Fine-tuning of the signalling network controlling morphogenesis and stem cell development in teeth

Tooth development is regulated by sequential and reciprocal interactions between epithelium and mesenchyme. The molecular mechanisms underlying this regulation are conserved and most of the participating molecules belong to several signalling families. Research focusing on mouse teeth has uncovered many aspects of tooth development, including molecular and evolutionary specifi cs, and in addition offered a valuable system to analyse the regulation of epithelial stem cells. In mice the spatial and temporal regulation of cell differentiation and the mechanisms of patterning during development can be analysed both in vivo and in vitro. Follistatin (Fst), a negative regulator of TGFβ superfamily signalling, is an important inhibitor during embryonic development. We showed the necessity of modulation of TGFβ signalling by Fst in three different regulatory steps during tooth development. First we showed that tinkering with the level of TGFβ signalling by Fst may cause variation in the molar cusp patterning and crown morphogenesis. Second, our results indicated that in the continuously growing mouse incisors asymmetric expression of Fst is responsible for the labial-lingual patterning of ameloblast differentiation and enamel formation. Two TGFβ superfamily signals, BMP and Activin, are required for proper ameloblast differentiation and Fst modulates their effects. Third, we identifi ed a complex signalling network regulating the maintenance and proliferation of epithelial stem cells in the incisor, and showed that Fst is an essential modulator of this regulation. FGF3 in cooperation with FGF10 stimulates proliferation of epithelial stem cells and transit amplifying cells in the labial cervical loop. BMP4 represses Fgf3 expression whereas Activin inhibits the repressive effect of BMP4 on the labial side. Thus, Fst inhibits Activin rather than BMP4 in the cervical loop area and limits the proliferation of lingual epithelium, thereby causing the asymmetric maintenance and proliferation of epithelial stem cells. In addition, we detected Lgr5, a Wnt target gene and an epithelial stem cell marker in the intestine, in the putative epithelial stem cells of the incisor, suggesting that Lgr5 is a marker of incisor stem cells but is not regulated by Wnt/β-catenin signalling in the incisor. Thus the epithelial stem cells in the incisor may not be directly regulated by Wnt/β-catenin signalling. In conclusion, we showed in the mouse incisors that modulating the balance between inductive and inhibitory signals constitutes a key mechanism regulating the epithelial stem cells and ameloblast differentiation. Furthermore, we found additional support for the location of the putative epithelial stem cells and for the stemness of these cells. In the mouse molar we showed the necessity of fi ne-tuning the signalling in the regulation of the crown morphogenesis, and that altering the levels of an inhibitor can cause variation in the crown patterning.

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.

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

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

Basement membrane and provisional matrix components (collagen type IV, laminins and von Willebrand factor) in rheumatoid arthritis and Sjögren s syndrome

The aim of this study was twofold- Firstly, to determine the composition of the type IV collagen which are the major components of the basement membrane (BM), in the synovial lining of the rheumatoid arthritis (RA) patient and in the BM in the labial salivary gland of the Sjögrens syndrome (SS) patient. Secondly, this thesis aimed to investigate the role of the BM component laminin α4 and laminin α5 in the migration of neutrophils from the blood vessels thorough the synovial lining layer into synovial fluid and the presence of vWF in the microvasculature of labial salivary gland in SS. Our studies showed that certain α chains type IV collagen are low in RA compared to control synovial linings, while laminin α5 exhibited a pattern of low expression regions at the synovial lining interface towards the joint cavity and fluid. Also, high numbers of macrophage-like lining cells containing MMP-9 were found in the lining. MMP-9 was also found in the synovial fluid. Collagen α1/2 (IV) mRNA was found to be present in high amount compared to the other α(IV) chains and also showed intense labelling in immunohistochemical staining in normal and SS patients. In healthy glands α5(IV) and α6(IV) chains were found to be continuous around ducts but discontinuous around acini. The α5(IV) and α6(IV) mRNAs were present in LSG explants and HSG cell line, while in SS these chains seemed to be absent or appear only in patches around the ductal BM and tended to be absent around acini in immunohistochemical staining, indicating that their synthesis and/or degradation seemed to be locally regulated around acinar cells. The provisional matrix component vWF serves as a marker of vascular damage. Microvasculature in SS showed signs of focal damage which in turn might impair arteriolar feeding, capillary transudation and venular drainage of blood. However, capillary density was not decreased but rather increased, perhaps as a result of angiogenesis compensatory to microvascular damage. Microvascular involvement of LSG may contribute to the pathogenesis of this syndrome. This twofold approach allows us to understand the intricate relation between the ECM components and the immunopathological changes that occur during the pathogenesis of these inflammatory rheumatic disease processes. Also notably this study highlights the importance of maintaining a healthy ECM to prevent the progression or possibly allow reversal of the disease to a considerable level. Furthermore, it can be speculated that a healthy BM could quarantine the inflamed region or in case of cancer cells barricade the movement of malignant cells thereby preventing further spread to the surrounding areas. This understanding can be further applied to design appropriate drugs which act specifically to maintain a proper BM/BM like intercellular matrix composition.

Sex steroids in Sjögren's syndrome

Sjögren s syndrome (SS) is a strongly female dominant autoimmune disease. SS targets mainly salivary and lacrimal glands and leads to loss of the secreting acinar cells of these glands. Accordingly, secretion of the affected glands is diminished and the main symptoms of SS, dryness of mouth and eyes, follow. In addition to these sicca symptoms, SS patients suffer from severe fatigue and can have various extraglandular symptoms. To date, the etiology of SS still remains unknown. Female dominance and the late onset of the disease simultaneously with remarkable hormonal changes in the body (menopause, adrenopause) encouraged us to hypothesize that sex steroids, especially androgens, are involved in the onset and progression of SS. We confirmed our hypothesis and showed that patients with SS suffer from androgen depletion both systemically and locally in the target tissue of SS, salivary glands. We especially focused on the local androgen environment in salivary glands and demonstrated that healthy salivary glands contain a complete enzymatic machinery for local synthesis of androgens and estrogens from pro-hormone dehydroepiandrosterone (DHEA). However, in SS salivary glands the enzymes catalyzing the local androgen synthesis are defective and, in a subgroup of patients, practically non-functional. Probably due to this local defect in DHEA processing, therapy with DHEA was found unbeneficial for SS patients in the treatment of fatigue. We also studied the effect of the local androgen depletion on salivary glands. We found that in salivary gland cells and healthy labial salivary glands androgens upregulate integrin subunits α1 and α2, which are important for the communication, differentiation and function of the acinar cells. On the contrary, in SS salivary glands DHEA failed to upregulate these signaling molecules, again probably due to defective processing of DHEA into active androgens. Our finding highlights the importance of the local androgen environment and local DHEA processing for the function and welfare of salivary glands. In conclusion, this study showed that patients with SS are androgen depleted both systemically and locally in salivary glands. SS patients also have a defective local sex steroid synthesizing enzymatic machinery further impairing the local androgen depletion. We also showed that the local androgen defect leads to decreased expression of acinar cell specific integrin molecules, which impairs the signaling between the acinar cells and basement membrane and might thus explain the acinar cell loss seen in SS salivary glands. By showing the importance of the local sex steroid imbalance in SS we have clarified some etiopathogenetic mechanisms of SS, which have thus far remained unknown.