Characterization of cytokines, matrix metalloproteinases and toll-like receptors in human periodontal tissue destruction

Periodontal Disease affects the supporting structures of the teeth and is initiated by a microbial biofilm called dental plaque. Severity ranges from superficial inflammation of the gingiva (gingivitis) to extensive destruction of connective tissue and bone leading to tooth loss (periodontitis). In periodontitis the destruction of tissue is caused by a cascade of microbial and host factors together with proteolytic enzymes. Matrix metalloproteinases (MMPs) are known to be central mediators of the pathologic destruction in periodontitis. Initially plaque bacteria provide pathogen-associated molecular patterns (PAMPs) which are sensed by Toll-like receptors (TLRs), and initiate intracellular signaling cascades leading to host inflammation. Our aim was to characterize TNF-α (tumor necrosis factor-alpha) and its type I and II receptors in periodontal tissues, as well as, the effects of TNF-α, IL-1β (interleukin-1beta) and IL-17 on the production and/or activation of MMP-3, MMP-8 and MMP-9. Furthermore we mapped the TLRs in periodontal tissues and assessed how some of the PAMPs binding to the key TLRs found in periodontal tissues affect production of TNF-α and IL-1β by gingival epithelial cells with or without combination of IL-17. TNF-α and its receptors were detected in pericoronitis. Furthermore, increased expression of interleukin-1β and vascular cell adhesion molecule-1 was found as a biological indicator of TNF-α ligand-receptor interaction. MMP-3, -8, and 9 were investigated in periodontitis affected human gingival crevicular fluid and gingival fibroblasts produced pro-MMP-3. Following that, the effect of IL-17 was studied on MMP and pro-inflammatory cytokine production. IL-17 was increased in periodontitis and up-regulated IL-1β, TNF-α, MMP-1 and MMP-3. We continued by demonstrating TLRs in gingival tissues, in which significant differences between patients with periodontitis and healthy controls were found. Finally, enzyme-linked immunosorbent assays were performed to show that the gingival cells response to inflammatory responses in a TLR-dependent manner. Briefly, this thesis demonstrates that TLRs are present in periodontal tissues and present differences in periodontitis compared to healthy controls. The cells of gingival tissues respond to inflammatory process in a TLR-dependent manner by producing pro-inflammatory cytokines. During the destruction of periodontal tissues, the release (IL-1β and TNF-α) and co-operation with other pro-inflammatory cytokines (IL-17), which in turn increase the inflammation and thus be more harmful to the host with the increased presence of MMPs (MMP-1, MMP-3, MMP-8, MMP-9) in diseased over healthy sites.

Matrix Metalloproteinases and their Tissue Inhibitors as Biomarkers in Ulcerative Colitis and Crohn s Disease

Matrix metalloproteinases (MMPs) represent a family of 23 metalloendopeptidases, collectively capable of degrading all components of the extracellular matrix. MMPs have been implicated in several inflammatory processes such as arthritis, atherosclerosis, and even carcinomas. They are also involved in several beneficial activities such as epithelial repair. MMPs are inhibited by endogenous tissue inhibitors of matrix metalloproteinases (TIMP). In this study, MMPs were investigated in intestinal mucosa of inflammatory bowel diseases (IBD), chronic intestinal disorders. The main focus was to characterize mucosal inflammation in the intestine, but also cutaneous pyoderma gangrenosum (PG), to assess similarites with IBD inflammation. MMPs and TIMPs were mainly examined in colonic mucosa, in adult Crohn s disease (CD), and paediatric CD, ulcerative colitis (UC), and indeterminate colitis (IC). Ileal pouch mucosa of proctocolectomized paediatric onset IBD patients was also investigated to characterize pouch mucosa. The focus was on finding specific MMPs that could act as markers to differentiate between different IBD disorders, and MMPs that could be implied as markers for tissue injury, potentially serving as targets for MMP-inhibitors. All examinations were performed using immunohistochemistry. The results show that immunosuppressive agents decrease stromal expression of MMP-9 and -26 that could serve as specific targets for MMP-inhibitors in treating CD. In paediatric colonic inflammation, MMP-10 and TIMP-3 present as molecular markers for IBD inflammation, and MMP-7 for CD. MMP expression in the the pouch mucosa could not be classified as strictly IBD- or non-IBD-like. For the first time, this study describes the expression of MMP-3, -7, -9, -12, and TIMP-2 and -3 in pouch mucosa. The MMP profile in PG bears resemblance to both intestinal IBD inflammation and cutaneous inflammation. Based on the results, MMPs and their inhibitors emerge as promising tools in the differential diagnosis of IBD and characterization of the disease subtype, although further research is necessary. Furthermore, the expression of several MMPs in pouch has been described for the first time. While further research is warranted, the findings contribute to a better understanding of events occurring in IBD mucosa, as well as pyoderma gangrenosum.

Associations among Emdogain®, human oral carcinoma cells and oral proteolytic enzymes

The Enamel matrix derivative Emdogain® (EMD) is a commercially available tissue extract preparation of porcine enamel origin. Studies have shown EMD to be clinically useful in promoting periodontal regeneration. EMD has been widely used in periodontal therapy for over ten years, but the mechanism of its action and the exact composition are not completely clear. EMD is predominantly amelogenin (>90%). However, unlike amelogenin, EMD has a number of growth factor-like effects and it has been shown to enhance the proliferation, migration and other cellular functions of periodontal ligament fibroblasts and osteoblasts. In contrast, the effects of EMD on epithelial cell lines and in particular on oral malignant cells have not been adequately studied. In addition, EMD has effects on the production of cytokines by several oral cell lines and the product is in constant interaction with different oral enzymes. Regardless of the various unknown properties of EMD, it is said to be clinically safe in regenerative procedures, also in medically compromised patients. The aim of the study was to examine whether gingival crevicular fluid (GCF), which contains several different proteolysis enzymes, could degrade EMD and alter its biological functions. In addition, the objective was to study the effects of EMD on carcinogenesis-related factors, in particular MMPs, using in vitro and in vivo models. This study also aimed to contribute to the understanding of the composition of EMD. GCF was capable of degrading EMD, depending on the periodontal status, with markedly more degradation in all states of periodontal disease compared to healthy controls. EMD was observed to stimulate the migration of periodontal ligament fibroblasts (PLF), whereas EMD together with GCF could not stimulate this proliferation. In addition, recombinant amelogenin, the main component of EMD, decreased the migration of PLFs. A comparison of changes induced by EMD and TGF-β1 in the gene profiles of carcinoma cells showed TGF-β1 to regulate a greater number of genes than EMD. However, both of the study reagents enhanced the expression of MMP-10 and MMP-9. Furthermore, EMD was found to induce several factors closely related to carcinogenesis on gene, protein, cell and in vivo levels. EMD enhanced the production of MMP-2, MMP-9 and MMP-10 proteins by cultured carcinoma cells. In addition, EMD stimulated the migration and in vitro wound closure of carcinoma cells. EMD was also capable of promoting metastasis formation in mice. In conclusion, the diseased GCF, containing various proteases, causes degradation of EMD and decreased proliferation of PLFs. Thus, this in vitro study suggests that the regenerative effect of EMD may decrease due to proteases present in periodontal tissues during the inflammation and healing of the tissues in vivo. Furthermore, EMD was observed to enhance several carcinoma-related factors and in particular the production of MMPs by benign and malignant cell lines. These findings suggest that the clinical safety of EMD with regard to dysplastic mucosal lesions should be further investigated.

The scientific basis and development of a matrix metalloproteinase (MMP) -8 specific chair-side test for monitoring of periodontal health and disease from gingival crevicular fluid

Matrix metalloproteinase (MMP) -8, collagenase-2, is a key mediator of irreversible tissue destruction in chronic periodontitis and detectable in gingival crevicular fluid (GCF). MMP-8 mostly originates from neutrophil leukocytes, the first line of defence cells which exist abundantly in GCF, especially in inflammation. MMP-8 is capable of degrading almost all extra-cellular matrix and basement membrane components and is especially efficient against type I collagen. Thus the expression of MMP-8 in GCF could be valuable in monitoring the activity of periodontitis and possibly offers a diagnostic means to predict progression of periodontitis. In this study the value of MMP-8 detection from GCF in monitoring of periodontal health and disease was evaluated with special reference to its ability to differentiate periodontal health and different disease states of the periodontium and to recognise the progression of periodontitis, i.e. active sites. For chair-side detection of MMP-8 from the GCF or peri-implant sulcus fluid (PISF) samples, a dip-stick test based on immunochromatography involving two monoclonal antibodies was developed. The immunoassay for the detection of MMP-8 from GCF was found to be more suitable for monitoring of periodontitis than detection of GCF elastase concentration or activity. Periodontally healthy subjects and individuals suffering of gingivitis or of periodontitis could be differentiated by means of GCF MMP-8 levels and dipstick testing when the positive threshold value of the MMP-8 chair-side test was set at 1000 µg/l. MMP-8 dipstick test results from periodontally healthy and from subjects with gingivitis were mainly negative while periodontitis patients sites with deep pockets ( 5 mm) and which were bleeding on probing were most often test positive. Periodontitis patients GCF MMP-8 levels decreased with hygiene phase periodontal treatment (scaling and root planing, SRP) and even reduced during the three month maintenance phase. A decrease in GCF MMP-8 levels could be monitored with the MMP-8 test. Agreement between the test stick and the quantitative assay was very good (κ = 0.81) and the test provided a baseline sensitivity of 0.83 and specificity of 0.96. During the 12-month longitudinal maintenance phase, periodontitis patients progressing sites (sites with an increase in attachment loss ≥ 2 mm during the maintenance phase) had elevated GCF MMP-8 levels compared with stable sites. General mean MMP-8 concentrations in smokers (S) sites were lower than in non-smokers (NS) sites but in progressing S and NS sites concentrations were at an equal level. Sites with exceptionally and repeatedly elevated MMP-8 concentrations during the maintenance phase were clustered in smoking patients with poor response to SRP (refractory patients). These sites especially were identified by the MMP-8 test. Subgingival plaque samples from periodontitis patients deep periodontal pockets were examined by polymerase chain reaction (PCR) to find out if periodontal lesions may serve as a niche for Chlamydia pneumoniae. Findings were compared with the clinical periodontal parameters and GCF MMP-8 levels to determine the correlation with periodontal status. Traces of C. pneumoniae were identified from one periodontitis patient s pooled subgingival plaque sample by means of PCR. After periodontal treatment (SRP) the sample was negative for C. pneumoniae. Clinical parameters or biomarkers (MMP-8) of the patient with the positive C. pneumoniae finding did not differ from other study patients. In this study it was concluded that MMP-8 concentrations in GCF of sites from periodontally healthy individuals, subjects with gingivitis or with periodontitis are at different levels. The cut-off value of the developed MMP-8 test is at an optimal level to differentiate between these conditions and can possibly be utilised in identification of individuals at the risk of the transition of gingivitis to periodontitis. In periodontitis patients, repeatedly elevated GCF MMP-8 concentrations may indicate sites at risk of progression of periodontitis as well as patients with poor response to conventional periodontal treatment (SRP). This can be monitored by MMP-8 testing. Despite the lower mean GCF MMP-8 concentrations in smokers, a fraction of smokers sites expressed very high MMP-8 concentrations together with enhanced periodontal activity and could be identified with MMP-8 specific chair-side test. Deep periodontal lesions may be niches for non-periodontopathogenic micro-organisms with systemic effects like C. pneumoniae and possibly play a role in the transmission from one subject to another.

Candida proteinases in the degradation of oral mucosal tissue components associated with Candida invasion

The aim of this thesis was to compare the degradation of human oral epithelial proteins by proteinases of different Candida yeast species. We focused on proteins associated with Candida invasion in the cell-to-cell junction, the basement membrane zone, the extracellular matrix, and local tissue inflammatory regulators. Another main objective was to evaluate the effect of the yeast/hyphal transition and pH on the degradative capability of Candida. The enzymatic activity of the Candida proteinases was verified by gelatin zymography. Laminins-332 (Lm-322) and -511(Lm-511) produced by human oral keratinocytes were gathered from the growth media, and E-cadherin (E-Cad) was isolated from the cell membrane of the keratinocytes by immunoprecipitation. The proteins were incubated with Candida cells and cell-free fractions, and degradation was detected by fluorography. Fibronectin degradation was visualised by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Matrix metalloproteinase-9 (MMP-9) activation and tissue inhibitor of metalloproteinase-1 (TIMP-1) fragmentation was detected by using the Western blot and enhanced chemoluminescence (ECL) techniques. Residual activity of TIMP-1 was evaluated by a casein degradation assay. A fluorimetric assay was used to detect and compare Candida proteinase activities with MMP-9. These studies showed that the ability of the different Candida yeast species to degrade human Lm-332, fibronectin, and E-Cad vary from strain to strain and that this degradation is pH-dependent. This indicates that local acidic pH in tissue may play a role in tissue destruction by activating Candida proteinases and aid invasion of Candida into deeper tissue. A potential correlation exists between the morphological form of the yeasts and the degradative ability; the C. albicans yeast form seems to be related to superficial infections, and hyphal forms can apparently invade deeper tissues between the epithelial cells by degradation of E-Cad. Basement membrane degradation is possible, especially in the junctional epithelium, which contains only Lm-332 as a structural component. Local tissue host inflammatory mediators, such as MMP-9, were activated, and TIMP-1 was degraded by certain Candida species, thus indicating the possibility of a weakened host tissue defence mechanism in vivo.

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

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

Latent TGF-beta Binding Proteins : Adhesive functions and matrix association of LTBP-2 and potential functions of LTBP-1 and LTBP-3 in mesothelioma

Latent transforming growth factor-beta (TGF-beta) binding proteins (LTBPs) -1, -3 and -4 are ECM components whose major function is to augment the secretion and matrix targeting of TGF-beta, a multipotent cytokine. LTBP-2 does not bind small latent TGF-beta but has suggested functions as a structural protein in ECM microfibrils. In the current work we focused on analyzing possible adhesive functions of LTBP-2 as well as on characterizing the kinetics and regulation of LTBP-2 secretion and ECM deposition. We also explored the role of TGF-beta binding LTBPs in endothelial cells activated to mimic angiogenesis as well as in malignant mesothelioma. We found that, unlike most adherent cells, several melanoma cell lines efficiently adhered to purified recombinant LTBP-2. Further characterization revealed that the adhesion was mediated by alpha3beta1 and alpha6beta1 integrins. Heparin also inhibited the melanoma cell adhesion suggesting a role for heparan sulphate proteoglycans. LTBP-2 was also identified as a haptotactic substrate for melanoma cell migration. We used cultured human embryonic lung fibroblasts to analyze the temporal and spatial association of LTBP-2 into ECM. By We found that LTBP-2 was efficiently assembled to the ECM only in confluent cultures following the deposition of fibronectin (FN) and fibrillin-1. In early, subconfluent cultures it remained primarily in soluble form after secretion. LTBP-2 colocalized transiently with FN and fibrillin-1. Silencing of fibrillin-1 expression by lentiviral shRNAs profoundly disrupted the deposition of LTBP-2 indicating that the ECM association of LTBP-2 depends on a pre-formed fibrillin-1 network. Considering the established role of TGF-beta as a regulator of angiogenesis we induced morphological activation of endothelial cells by phorbol 12-myristate 13-acetate (PMA) and followed the fate of LTBP-1 in the endothelial ECM. This resulted in profound proteolytic processing of LTBP-1 and release of latent TGF-beta complexes from the ECM. The processing was coupled with increased activation of MT-MMPs and specific upregulation of MT1-MMP. The major role of MT1-MMP in the proteolysis of LTBP-1 was confirmed by suppressing the expression with lentivirally induced short-hairpin RNAs as well as by various metalloproteinases inhibitors. TGF-beta can promote tumorigenesis of malignant mesothelioma (MM), which is an aggressive tumor of the pleura with poor prognosis. TGF-beta activity was analyzed in a panel of MM tumors by immunohistochemical staining of phosphorylated Smad-2 (P-Smad2). The tumor cells were strongly positive for P-Smad2 whereas LTBP-1 immunoreactivity was abundant in the stroma, and there was a negative correlation between LTBP-1 and P-Smad2 staining. In addition, the high P-Smad2 immunoreactivity correlated with shorter survival of patients. mRNA analysis revealed that TGF-beta1 was the most highly expressed isoform in both normal human pleura and MM tissue. LTBP-1 and LTBP-3 were both abundantly expressed. LTBP-1 was the predominant isoform in established MM cell lines whereas the expression of LTBP-3 was high in control cells. Suppression of LTBP-3 expression by siRNAs resulted in increased TGF-beta activity in MM cell lines accompanied by decreased proliferation. Our results suggest that decreased expression of LTBP-3 in MM could alter the targeting of TGF-beta to the ECM and lead to its increased activation. The current work emphasizes the coordinated process of the assembly and appropriate targeting of LTBPs with distinct adhesive or cytokine harboring properties into the ECM. The hierarchical assembly may have implications in the modulation of signaling events during morphogenesis and tissue remodeling.

Novel matrix metalloproteinases in intestinal inflammation and in cancer of the gastrointestinal tract

Matrix metalloproteinases (MMPs) comprise a family of 23 zinc-dependent human endopeptidases that can degrade virtually all components of the extracellular matrix (ECM). They are classified into eight subgroups according to their structure and into six subgroups based on their substrate-specificity. MMPs have been implicated in inflammation, tissue destruction, cell migration, arthritis, vascular remodeling, angiogenesis, and tumor growth and invasion. MMPs are inhibited by their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs). Different MMPs function in the same tasks depending on the tissue or cancer subtype. I investigated the role of recently discovered MMPs, especially MMPs-19 and -26, in intestinal inflammation, in intestinal and cutaneous wound healing, and in intestinal cancer. Several MMPs and TIMPs were studied to determine their exact location at tissue level and to obtain information on possible functions of MMPs in such tissues and diseases as the healthy intestine, inflammatory bowel disease (IBD), neonatal necrotizing enterocolitis (NEC), pyoderma gangrenosum (PG), and colorectal as well as pancreatic cancers. In latent celiac disease (CD), I attempted to identify markers to predict later onset of CD in children and adolescents. The main methods used were immunohistochemistry, in situ hybridization, and Taqman RT-PCR. My results show that MMP-26 is important for re-epithelialization in intestinal and cutaneous wound healing. In colon and pancreatic cancers, MMP-26 seems to be a marker of invasive potential, although it is not itself expressed at the invasive front. MMP-21 is upregulated in pancreatic cancer and may be associated with tumor differentiation. MMPs-19 and -28 are associated with normal tissue turnover in the intestine, but they disappear in tumor progression as if they were protective markers . MMP-12 is an essential protease in intestinal inflammation and tissue destruction, as seen here in NEC and in previous CD studies. In patients with type 1 diabetes (T1D), MMPs-1, -3, and -12 were upregulated in the intestinal mucosa. Furthermore, MMP-7 was strongly elevated in NEC. In a model of aberrant wound repair, PG, MMPs-8, -9, and 10 and TNFα may promote ECM destruction, while absence of MMP-1 and MMP-26 from keratinocytes retards re-epithelialization. Based on my results, I suggest MMP-26 to be considered a putative marker for poor prognosis in pancreatic and colon cancer. However, since it functions differently in various tissues and tumor subtypes, this use cannot be generalized. Furthermore, MMP-26 is a beneficial marker for wound healing if expressed by migrating epithelial cells. MMP-12 expression in latent CD patients warrants research in a larger patient population to confirm its role as a specific marker for CD in pathologically indistinct cases. MMP-7 should be considered one of the most crucial proteases in NEC-associated tissue destruction; hence, specific inhibitors of this MMP are worth investigating. In PG, TNFα inhibitors are potential therapeutic agents, as shown already in clinical trials. In conclusion, studies of several MMPs in specific diseases and in healthy tissues are needed to elucidate their roles at the tissue level. MMPs and TIMPs are not exclusively destructive or reparative in tissues. They seem to function differently in different tissues. To identify selective MMP inhibitors, we must thoroughly understand the MMP profile (degradome) and their functions in various organs not to interfere with normal reparative functions during wound repair or beneficial host-response effects during cancer initiation and growth.

Matrix metalloproteinases as biomarkers in premalignant and malignant tumors of the human skin

The incidence of non-melanoma skin cancer is increasing worldwide. Basal cell carcinoma followed by squamous cell carcinoma and malignant melanoma are the most frequent skin tumors. Immunosuppressed patients have an increased risk of neoplasia, of which non-melanoma skin cancer is the most common. Matrix metalloproteinases (MMPs) are proteolytic enzymes that collectively are capable of degrading virtually all components of the extracellular matrix. MMPs can also process substrates distinct from extracellular matrix proteins and influence cell proliferation, differentiation, angiogenesis, and apoptosis. MMP activity is regulated by their natural inhibitors, tissue inhibitors of metallopro-teinases (TIMPs). In this study, the expression patterns of MMPs, TIMPs, and certain cancer-related molecules were investigated in premalignant and malignant lesions of the human skin. As methods were used immunohistochemisty, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR) from the cell cultures. Our aim was to evaluate the expression pattern of MMPs in extramammary Paget's disease in order to find markers for more advanced tumors, as well as to shed light on the origin of this rare neoplasm. Novel MMPs -21, -26, and -28 were studied in melanoma cell culture, in primary cutaneous melanomas, and their sentinel nodes. The MMP expression profile in keratoacanthomas and well-differentiated squamous cell carcinomas was analyzed to find markers to differentiate benign keratinocyte hyperproliferation from malignantly transformed cells. Squamous cell carcinomas of immunosuppressed organ transplant recipients were compared to squamous cell carcinomas of matched immunocompetent controls to investigate the factors explaining their more aggressive nature. We found that MMP-7 and -19 proteins are abundant in extramammary Paget's disease and that their presence may predict an underlying adenocarcinoma in these patients. In melanomas, MMP-21 was upregulated in early phases of melanoma progression, but disappeared from the more aggressive tumors with lymph node metastases. The presence of MMP-13 in primary melanomas and lymph node metastases may relate to more aggressive disease. In keratoacanthomas, the expression of MMP-7 and -9 is rare and therefore should raise a suspicion of well-differentiated squamous cell carcinomas. Furthermore, MMP-19 and p16 were observed in benign keratinocyte hyperproliferation of keratoacanthomas, whereas they were generally lost from malignant keratinocytes of SCCs. MMP-26 staining was significantly stronger in squamous cell carcinomas and Bowen s disease samples of organ transplant recipients and it may contribute to the more aggressive nature of squamous cell carcinomas in immunosuppressed patients. In addition, the staining for MMP-9 was significantly stronger in macrophages surrounding the tumors of the immunocompetent group and in neutrophils of those patients on cyclosporin medication. In conclusion, based on our studies, MMP-7 and -19 might serve as biomarkers for more aggressive extramammary Paget's disease and MMP-21 for malignant transformation of melanocytes. MMP -7, -9, and -26, however, could play an important role in the pathobiology of keratinocyte derived malignancies.

Pathological changes at the target tissue level in Sjögren's syndrome and their effect on the exocrine function of the salivary glands

Sjögren s syndrome (SS) is a common autoimmune disease affecting the lacrimal and salivary glands. SS is characterized by a considerable female predominance and a late age of onset, commonly at the time of adreno- and menopause. The levels of the androgen prohormone dehydroepiandrosterone-sulphate (DHEA-S) in the serum are lower in patients with SS than in age- and sex-matched healthy control subjects. The eventual systemic effects of low androgen levels in SS are not currently well understood. Basement membranes (BM) are specialized layers of extracellular matrix and are composed of laminin (LM) and type IV collagen matrix networks. BMs deliver messages to epithelial cells via cellular LM-receptors including integrins (Int) and Lutheran blood group antigen (Lu). The composition of BMs and distribution of LM-receptors in labial salivary glands (LSGs) of normal healthy controls and patients with SS was assessed. LMs have complex and highly regulated distribution in LSGs. LMs seem to have specific tasks in the dynamic regulation of acinar cell function. LM-111 is important for the normal acinar cell differentiation and its expression is diminished in SS. Also LM-211 and -411 seem to have some acinar specific functional tasks in LSGs. LM-311, -332 and -511 seem to have more general structure maintaining and supporting roles in LSGs and are relatively intact also in SS. Ints α3β1, α6β1, α6β4 and Lu seem to supply structural basis for the firm attachment of epithelial cells to the BM in LSGs. The expression of Ints α1β1 and α2β1 differed clearly from other LM-receptors in that they were found almost exclusively around the acini and intercalated duct cells in salivons suggesting some type of acinar cell compartment-specific or dominant function. Expression of these integrins was lower in SS compared to healthy controls suggesting that the LM-111 and -211-to-Int α1β1 and α2β1 interactions are defective in SS and are crucial to the maintenance of the acini in LSGs. DHEA/DHEA-S concentration in serum and locally in saliva of patients with SS seems to have effects on the salivary glands. These effects were first detected using the androgen-dependent CRISP-3 protein, the production and secretion of which were clearly diminished in SS. This might be due to the impaired function of the intracrine DHEA prohormone metabolizing machinery, which fails to successfully convert DHEA into its active metabolites in LSGs. The progenitor epithelial cells from the intercalated ductal area of LSGs migrate to the acinar compartment and then undergo a phenotype change into secretory acinar cells. This migration and phenotype change seem to be regulated by the LM-111-to-Int α1β1/Int α2β1 interactions. Lack of these interactions could be one factor limiting the normal remodelling process. Androgens are effective stimulators of Int α1β1 and α2β1 expression in physiologic concentrations. Addition of DHEA to the culture medium had effective stimulating effect on the Int α1β1 and α2β1 expression and its effect may be deficient in the LSGs of patients with SS.

Growth factor induction in intervertebral disc tissue: Observations in basic mechanisms of disc degeneration and rearrangement

The intervertebral disc is composed of concentrically arranged components: annulus fibrosus, the transition zone, and central nucleus pulposus. The major disc cell type differs in various parts of the intervertebral disc. In annulus fibrosus a spindle shaped fibroblast-like cell mainly dominates, whereas in central nucleus pulposus the more rounded chondrocyte-like disc cell is the major cell type. At birth the intervertebral disc is well vascularized, but during childhood and adolescence blood vessels become smaller and less numerous. The adult intervertebral disc is avascular and is nourished via the cartilage endplates. On the other hand, degenerated and prolapsed intervertebral discs are again vascularized, and show many changes compared to normal discs, including: nerve ingrowth, change in collagen turnover, and change in water content. Furthermore, the prolapsed intervertebral disc tissue has a tendency to decrease in size over time. Growth factors are polypeptides which regulate cell growth, extracellular matrix protease activity, and vascularization. Oncoproteins c-Fos and c-Jun heterodimerize, forming the AP-1 transcription factor which is expressed in activated cells. In this thesis the differences of growth factor expression in normal intervertebral disc, the degenerated intervertebral disc and herniated intervertebral disc were analyzed. Growth factors of particular interest were basic fibroblast growth factor (bFGF or FGF-2), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and transforming growth factor beta (TGFβ). Cell activation was visualized by the expression of the AP-1 transcription promoters c-Fos and c-Jun. The expression was shown with either mono- or polyclonal antibodies by indirect avidin-biotin-peroxidase immunohistochemical staining method. The normal control material was collected from a tissue bank of five organ donors. The degenerated disc material was from twelve patients operated on for painful degenerative disc disease, and herniated disc tissue material was obtained from 115 patients operated on for sciatica. Normal control discs showed only TGFβ immunopositivity. All other factors studied were immunonegative in the control material. Prolapsed disc material was immunopositive for all factors studied, and this positivity was located either in the disc cells or in blood vessels. Furthermore, neovascularization was noted. Disc cell immunoreaction was shown in chondrocyte-like disc cells or in fibroblast-like disc cells, the former being expressed especially in conglomerates (clusters of disc cells). TGFβ receptor induction was prominent in prolapsed intervertebral disc tissue. In degenerated disc material, the expression of growth factors was analyzed in greater detail in various parts of the disc: nucleus pulposus, anterior annulus fibrosus and posterior annulus fibrosus. PDGF did not show any immunoreactivity, whereas all other studied growth factors were localized either in chondrocyte-like disc cells, often forming clusters, in fibroblast-like disc cells, or in small capillaries. Many of the studied degenerated discs showed tears in the posterior region of annulus fibrosus, but expression of immunopositive growth factors was detected throughout the entire disc. Furthermore, there was a difference in immunopositive cell types for different growth factors. The main conclusion of the thesis, supported by all substudies, is the occurrence of growth factors in disc cells. They may be actively participating in a network regulating disc cell growth, proliferation, extracellular matrix turnover, and neovascularization. Chondrocyte-like disc cells, in particular, expressed growth factors and oncoproteins, highlighting the importance of this cell type in the basic pathophysiologic events involved in disc degeneration and disc rearrangement. The thesis proposes a hypothesis for cellular remodelling in intervertebral disc tissue. In summary, the model presents an activation pattern of different growth factors at different intervertebral disc stages, mechanisms leading to neovascularization of the intervertebral disc in pathological conditions, and alteration of disc cell shape, especially in annulus fibrosus. Chondrocyte-like disc cells become more numerous, and these cells are capable of forming clusters, which appear to be regionally active within the disc. The alteration of the phenotype of disc cells expressing growth factors from fibroblast-like disc cells to chondrocyte-like cells in annulus fibrosus, and the numerous expression of growth factor expressing disc cells in nucleus pulposus, may be a key element both during pathological degeneration of the intervertebral disc, and during the healing process after trauma.

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.

The effects of organic environmental toxicants on hard tissue formation in developing tooth : An in vitro study in mice

Dioxins are organic toxicants that are known to impair tooth development, especially dental hard tissue formation. The most toxic dioxin congener is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Further, clinical studies suggest that maternal smoking during pregnancy can affect child s tooth development. One of the main components of tobacco smoke is the group of non-halogenated polycyclic aromatic hydrocarbons (PAHs), a representative of which is 7,12-dimethylbenz[a]anthracene (DMBA). Tributyltin (TBT), an organic tin compound, has been shown to impair bone mineralization in experimental animals. In addition to exposure to organic toxicants, a well-established cause for enamel hypomineralization is excess fluoride intake. The principal aim of this thesis project was to examine in vitro if, in addition to dioxins, other organic environmental toxicants, like PAHs and organic tin compounds, have adverse effects on tooth development, specifically on formation and mineralization of the major dental hard tissues, the dentin and the enamel. The second aim was to investigate in vitro if fluoride could intensify the manifestation of the detrimental developmental dental effects elicited by TCDD. The study was conducted by culturing mandibular first and second molar tooth germs of E18 NMRI mouse embryos in a Trowell-type organ culture and exposing them to DMBA, TBT, and sodium fluoride (NaF) and/or TCDD at various concentrations during the secretory and mineralization stages of development. Specific methods used were HE-staining for studying cell and tissue morphology, BrdU-staining for cell proliferation, TUNEL-staining for apoptosis, and QPCR, in situ hybridization and immunohistochemistry for the expressions of selected genes associated with mineralization. This thesis work showed that DMBA, TBT, TCDD and NaF interfere with dentin and enamel formation of embryonic mouse tooth in vitro, and that fluoride can potentiate the harmful effect of TCDD. The results suggested that adverse effects of TBT involve altered expression of genes associated with mineralization, and that DMBA and TBT as well as NaF and TCDD together primarily affect dentin mineralization. Since amelogenesis does not start until mineralization of dentin begins, impaired enamel matrix secretion could be a secondary effect. Dioxins, PAHs and organotins are all liposoluble and can be transferred to the infant by breast-feeding. Since doses are usually very low, developmental toxicity on most of the organs is difficult to indentify clinically. However, tooth may act as an indicator of exposure, since the major dental hard tissues, the dentin and the enamel, are not replaced once they have been formed. Thus, disturbed dental hard tissue formation raises the question of more extensive developmental toxicity.