentrations were stable whereas mean endostatin and VEGF-C concentrations decreased. Higher concentrations of endostatin and VEGF-C correlated with lower birth weight (BW) and associated with administration of antenatal betamethasone. Parameters reflecting prenatal lung inflammation associated with lower PlGF, endostatin and VEGF-C concentrations. A higher mean supplemental fraction of inspired oxygen during the first 2 postnatal weeks (FiO2) correlated with higher endostatin concentrations. III: Endostatin concentrations in term infants were significantly higher than in VLBW infants. In VLBW infants higher endostatin concentrations associated with the development of BPD, this association remained significant after logistic regression analysis. We conclude that PlGF, endostatin and VEGF-C all have a physiological role in the developing lung. Also, the VEGFR-2 expression profile seems to reflect the ongoing differentiation of endothelia during development. Both endostatin and VEGFR-2 seem to be important in the development of BPD. During the latter part of the first postnatal week, preterm infants developing BPD have lower concentrations of VEGF-A in TAF. Our findings of disrupted VEGFR-2 staining in capillary and septal endothelium seen in the BPD group, as well as the increase in endostatin concentrations both in TAF and cord plasma associated with BPD, seem to strengthen the notion that there is a shift in the angiogenic balance towards a more antiangiogenic environment in BPD. These findings support the vascular hypothesis of BPD.

Matrix proteinases in lung injury in the preterm infant

During inflammation, excess production and release of matrix proteinases, including matrix metalloproteinases (MMPs) and serine proteinases, may result in dysregulated extracellular proteolysis leading to development of tissue damage. Pulmonary inflammation may play an important role in the pathogenesis of lung injury in the preterm infant. The aims of this study were to evaluate involvement of MMPs and serine proteinase trypsin in acute and chronic lung injury in preterm infants and to study the role of these enzymes in acute lung injury by means of an animal model of hyperoxic lung injury. Molecular forms and levels of MMP-2, -8, and -9, and their specific inhibitor, tissue inhibitor of metalloproteinases (TIMP)-2, as well as trypsin were studied in tracheal aspirate fluid (TAF) samples collected from preterm infants with respiratory distress. Expression and distribution of trypsin-2 and proteinase-activated receptor 2 (PAR2) was examined in autopsy lung specimens from fetuses, from preterm infants with respiratory distress syndrome (RDS) or bronchopulmonary dysplasia (BPD), and from newborn infants without lung injury. We detected higher MMP-8 and trypsin-2 and lower TIMP-2 in TAF from preterm infants with more severe acute respiratory distress. Infants subsequently developing BPD had higher levels of MMP-8 and trypsin-2 early postnatally than did those who survived without this chronic lung injury. Immunohistochemically, trypsin-2 was mainly detectable in bronchial epithelium, but also in alveolar epithelium, and its expression was strongest in prolonged RDS. Since trypsin-2 is potent activator of PAR2, a G-protein coupled receptor involved in inflammation, we studied PAR2 expression in the lung. PAR2 co-localized with trypsin-2 in bronchoalveolar epithelium and its expression was significantly higher in bronchoalveolar epithelium in preterm infants with prolonged RDS than in newborn controls. In the experimental study, rats were exposed to >95% oxygen for 24, 48, and 60 hours, or room air. At 48 hours of hyperoxia, MMP-8 and trypsin levels sharply increased in bronchoalveolar lavage fluid, and expression of trypsin appeared in alveolar epithelium, and MMP-8 predominantly in macrophages. In conclusion, high pulmonary MMP-8 and trypsin-2 early postnatally are associated with severity of acute lung injury and subsequent development of BPD in preterm infants. In the injured preterm lung, trypsin-2 co-localizes with PAR2 in bronchoalveolar epithelium, suggesting that PAR2 activated by high levels of trypsin-2 is involved in lung inflammation associated with development of BPD. Marked increase in MMP-8 and trypsin early in the course of experimental hyperoxic lung injury suggests that these enzymes play a role in the pathogenesis of acute lung injury. Further exploration of the roles of trypsin and MMP-8 in lung injury may offer new targets for therapeutic intervention.

Innate Immune Recognition of RNA-virus infection in human macrophages

Innate immunity and host defence are rapidly evoked by structurally invariant molecular motifs common to microbial world, called pathogen associated molecular patterns (PAMPs). In addition to PAMPs, endogenous molecules released in response to inflammation and tissue damage, danger associated molecular patterns (DAMPs), are required for eliciting the response. The most important PAMPs of viruses are viral nucleic acids, their genome or its replication intermediates, whereas the identity and characteristics of virus infection-induced DAMPs are poorly defined. PAMPs and DAMPs engage a limited set of germ-line encoded pattern recognition receptors (PRRs) in immune and non-immune cells. Membrane-bound Toll-like receptors (TLRs), cytoplasmic retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs) and nucleotide-binding oligomerization domain-like receptor (NLRs) are important PRRs involved in the recognition of the molecular signatures of viral infection, such as double-stranded ribonucleic acids (dsRNAs). Engagement of PRRs results in local and systemic innate immune responses which, when activated against viruses, evoke secretion of antiviral and pro-inflammatory cytokines, and programmed cell death i.e., apoptosis of the virus-infected cell. Macrophages are the central effector cells of innate immunity. They produce significant amounts of antiviral cytokines, called interferons (IFNs), and pro-inflammatory cytokines, such as interleukin (IL)-1β and IL-18. IL-1β and IL-18 are synthesized as inactive precursors, pro-IL-1β and pro-IL-18, that are processed by caspase-1 in a cytoplasmic multiprotein complex, called the inflammasome. After processing, these cytokines are biologically active and will be secreted. The signals and secretory routes that activate inflammasomes and the secretion of IL-1β and IL-18 during virus infections are poorly characterized. The main goal of this thesis was to characterize influenza A virus-induced innate immune responses and host-virus interactions in human primary macrophages during an infection. Methodologically, various techniques of cellular and molecular biology, as well as proteomic tools combined with bioinformatics, were utilized. Overall, the thesis provides interesting insights into inflammatory and antiviral innate immune responses, and has characterized host-virus interactions during influenza A virus-infection in human primary macrophages.

Macrophages in regressed and progressed uveal melanoma

Uveal melanoma (UM) is the most common primary ocular malignancy in adults. In Finland, approximately 50 new cases are diagnosed yearly. Up to 50% of UM metastasize, mostly to the liver, although other organs are also affected. Despite improvements in the management of the primary tumour, the survival rates of patients with metastatic UM are poor. Until the 1970s, UMs were treated by enucleation i.e. removal of the eye. Currently, UM is usually treated by brachytherapy, which is known to influence tumour cells and blood vessels. UMs enucleated both primarily and secondarily after brachytherapy contain tumour-infiltrating macrophages, and a high number of macrophages in primary UM is associated with a shorter survival and a higher microvascular density (MVD) within the tumour tissue. The latter is independently associated with a shorter time to metastatic death. Macrophages have several diverse roles depending on their response to variable signals from the surrounding microenvironment. They function as scavengers, as producers of angiogenic and growth factors as well as proteases, which modulate extracellular matrix. Thus, tumour invasiveness and the risk for metastasis increase with increasing macrophage density. The aim of this study was to evaluate the effects of regression and progression of UM on macrophage numbers and microcirculation factors. Tumour regression is induced by primary brachytherapy, and tumour progression is evidenced by the development of metastases. Understanding the biological behaviour of UMs in the both states may help us in finding new treatment modalities against this disease. To achieve these aims case-control analyses of irradiated UMs and primarily-enucleated eyes (34 matched pairs) were performed. UMs were stained immunohistochemically to detect macrophages, extravascular matrix (EVM) loops and networks, and MVD. Following brachytherapy, a lower MVD was observed. The average number of macrophages remained unchanged. Considering that irradiated melanomas may still contain proliferating tumour cells, a clinically-relevant consequence of my study would be the reassurance that the risk for metastasis is likely to be reduced, given that the low MVD in untreated UMs indicates a favourable prognosis. The effect of progression on macrophages was studied in a paired analysis of primarily-enucleated UM and their corresponding hepatic metastases (48 pairs). A cross-sectional histopathological analysis of these pairs was carried out by staining both specimens in a similar way to the first study. MVD was greater in hepatic metastases than in corresponding primary tumours, and the survival of the patient tended to be shorter if hepatic metastases had a higher MVD. Hepatic metastases had also more dendritic macrophages than the primary UMs. Thus, the progression to metastasis seems to alter the inflammatory status within the tumour. Furthermore, determining MVD of biopsied hepatic metastases may serve as a supplementary tool in estimating the prognosis of patients with metastatic uveal melanoma. After irradiation, the majority of treated eyes have been clinically observed to have pigmented episcleral deposits. A noncomparative clinical case series of 211 irradiated UM eyes were studied by recording the number and location of pigmented episcleral deposits during follow-up visits after brachytherapy. For the first time, the study described pigmented episcleral deposits, which are found in the most UM eyes after brachytherapy, and proved them to consist of macrophages full with engulfed melanin particles. This knowledge may save patients from unnecessary enucleation, because episcleral pigmented deposits might be mistaken for extrascleral tumour growth. The presence of pigmented macrophage-related episcleral deposits was associated with plaque size and isotope rather than with tumour size, suggesting that, in addition to tumour regression, radiation atrophy of retinal pigment epithelium and choroid contributes to the formation of the deposits. In the paired (the same 34 pairs as in the first study) cross-sectional study of irradiated and non-irradiated UMs, clinically-visible episcleral deposits and migrating macrophages in other extratumoral tissues were studied histopathologically. Resident macrophages were present in extratumoral tissues in eyes with both irradiated and non-irradiated UM. Irradiation increased both the number of CD68+ macrophages in the sclera beneath the tumour and the number of clinically-observed episcleral macrophages aggregates. Brachytherapy seemed to alter the route of migration of macrophages: after irradiation, macrophages migrated preferentially through the sclera while in non-irradiated UMs they seemed to migrate more along the choroid. In order to understand the influence of these routes on tumour progression and regression in the future, labelling and tracking of activated macrophages in vivo is required.

Experimental and computational studies on lipoprotein lipolysis at acidic pH

Atherosclerosis is a disease of the arteries; its characteristic features include chronic inflammation, extra- and intracellular lipid accumulation, extracellular matrix remodeling, and an increase in extracellular matrix volume. The underlying mechanisms in the pathogenesis of advanced atherosclerotic plaques, that involve local acidity of the extracellular fluid, are still incompletely understood. In this thesis project, my co-workers and I studied the different mechanisms by which local extracellular acidity could promote accumulation of the atherogenic apolipoprotein B-100 (apoB-100)-containing plasma lipoprotein particles in the inner layer of the arterial wall, the intima. We found that lipolysis of atherogenic apoB-100-containing plasma lipoprotein particles (LDL, IDL, and sVLDL) by the secretory phospholipase A2 group V (sPLA2-V) enzyme, was increased at acidic pH. Also, the binding of apoB-100-containing plasma lipoprotein particles to human aortic proteoglycans was dramatically enhanced at acidic pH. Additionally, lipolysis by sPLA2-V enzyme further increased this binding. Using proteoglycan-affinity chromatography, we found that sVLDL lipoprotein particles consist of populations, differing in their affinities toward proteoglycans. These populations also contained different amounts of apolipoprotein E (apoE) and apolipoprotein C-III (apoC-III); the amounts of apoC-III and apoE per particle were highest in the population with the lowest affinity toward proteoglycans. Since PLA2-modification of LDL particles has been shown to change their aggregation behavior, we also studied the effect of acidic pH on the monolayer structure covering lipoprotein particles after PLA2-induced hydrolysis. Using molecular dynamics simulations, we found that, in acidity, the monolayer is more tightly packed laterally; moreover, its spontaneous curvature is negative, suggesting that acidity may promote lipoprotein particles fusion. In addition to extracellular lipid accumulation, the apoB-100-containing plasma lipoprotein particles can be taken up by inflammatory cells, namely macrophages. Using radiolabeled lipoprotein particles and cell cultures, we showed that sPLA2-V-modification of LDL, IDL, and sVLDL lipoproteins particles, at neutral or acidic pH, increased their uptake by human monocyte-derived macrophages.

ADAMs, Osteoclastogenesis and Bone Destruction in the Loosening of the Totally Replaced Hip

Total hip replacement is the golden standard treatment for severe osteoarthritis refractory for conservative treatment. Aseptic loosening and osteolysis are the major long-term complications after total hip replacement. Foreign body giant cells and osteoclasts are locally formed around aseptically loosening implants from precursor cells by cell fusion. When the foreign body response is fully developed, it mediates inflammatory and destructive host responses, such as collagen degradation. In the present study, it was hypothesized that the wear debris and foreign body inflammation are the forces driving local osteoclast formation, peri-implant bone resorption and enhanced tissue remodeling. Therefore the object was to characterize the eventual expression and the role of fusion molecules, ADAMs (an abbreviation for A Disintegrin And Metalloproteinase, ADAM9 and ADAM12) in the fusion of progenitor cells into multinuclear giant cells. For generation of such cells, activated macrophages trying to respond to foreign debris play an important role. Matured osteoclasts together with activated macrophages mediate bone destruction by secreting protons and proteinases, including matrix metalloproteinases (MMPs) and cathepsin K. Thus this study also assessed collagen degradation and its relationship to some of the key collagenolytic proteinases in the aggressive synovial membrane-like interface tissue around aseptically loosened hip replacement implants. ADAMs were found in the interface tissues of revision total hip replacement patients. Increased expression of ADAMs at both transcriptional and translational levels was found in synovial membrane-like interface tissue of revision total hip replacement (THR) samples compared with that in primary THR samples. These studies also demonstrate that multinucleate cell formation from monocytes by stimulation with macrophage-colony stimiulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) is characterized by time dependent changes of the proportion of ADAMs positive cells. This was observed both in the interface membrane in patients and in two different in vitro models. In addition to an already established MCS-F and RANKL driven model, a new virally (parainfluenza 2) driven model (of human salivary adenocarcinoma (HSY) cells or green monkey kidney (GMK) cells) was developed to study various fusion molecules and their role in cell fusion in general. In interface membranes, collagen was highly degraded and collagen degradation significantly correlated with the number of local cells containing collagenolytic enzymes, particularly cathepsin K. As a conclusion, fusion molecules ADAM9 and ADAM12 seem to be dynamically involved in cell-cell fusion processes and multinucleate cell formation. The highly significant correlation between collagen degradation and collagenolytic enzymes, particularly cathepsin K, indicates that the local acidity of the interface membrane in the pathologic bone and soft tissue destruction. This study provides profound knowledge about cell fusion and mechanism responsible for aseptic loosening as well as increases knowledge helpful for prevention and treatment.

Cellular Cholesterol Accumulation and Egress from Endosomal Compartments

One of the most important factors determining the development of atherosclerosis is the amount of LDL particles in the circulation. In general, LDL particles are clinically regarded as “bad cholesterol” since these particles get entrapped within the vascular wall, leading to atherosclerosis. Circulating HDL particles are conversely regarded as “good cholesterol” because of their ability to transport cholesterol from peripheral tissues to the liver for secretion as bile salts. Once inside the artery wall LDL particles are engulfed by macrophages, resulting in macrophage foam cells. If the macrophage foam cells are not able to efflux the cholesterol back into the bloodstream, the excessive cholesterol ultimately leads to cell death, and the deposition of cellular debris within the atherosclerotic lesion. The cells ability to secrete cholesterol is mainly dependent on the ABCA1 transporter (ATP-binding cassette transporter A1) which transfers cellular cholesterol to extracellular apoA-I (apolipoprotein A-I) particles, leading to the generation of nascent HDL particles. The process of atherosclerotic plaque development is therefore to a large extent a cellular one, in which the capacity of the macrophages in handling the excessive cholesterol load determines the progression of lesion development. In this work we have studied the cellular mechanisms that regulate the trafficking of LDL-derived cholesterol from endosomal compartments to other parts of the cell. As a basis for the study we have utilized cells from patients with Niemann-Pick type C disease, a genetic disorder resulting from mutations in the NPC1 and NPC2 genes. In these cells, cholesterol is entrapped within the endosomal compartment, and is not available for efflux. By identifying proteins that bypass the cholesterol trafficking defect, we were able to identify the small GTPase Rab8 as an important protein involved in ABCA1 dependent cholesterol efflux. In the study, we show that Rab8 regulates cholesterol efflux in human macrophages by facilitating intracellular cholesterol transport, as well as by regulating the plasma membrane availability of ABCA1. Collectively, these results give new insight in to atherosclerotic lesion development and intracellular cholesterol processing.

The influence of HIV infection to the periodontium; a clinical, microbiological, and enzymological study

The main targets of human immunodeficiency virus (HIV) are CD4 receptors of CD4+ lymphocytes and many other cells such as monocytes/macrophages, megakaryocytes, peripheral blood dendritic cells, follicular dendritic cells (DC), epidermal Langerhans cells, and astrocytes. Infection and killing of CD4+ lymphocytes or false reaction of the body to HIV infection and the spontaneous apoptosis of CD4+ lymphocytes decrease CD4+ lymphocyte counts leading to immunosuppression, further disease progression, and appearance of opportunistic infections and malignancies. Oral manifestations are considered to be among the first signs of HIV infection. Enhanced degradation of extracellular matrix and basement membrane components in oral diseases including periodontitis is caused by Zn-dependent enzymes called matrix metalloproteinases (MMPs). The levels and degrees of activation of MMP-1, -2, -3, -7, -8, -9, -25, -26, tissue inhibitors of MMPs (TIMP)-1 and -2, and myeloperoxidase (MPO) and collagenolytic/gelatinolytic activities, and also Ig A, -G, and -M, total protein, and albumin levels in a two-year follow-up were studied from salivary samples. The expression of MMP-7, -8, -9, -25, and -26 immunoreactivities in gingival tissue specimens were studied. Healthy HIV-negative subjects served as controls. All studied clinical periodontal parameters and microbiological evaluation of the periodontopathogens showed that periodontal health of the HIV-positive patients was moderately decreased in comparison to the healthy controls. The levels of Candida in the periodontal pockets and salivary MPO increased with the severity of HIV infection. Immunoreactivities and levels of MMPs and TIMPs, and MMP activities (collagenase, gelatinase) were enhanced in the HIV-positive patient salivary samples relative to the healthy controls regardless of the phase of HIV infection. However, these parameters did not reflect periodontal status in a similar way as in the generally healthy periodontitis patients. Salivary total protein, albumin, IgA, -G, and -M levels were significantly higher in all phases of HIV infection compared to the controls, and salivary total protein, IgG and IgM levels remained higher after two years follow-up, partly correlating with the disease progression and which may reflect the leakage of serum components into the mouth and thus a decreased mucosal barrier. Salivary analyses of MMPs and TIMPs with immunohistochemical analyses showed that HIV infection could predispose to periodontal destruction when compared with healthy controls or the body s defence reactions associated with HIV infection may have been reflected or mediated by MMPs.

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.

Cone beam computed tomography in oral radiology

In dentistry, basic imaging techniques such as intraoral and panoramic radiography are in most cases the only imaging techniques required for the detection of pathology. Conventional intraoral radiographs provide images with sufficient information for most dental radiographic needs. Panoramic radiography produces a single image of both jaws, giving an excellent overview of oral hard tissues. Regardless of the technique, plain radiography has only a limited capability in the evaluation of three-dimensional (3D) relationships. Technological advances in radiological imaging have moved from two-dimensional (2D) projection radiography towards digital, 3D and interactive imaging applications. This has been achieved first by the use of conventional computed tomography (CT) and more recently by cone beam CT (CBCT). CBCT is a radiographic imaging method that allows accurate 3D imaging of hard tissues. CBCT has been used for dental and maxillofacial imaging for more than ten years and its availability and use are increasing continuously. However, at present, only best practice guidelines are available for its use, and the need for evidence-based guidelines on the use of CBCT in dentistry is widely recognized. We evaluated (i) retrospectively the use of CBCT in a dental practice, (ii) the accuracy and reproducibility of pre-implant linear measurements in CBCT and multislice CT (MSCT) in a cadaver study, (iii) prospectively the clinical reliability of CBCT as a preoperative imaging method for complicated impacted lower third molars, and (iv) the tissue and effective radiation doses and image quality of dental CBCT scanners in comparison with MSCT scanners in a phantom study. Using CBCT, subjective identification of anatomy and pathology relevant in dental practice can be readily achieved, but dental restorations may cause disturbing artefacts. CBCT examination offered additional radiographic information when compared with intraoral and panoramic radiographs. In terms of the accuracy and reliability of linear measurements in the posterior mandible, CBCT is comparable to MSCT. CBCT is a reliable means of determining the location of the inferior alveolar canal and its relationship to the roots of the lower third molar. CBCT scanners provided adequate image quality for dental and maxillofacial imaging while delivering considerably smaller effective doses to the patient than MSCT. The observed variations in patient dose and image quality emphasize the importance of optimizing the imaging parameters in both CBCT and MSCT.

Cervical headgear in Class II division 1 correction in children : Academic Dissertation

Class II division 1 malocclusion occurs in 3.5 to 13 percent of 7 12 year-old children. It is the most common reason for orthodontic treatment in Finland. Correction is most commonly performed using headgear treatment. The aim of this study was to investigate the effects of cervical headgear treatment on dentition, facial skeletal and soft tissue growth, and upper airway structure, in children. 65 schoolchildren, 36 boys and 29 girls were studied. At the onset of treatment a mean age was 9.3 (range 6.6 12.4) years. All the children were consequently referred to an orthodontist because of Class II division 1 malocclusion. The included children had protrusive maxilla and an overjet of more than 2mm (3 to 11 mm). The children were treated with a Kloehn-type cervical headgear as the only appliance until Class I first molar relationships were achieved. The essential features of the headgear were cervical strong pulling forces, a long upward bent outer bow, and an expanded inner bow. Dental casts and lateral and posteroanterior cephalograms were taken before and after the treatment. The results were compared to a historical, cross-sectional Finnish cohort or to historical, age- and sex-matched normal Class I controls. The Class I first molar relationships were achieved in all the treated children. The mean treatment time was 1.7 (range 0.3-3.1) years. Phase 2 treatments were needed in 52% of the children, most often because of excess overjet or overbite. The treatment decreased maxillary protrusion by inhibiting alveolar forward growth, while the rest of the maxilla and mandible followed normal growth. The palate rotated anteriorly downward. The expansion of the inner bow of the headgear induced widening of the maxilla, nasal cavity, and the upper and lower dental arches. Class II malocclusion was associated with narrower oro- and hypopharyngeal space than in the Class I normal controls. The treatment increased the retropalatal airway space, while the rest of the airway remained unaffected. The facial profile improved esthetically, while the facial convexity decreased. Facial soft tissues masked the facial skeletal convexity, and the soft tissue changes were smaller than skeletal changes. In conclusion, the headgear treatment with the expanded inner bow may be used as an easy and simple method for Class II correction in growing children.

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.

Microbe-induced Innate Immune Responses in Human Dendritic Cells

Two types of antigen-presenting cells (APCs), macrophages and dendritic cells (DCs), function at the interface of innate and adaptive immunity. Through recognition of conserved microbial patterns, they are able to detect the invading pathogens. This leads to activation of signal transduction pathways that in turn induce gene expression of various molecules required for immune responses and eventually pathogen clearance. Cytokines are among the genes induced upon detection of microbes. They play an important role in regulating host immune responses during microbial infection. Chemotactic cytokines, chemokines, are involved in migratory events of immune cells. Cytokines also promote the differentiation of distinct T cell responses. Because of the multiple roles of cytokines in the immune system, the cytokine network needs to be tightly regulated. In this work, the induction of innate immune responses was studied using human primary macrophages or DCs as cell models. Salmonella enterica serovar Typhimurium served as a model for an intracellular bacterium, whereas Sendai virus was used in virus experiments. The starting point of this study was that DCs of mouse origin had recently been characterized as host cells for Salmonella. However, only little was known about the immune responses initiated in Salmonella-infected human DCs. Thus, cellular responses of macrophages and DCs, in particular the pattern of cytokine production, to Salmonella infection were compared. Salmonella-induced macrophages and DCs were found to produce multiple cytokines including interferon (IFN) -gamma, which is conventionally produced by T and natural killer (NK) cells. Both macrophages and DCs also promoted the intracellular survival of the bacterium. Phenotypic maturation of DCs as characterized by upregulation of costimulatory and human leukocyte antigen (HLA) molecules, and production of CCL19 chemokine, were also detected upon infection with Salmonella. Another focus of this PhD work was to unravel the regulatory events controlling the expression of cytokine genes encoding for CCL19 and type III IFNs, which are central to DC biology. We found that the promoters of CCL19 and type III IFNs contain similar regulatory elements that bind nuclear factor kappaB (NF-kappaB) and interferon regulatory factors (IRFs), which could mediate transcriptional activation of the genes. The regulation of type III IFNs in virus infection resembled that of type I IFNs a cytokine class traditionally regarded as antiviral. The induction of type I and type III IFNs was also observed in response to bacterial infection. Taken together, this work identifies new details about the interaction of Salmonella with its phagocytic host cells of human origin. In addition, studies provide information on the regulatory events controlling the expression of CCL19 and the most recently identified IFN family genes, type III IFN genes.

GPRA and the asthma locus on chromosome 7p14-p15

The basis of this work was the identification of a genomic region on chromosome 7p14-p15 that strongly associated with asthma and high serum total immunoglobulin E in a Finnish founder population from Kainuu. Using a hierarchical genotyping approach the linkage region was narrowed down until an evolutionary collectively inherited 133-kb haplotype block was discovered. The results were confirmed in two independent data sets: Asthma families from Quebec and allergy families from North-Karelia. In all the three cohorts studied, single nucleotide polymorphisms tagging seven common gene variants (haplotypes) were identified. Over half of the asthma patients carried three evolutionary closely related susceptibility haplotypes as opposed to approximately one third of the healthy controls. The risk effects of the gene variants varied from 1.4 to 2.5. In the disease-associated region, there was one protein-coding gene named GPRA (G Protein-coupled Receptor for Asthma susceptibility also known as NPSR1) which displayed extensive alternative splicing. Only the two isoforms with distinct intracellular tail sequences, GPRA-A and -B, encoded a full-length G protein-coupled receptor with seven transmembrane regions. Using various techniques, we showed that GPRA is expressed in multiple mucosal surfaces including epithelial cells throughout the respiratory tract. GPRA-A has additional expression in respiratory smooth muscle cells. However, in bronchial biopsies with unknown haplotypes, GPRA-B was upregulated in airways of all patient samples in contrast to the lack of expression in controls. Further support for GPRA as a common mediator of inflammation was obtained from a mouse model of ovalbumin-induced inflammation, where metacholine-induced airway hyperresponsiveness correlated with elevated GPRA mRNA levels in the lung and increased GPRA immunostaining in pulmonary macrophages. A novel GPRA agonist, Neuropeptide S (NPS), stimulated phagocytosis of Esterichia coli bacteria in a mouse macrophage cell line indicating a role for GPRA in the removal of inhaled allergens. The suggested GPRA functions prompted us to study, whether GPRA haplotypes associate with respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) in infants sharing clinical symptoms with asthma. According to the results, near-term RDS and asthma may also share the same susceptibility and protective GPRA haplotypes. As in asthma, GPRA-B isoform expression was induced in bronchial smooth muscle cells in RDS and BPD suggesting a role for GPRA in bronchial hyperresponsiveness. In conclusion, the results of the present study suggest that the dysregulation of the GPRA/NPS pathway may not only be limited to the individuals carrying the risk variants of the gene but is also involved in the regulation of immune functions of asthma.

Microbe-induced activation of inflammatory cytokine response in human cells

Human body is in continuous contact with microbes. Although many microbes are harmless or beneficial for humans, pathogenic microbes possess a threat to wellbeing. Antimicrobial protection is provided by the immune system, which can be functionally divided into two parts, namely innate and adaptive immunity. The key players of the innate immunity are phagocytic white blood cells such as neutrophils, monocytes, macrophages and dendritic cells (DCs), which constantly monitor the blood and peripheral tissues. These cells are armed for rapid activation upon microbial contact since they express a variety of microbe-recognizing receptors. Macrophages and DCs also act as antigen presenting cells (APCs) and play an important role in the development of adaptive immunity. The development of adaptive immunity requires intimate cooperation between APCs and T lymphocytes and results in microbe-specific immune responses. Moreover, adaptive immunity generates immunological memory, which rapidly and efficiently protects the host from reinfection. Properly functioning immune system requires efficient communication between cells. Cytokines are proteins, which mediate intercellular communication together with direct cell-cell contacts. Immune cells produce inflammatory cytokines rapidly following microbial contact. Inflammatory cytokines modulate the development of local immune response by binding to cell surface receptors, which results in the activation of intracellular signalling and modulates target cell gene expression. One class of inflammatory cytokines chemokines has a major role in regulating cellular traffic. Locally produced inflammatory chemokines guide the recruitment of effector cells to the site of inflammation during microbial infection. In this study two key questions were addressed. First, the ability of pathogenic and non-pathogenic Gram-positive bacteria to activate inflammatory cytokine and chemokine production in different human APCs was compared. In these studies macrophages and DCs were stimulated with pathogenic Steptococcus pyogenes or non-pathogenic Lactobacillus rhamnosus. The second aim of this thesis work was to analyze the role of pro-inflammatory cytokines in the regulation of microbe-induced chemokine production. In these studies bacteria-stimulated macrophages and influenza A virus-infected lung epithelial cells were used as model systems. The results of this study show that although macrophages and DCs share several common antimicrobial functions, these cells have significantly distinct responses against pathogenic and non-pathogenic Gram-positive bacteria. Macrophages were activated in a nearly similar fashion by pathogenic S. pyogenes and non-pathogenic L. rhamnosus. Both bacteria induced the production of similar core set of inflammatory chemokines consisting of several CC-class chemokines and CXCL8. These chemokines attract monocytes, neutrophils, dendritic cells and T cells. Thus, the results suggest that bacteria-activated macrophages efficiently recruit other effector cells to the site of inflammation. Moreover, macrophages seem to be activated by all bacteria irrespective of their pathogenicity. DCs, in contrast, were efficiently activated only by pathogenic S. pyogenes, which induced DC maturation and production of several inflammatory cytokines and chemokines. In contrast, L. rhamnosus-stimulated DCs matured only partially and, most importantly, these cells did not produce inflammatory cytokines or chemokines. L. rhamnosus-stimulated DCs had a phenotype of "semi-mature" DCs and this type of DCs have been suggested to enhance tolerogenic adaptive immune responses. Since DCs have an essential role in the development of adaptive immune response the results suggest that, in contrast to macrophages, DCs may be able to discriminate between pathogenic and non-pathogenic bacteria and thus mount appropriate inflammatory or tolerogenic adaptive immune response depending on the microbe in question. The results of this study also show that pro-inflammatory cytokines can contribute to microbe-induced chemokine production at multiple levels. S. pyogenes-induced type I interferon (IFN) was found to enhance the production of certain inflammatory chemokines in macrophages during bacterial stimulation. Thus, bacteria-induced chemokine production is regulated by direct (microbe-induced) and indirect (pro-inflammatory cytokine-induced) mechanisms during inflammation. In epithelial cells IFN- and tumor necrosis factor- (TNF-) were found to enhance the expression of PRRs and components of cellular signal transduction machinery. Pre-treatment of epithelial cells with these cytokines prior to virus infection resulted in markedly enhanced chemokine response compared to untreated cells. In conclusion, the results obtained from this study show that pro-inflammatory cytokines can enhance microbe-induced chemokine production during microbial infection by providing a positive feedback loop. In addition, pro-inflammatory cytokines can render normally low-responding cells to high chemokine producers via enhancement of microbial detection and signal transduction.