The prevalence of celiac disease in patients with geographic tongue.

BACKGROUND: Geographic tongue (GT) is a benign inflammatory condition usually involving the dorsal surface and lateral borders of the tongue. Numerous etiological factors of GT have been suggested, including immunological factors; genetic; atopic or allergic predisposition; emotional stress; and hormonal disturbances. GT may also coexist as one of the possible manifestations of celiac disease (CD). Therefore, the aim of this study was to investigate the prevalence of CD, positive serologic tests for CD screening, and HLA-DQ presence in patients with GT. METHODS: Tissue transglutaminase antibodies (anti-tTG), antibodies against gliadin (AGA), and human leukocyte antigen (HLA) typing were assessed for 60 GT patients and 60 healthy control subjects. The duodenal biopsy was performed in patients with positive serologic tests. RESULTS: We found that 9 (15%) GT patients were positive for IgA tTG, and in those patients histological changes consistent with CD were confirmed by duodenal biopsy. Only two of them reported the presence of gastrointestinal symptoms. There were statistically significant differences between the GT patients and control group for immunoglobulin (Ig) A tTG (P = 0.03), IgG tTG (P = 0.04), IgA AGA (P = 0.04), and IgG AGA (P = 0.02). CONCLUSION: The results of our study demonstrated the increased prevalence of CD in patients with GT. Therefore, the clinical oral examination should be considered a diagnostic tool, especially in atypical or silent forms of CD, since it may contribute to provide an early diagnosis.

The Odds of One-Year Mortality in Bedridden Geriatric Patients Discharged from Acute Rehabilitation Ward Are Increased Eightfold If the Patients Have Three or More Complications.

Low muscle strength, functional score at discharge, and complications during a ten-day rehabilitation hospital stay can affect mortality rates in bedridden geriatric patients. This was a prospective observational study in a cohort of 105 bedridden geriatric patients admitted to the Rehabilitation ward after a major illness or surgery. All participants had a severe dependency on another person (Barthel's Index < 60). The one-year mortality rate in this cohort was 15.2%, with further subdivision according to the number of complications: 61.5% in patients with ≥3 complications during hospitalization, 17.6% in patients with two complications, 9.5% with one complication, and 3% in patients with no complications. The Barthel Index at discharge (OR = 0.95; p = 0.003) and ≥3 medical complications (OR = 8.33; p = 0.005) during rehabilitation ward stay were significant predictors for one-year mortality. The odds of one-year mortality after discharge increased eightfold in patients with ≥3 medical complications. Sarcopenia, age, and sex were not significant predictors of mortality in this cohort. The 10-day acute rehabilitation was too short to achieve progress from severe to moderate independence in 60% of patients. The Barthel Index at discharge and a number of complications affect the mortality rate. These findings provide valuable insights into the complex dynamics of mortality and functional outcomes in bedridden geriatric patients.

Clinical Phenotype of HLA B*44 Patients in a Rheumatology Outpatient Clinic Favors Peripheral Arthropathies.

Objective: The genetic background of HLA-B*27 in spondyloarthritis is known, and the search for another gene with similar role is ongoing. We wanted to investigate clinical presentations of HLA-B*44 patients in rheumatology practice. Methods: A cross-sectional retrospective study of 303 HLA-B*44 adult patients from the outpatient rheumatology clinic from 5/2018-5/2024. Clinical phenotype, confirmed or excluded rheumatic diagnosis, therapy used, and data on HLA A, B, and DR alleles inherited with B*44 were analyzed. Results: A female predominance of 2.79:1 was noted. A total of 150 [49.5%] patients were referred due to peripheral joint pain, 77 [25.4%] due to combined spine and peripheral joint pain or spine alone (57 [18.8%]). A total of 19 [6.3%] patients had no symptoms of the musculoskeletal system. Statistically significant peripheral joint affection was proved in females but not in males (p = 0.04). A total of 121 [40%] patients from B*44 group had established rheumatic disease, with the rest being excluded or under observation. The most common working diagnoses were polyarthritis (32 [10.5%]) and mono-oligoarthritis (14 [4.6%]). A second allele in addition to HLA B*44 showed a similar frequency to the general population. Patients with HLA B*44/44 and B*27/44 genotypes were at the most risk for having definitive rheumatic disease (>60%). Conventional synthetic disease-modifying anti-rheumatic drugs (DMARDs) were used in 38.6% of patients, non-steroidal anti-inflammatory drugs were used in 31.6% of patients, biologic DMARDs were used in 8.9% of patients, and corticosteroids were used in 7.3% of patients. Conclusions: The most common presentation in HLA-B*44 patients is peripheral joint affection. Most patients with HLA-B*27/44 and B*44/44 genotypes had definitive rheumatic disease. B*44 homozygosity or B*27/44 might be risk factors for arthritis development.

Cell proliferation and apoptosis in the fusion of human primary and secondary palates.

The markers of cell proliferation (Ki-67) and apoptosis [caspase-3, TdT-mediated biotin-dUTP nick-end labelling (TUNEL)] and the expression of syndecan-1 and heat shock protein 70 (Hsp70) were analyzed immunohistochemically in 11 developing human palates, from developmental weeks 6 to 10. During fusion of the primary palate, the proportion of proliferating cells decreased from 42 to 32% and the proportion of apoptotic cells decreased from 11 to 7% in the medial-edge epithelium. At later stages, the proportions of both types of cells decreased in the ectomesenchyme, except for proliferating cells in its non-condensing part. At developmental weeks 9-10, the epithelial seam in the secondary palate comprised 28% proliferative cells and 5% apoptotic cells. While condensing ectomesenchyme contained more apoptotic cells than proliferating cells, the opposite was observed for the non-condensing ectomesenchyme. Co-expression of syndecan-1 and Hsp70 was detected in cells budding from the epithelial seam. Our study indicates similar principles for human primary palate and secondary palate fusion, and parallel persistence of proliferation and apoptotic activity. While proliferation enables growth and fusion of different palatal primordia, apoptosis results in the removal of of large numbers epithelial cells at the fusion point. The disintegration of seam remnants seems to be executed through the processes of change in protein content and cell migration, probably leading to cell death as their final outcome.

Heparan Sulfate Glycosaminoglycan Is Predicted to Stabilize Inflammatory Infiltrate Formation and RANKL/OPG Ratio in Severe Periodontitis in Humans.

Since chronically inflamed periodontal tissue exhibits extracellular matrix (ECM) degradation, the possible alternative to standard periodontitis treatment is to restore ECM by supplementing its components, including heparan sulfate glycosaminoglycan (HS GAG). Supplementation of the degraded ECM with synthetic derivatives of HS GAGs has been shown to be effective for periodontal tissue regeneration in experimental animal models of periodontitis. However, the potential of HS GAG supplementation for the treatment of periodontal disease in humans is still unknown. Here, we used a statistical model to investigate the role of HS GAG on inflammatory infiltrate formation and alveolar bone resorption in humans with severe periodontitis. The model was based on data from immunofluorescence staining (IF) of human gingiva samples, and reconstruction of a subset of HS GAG -related proteins from STRING reactome database. According to predictions, increased expression of native HS GAG might stabilize the accumulation of gingival inflammatory infiltrate (represented by the general inflammatory cell marker CD45) and alveolar bone resorption (represented by Receptor Activator of Nuclear ΚΒ ligand (RANKL) and osteoprotegerin (OPG) ratio) but could not restore them to healthy tissue levels. Therefore, supplementation of native HS GAG may be of limited benefits for the treatment of sever periodontitis in humans.

Novel approach for quantification of multiple immunofluorescent signals using histograms and 2D plot profiling of whole-section panoramic images.

We describe a novel approach for quantification and colocalization of immunofluorescence (IF) signals of multiple markers on high-resolution panoramic images of serial histological sections utilizing standard staining techniques and readily available software for image processing and analysis. Human gingiva samples stained with primary antibodies against the common leukocyte antigen CD45 and factors related to heparan sulfate glycosaminoglycans (HS GAG) were used. Expression domains and spatial gradients of IF signals were quantified by histograms and 2D plot profiles, respectively. The importance of histomorphometric profiling of tissue samples and IF signal thresholding is elaborated. This approach to quantification of IF staining utilizes pixel (px) counts and comparison of px grey value (GV) or luminance. No cell counting is applied either to determine the cellular content of a given histological section nor the number of cells positive to the primary antibody of interest. There is no selection of multiple Regions-Of-Interest (ROIs) since the entire histological section is quantified. Although the standard IF staining protocol is applied, the data output enables colocalization of multiple markers (up to 30) from a given histological sample. This can serve as an alternative for colocalization of IF staining of multiple primary antibodies based on repeating cycles of staining of the same histological section since those techniques require non standard staining protocols and sophisticated equipment that can be out of reach for small laboratories in academic settings. Combined with the data from ontological bases, this approach to quantification of IF enables creation of in silico virtual disease models.

Syndecans and Enzymes for Heparan Sulfate Biosynthesis and Modification Differentially Correlate With Presence of Inflammatory Infiltrate in Periodontitis.

Periodontitis is a common degenerative disease initiated by the bacteria in subgingival biofilm. The exposure to bacterial biofilm triggers host inflammatory response whose dysregulation is ultimately responsible for the destruction of hard and soft periodontal tissues resulting in tooth loss. To date, significant effort has been invested in the research of the involvement of host cells and inflammatory mediators in regulation of inflammatory response in periodontitis. Syndecans (Sdcs) belong to a four-member family of heparan sulfate proteoglycans (HSPGs). Sdcs are compound molecules comprised of the core protein to which several heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. The role of Sdcs in pathogenesis of periodontitis is poorly investigated despite the numerous reports from experimental studies about the critical involvement of these factors in modulation of various aspects of inflammatory response, such as the formation of inflammatory mediators gradients, leukocyte recruitment and extracellular matrix remodeling in resolution of inflammation. Most of these functions of Sdcs are HS-related and, thus, dependent upon the structure of HS. This, in turn, is determined by the combinatorial action of enzymes for biosynthesis and modification of HS such as exostosis (EXTs), sulfotransferases (NDSTs), and heparanase 1 (HPSE1). The data presented in this study clearly indicate that some Sdcs display different expression profiles in healthy and diseased periodontal tissue. Additionally, the differences in expression profiles of HS GAG biosynthesis and modification enzymes (EXTs, NDSTs, and HPSE1) in healthy and diseased periodontal tissue imply that changes in HS GAG content and structure might also take place during periodontitis. Most notably, expression profiles of Sdcs, EXTs, NDSTs, and HPSE1 differentially correlate with the presence of inflammatory infiltrate in healthy and diseased periodontal tissue, which might imply that these factors could also be involved in modulation of inflammatory response in periodontitis.

Time course and expression pattern of the neuronal markers in the developing human spinal cord.

The aim of this study was to examine the spatio-temporal appearance of different neuronal cell subtypes by analyzing expression patterns of several neuronal markers (calretinin, neurofilament 200 (NF200), vanilloid receptor 1(VR1) and calcitonin gene-related peptide (CGRP)) of the embryonic human spinal cord (SC). Developing human SCs from 11 human conceptuses beetwen 5-10 developmental weeks (DW) were examined by light and electron microscopy and immunofluorescence. Light and electron microscopy revealed different embryonic stages of recognizable structure of the SC. NF200, CGRP and VR1 positive cells were observed in SCs during 5th-6th DW. NF200 was predominantly expressed in the ventral part, indicating presence of motoneurons. As development advanced, NF200 was mainly expressed in the marginal zone. Expression of CGRP was intense during all of the investigated periods, predominantly during the 5th-6th DW pointing to neural sensory differentiation, as opposed to the last DW when reduced expression of CGRP in the marginal layer indicated the terminations of the sensory afferents. Expression of VR1 was highest in the intermediate zone, at the beginning and at the end of the investigated periods, pointing to VR1 spatial pattern in the visceral afferents in the grey matter, while the first signs of calretinin were found in the 9th-10th DW ventrally. Delineating the relationships between factors involved in processes of neuronal differentiation as well as spatial and temporal arrangement of SC interrelated neurons can provide a useful information about normal SC development as well as the insight in possible causes of anomalies and disorders during embryonic life.

Syndecans and Enzymes Involved in Heparan Sulfate Biosynthesis and Degradation Are Differentially Expressed During Human Odontogenesis.

Syndecans belong to a four-member family of cell surface heparan sulfate proteoglycans (HSPGs) abundantly present in various tissues. They are primarily recognized as extracellular matrix (ECM) receptors able to bind various ECM components and form gradients of morphogens and growth factors. Syndecans are composed of core protein with distinctive cytoplasmic, transmembrane, and extracellular domains to which several HS glycosaminoglycan (GAG) chains are covalently attached. In development of composite organs, such as teeth, expression patterns of syndecans display temporo-spatial shifts between epithelial and mesenchymal tissue compartments. Along with diverse functional properties of syndecans and generally large number of their interactors due to HS GAG chain content, this suggests possible involvement of syndecans in modulation of epithelial-to-mesenchymal crosstalk. Functional versatility of syndecans greatly depends upon the biochemical properties of attached HS GAG chains. These are specifically determined during the HS biosynthesis by the combinatorial action of glycosyl-transferases (Exts/EXTs) and bi-functional sulfotransferases (Ndsts/NDSTs), as well as by post-biosynthetic enzymatic cleavage of HS by the only active endoglucuronidase in mammals, heparanase 1 (Hpse1/HPSE1). Matching the essential requirement for HS during organogenesis, null-mutant animals for genes encoding these enzymes display severe developmental anomalies of mineralized tissues (including teeth) with embryonic or perinatal lethality. In this study, we analyzed expression of syndecan HSPGs (syndecans 1, 2, and 4), enzymes involved in HS biosynthesis (EXT1, NDST1, NDST2) and HS cleavage (HPSE1) in human tooth germs during the early stages of odontogenesis. All of the investigated factors displayed temporo-spatial differences in expression patterns, and some of them showed distinctive asymmetries of expression domains. Our findings suggest that these factors might be differentially involved in cellular processes which take place during the early odontogenic sequence in humans.

Growth factors FGF8 and FGF2 and their receptor FGFR1, transcriptional factors Msx-1 and MSX-2, and apoptotic factors p19 and RIP5 participate in the early human limb development.

The expression pattern of fibroblast growth factors FGF8 and FGF2 and their receptor FGFR1, transcription factors MSX-1 and MSX-2, as well as cell proliferation (Ki-67) and cell death associated caspase-3, p19 and RIP5 factors were analyzed in histological sections of eight 4th-9th-weeks developing human limbs by immunohistochemistry and semi-thin sectioning. Increasing expression of all analyzed factors (except FGF8) characterized both the multilayered human apical ectodermal ridge (AER), sub-ridge mesenchyme (progress zone) and chondrocytes in developing human limbs. While cytoplasmic co-expression of MSX-1 and MSX-2 was observed in both limb epithelium and mesenchyme, p19 displayed strong cytoplasmic expression in non-proliferating cells. Nuclear expression of Ki-67 proliferating cells, and partly of MSX-1 and MSX-2 was detected in the whole limb primordium. Strong expression of factors p19 and RIP5, both in the AER and mesenchyme of human developing limbs indicates their possible involvement in control of cell senescence and cell death. In contrast to animal studies, expression of FGFR1 in the surface ectoderm and p19 in the whole limb primordium might reflect interspecies differences in limb morphology. Expression of FGF2 and downstream RIP5 gene, and transcription factors Msx-1 and MSX-2 did not show human-specific changes in expression pattern. Based on their spatio-temporal expression during human limb development, our study indicates role of FGFs and Msx genes in stimulation of cell proliferation, limb outgrowth, digit elongation and separation, and additionally MSX-2 in control of vasculogenesis. The cascade of orchestrated gene expressions, including the analyzed developmental factors, jointly contribute to the complex human limb development.

Antimicrobial effectiveness of polyhexamethylene biguanide on Enterococcus faecalis, Staphylococcus epidermidis and Candida albicans.

Aim To evaluate the antimicrobial activity of 0.2% polyhexamethilene biguanide (PHMB) in root canal models infected with Enterococcus faecalis, Candida albicans and Staphylococcus epidermidis. PHMB was compared in these tests with 2.5% NaOCl and 0.2% CHX. Methods Prepared models of 50 human root canals (n=50) were immerged in mixed, four- weeks old culture that consisted of E .faecalis, S. epidermidis and C. albicans. Roots were randomly divided into three groups: one with 30 (n=30) and two with 10 (n=10) samples. Samples were treated with polyhexamethylene biguanide (PHMB) (0.2%), sodium hypochlorite (NaOCl) (2.5%) and chlorhexidine (CHX) (0.2%), respectively. Root dentin was sampled before and after the tretment with these solutions. Colony- forming units (CFU) were counted to asses the antimicrobial effects of three solutions on viability of selected microrganisms in specimens before and after the treatment.T-test was used for comparison of results between specimens before and after the treatment, while Newman-Keuls test was used for pairwise comparison at p=0.05. Results The PHMB was significantly more efficient in reducing the number of all three tested microorganisms. NaOCl and CHX made only statistically significant (p<0.05) difference in case of E. faecalis and S. epidermidis. In the case of C. albicans, this difference was not statistically significant due to the small number of positive samples and high initial dispersion of results. Conclusion Both solutions PHMB and NaOCl were successful in eliminating E. faecalis and S. epidermidis from the mature dentin biofilm, CHX was not successful enough.

Glomeruli from patients with nephrin mutations show increased number of ciliated and poorly differentiated podocytes.

BACKGROUND: Podocytes are postmitotic, highly specialized cells which maintain the glomerular filtration barrier (GFB). Their injury is characterized by foot processes effacement and change in protein expression leading to proteinuria and end-stage kidney disease. METHODS: Our study focuses on the morphological and immunohistochemical changes of human podocytes during normal development and postnatal period, compared to congenital nephrotic syndrome of the Finnish type (CNF). Kidney tissues taken from 17 human conceptuses 8th-38th weeks old, two healthy and three CNF kidneys were embedded in paraffin for immunohistochemical or double immunofluorescence methods, or were embedded in resin for electron microscopy. Paraffin sections were stained with markers for proliferation (Ki-67), proteins nephrin and nestin, and alpha-tubulin. Quantification of positive cells were performed using Mann Whitney and Kruskal-Wallis test. RESULTS: Tissue analysis showed that proliferation of podocytes gradually decreased during development and disappeared in postnatal period. Decrease in number of ciliated glomerular cells and visceral podocytes (from 47% to 3%), and parietal epithelial cells (from 32% to 7%) characterized normal development. Nestin and nephrin co-expressed in developing podocytes in different cellular compartments. During development, nephrin expression increased (from 17% to 75%) and postnatally changed its pattern, while nestin positive glomerular cells decreased from 98% to 40%. CNF glomeruli displayed increased number of immature ciliated podocytes (6%) and parietal epithelial cells (9%). CONCLUSION: Changes in cytoplasmic alpha-tubulin expression and reduced nephrin expression (20%) indicating association of incomplete podocyte maturation with failure of GFB function and appearance of prenatal proteinuria in CNF patients.

Regulation of proliferation in developing human tooth germs by MSX homeodomain proteins and cyclin-dependent kinase inhibitor p19INK4d.

Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19INK4d. p19INK4d induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19INK4d by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19INK4d in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19INK4d throughout the investigated period indicates that p19INK4d plays active role during human tooth development. Furthermore, comparison of expression domains of p19INK4d with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.

Involvement of IGF-2, IGF-1R, IGF-2R and PTEN in development of human tooth germ - an immunohistochemical study.

Insulin-Like Growth Factor 2 (IGF-2) is a peptide hormone essential for prenatal growth and development. IGF-2 exerts its mitogenic effects via Insulin-Like Growth Factor 1 Receptor (IGF-1R), and is eliminated by binding to Insulin-Like Growth Receptor 2 (IGF-2R). IGF-2 is also negatively regulated by Phosphatase and Tensin Homolog (PTEN), a phosphatase mutated in various tumors. Not much is known about the interplay between these factors during human odontogenesis. In this study, expression patterns of IGF-2, IGF-1R, IGF-2R and PTEN were analyzed by double immunofluorescence in incisor human tooth germs during the foetal period of development between the 7th and 20th gestational week. Throughout the investigated period, IGF-2 was mostly expressed in enamel organ, whereas mild to moderate expression of PTEN could be seen in dental papilla and parts of enamel organ. Expression of IGF-1R was ubiquitous and displayed strong intensity throughout the entire enamel organ. In contrast, expression of IGF-2R had rather erratic pattern in enamel organ and dental papilla alike. Expression patterns of IGF-2, IGF-1R, IGF-2R and PTEN in highly proliferative cervical loops, as well as in differentiating pre-ameloblasts and pre-odontoblasts of cusp tip region during the early and late bell stages when enamel organ acquires definitive shape, indicate importance of these factors in crown morphogenesis of human incisor. Taken together, our data suggest the involvement of IGF-2, IGF-1R, IGF-2R and PTEN in temporo-spatial patterning of basic cellular processes (proliferation, differentiation) during normal tooth development. They are also relevant for improving knowledge of molecular basis of human odontogenesis.

Comparison of proliferation, apoptosis and expression of syndecan-1 and α-SMA in edentulous ridge oral mucosa of successful and early failed submerged dental implants--An immunohistochemical study.

AIMS: Pathogenic mechanisms involved in early submerged implant failure are poorly understood. In this study we immunohistochemically analyse differences in proliferation, apoptosis and inflammation in edentulous ridge oral mucosa (ERM) of successful and early failed submerged implants. MATERIALS AND METHODS: 30 samples of ERM covering successful and early failed submerged implants were obtained at the end of osseointegration period along with control samples of healthy ERM. Sections were stained with Ki-67 (proliferation), caspase-3 (apoptosis) and syndecan-1 (epithelial marker). Percentage of positive cells was analysed by Kruskal-Wallis test and Dunn's post hoc test. Co-localization of Ki-67 and caspase-3 with α-SMA, CD68 and TGF-ß was done by double immunofluorescence. RESULTS: There was no significant difference in number of Ki-67 positive cells within surface epithelium (SE) in all groups. Proliferation was significantly higher in underlying connective tissue (UCT) of ERM of early failed submerged implants (26%) compared to ERM of successful submerged implants (3%) and controls (4%). More apoptotic cells appeared in UCT of early failed submerged implants (8%) compared to UCT of successful submerged implants (4%) and UCT of control ERM (3%). Co-localization of Ki-67 and α-SMA in ERM of early failed submerged implants disclosed proliferating fibroblasts and pericytes of blood vessels. Macrophages and cells expressing TGF-ß appeared in UCT of failed implants. Expression of syndecan-1 was significantly weaker in SE of early failed submerged implants. CONCLUSIONS: Imbalance between proliferation and apoptosis, changes in syndecan-1 expression and inflammation are histopathological features of ERM of early failed submerged implants.

Analysis of expression patterns of IGF-1, caspase-3 and HSP-70 in developing human tooth germs.

AIMS: To analyze expression patterns of IGF-1, caspase-3 and HSP-70 in human incisor and canine tooth germs during the late bud, cap and bell stages of odontogenesis. MATERIALS AND METHODS: Head areas or parts of jaw containing teeth from 10 human fetuses aged between 9th and 20th developmental weeks were immunohistochemically analyzed using IGF-1, active caspase-3 and HSP-70 markers. Semi-quantitative analysis of each marker's expression pattern was also performed. RESULTS: During the analyzed period, IGF-1 and HSP-70 were mostly expressed in enamel organ. As development progressed, expression of IGF-1 and HSP-70 became more confined to differentiating tissues in the future cusp tip area, as well as in highly proliferating cervical loops. Few apoptotic bodies highly positive to active caspase-3 were observed in enamel organ and dental papilla from the cap stage onward. However, both enamel epithelia moderately expressed active caspase-3 throughout the investigated period. CONCLUSIONS: Expression patterns of IGF-1, active caspase-3 and HSP-70 imply importance of these factors for early human tooth development. IGF-1 and HSP-70 have versatile functions in control of proliferation, differentiation and anti-apoptotic protection of epithelial parts of human enamel organ. Active caspase-3 is partially involved in formation and apoptotic removal of primary enamel knot, although present findings might reflect its ability to perform other non-death functions such as differentiation of hard dental tissues secreting cells and guidance of ingrowth of proliferating cervical loops.

Expression of Ki-67, Oct-4, γ-tubulin and α-tubulin in human tooth development.

AIMS: To analyze factors controlling cell proliferation and differentiation, and appearance of primary cilia during the cap and bell stages of incisor or/and canine human enamel organs. MATERIALS AND METHODS: Qualitative and quantitative analysis of proliferating Ki-67 positive cells and expression of γ-tubulin, α-tubulin and Oct-4 was immunohistochemically analyzed in the cap an bell stages of 10 developing human incisor and canine germs, 8-21 weeks old. RESULTS: During the analyzed period, ratio of Ki-67 positive cells changed in outer enamel epithelium from 48.86% to 24.52%, in inner enamel epithelium increased from 56.11% to 60.06% and then dropped to 44.24%. While in dental papilla proliferation first increased from 46.26% to 55.45%, and then dropped to 22.08%, a constant decrease of proliferation characterized enamel reticulum (from 46.26% to 15.49%). Strong cytoplasmic Oct-4 expression characterized epithelial parts of enamel organ, particularly the differentiating ameloblasts. During further development, Oct-4 expression shifted to both nuclear and cytoplasmic expression in mesenchymal tooth components. Primary cilia characterized most of the cells in developing enamel organ. While non-ciliated (proliferating) cells mainly contained two centrioles (γ-tubulin), the primary cilia (α-tubulin) were arising from basal bodies (γ-tubulin) of non-proliferating cells. CONCLUSIONS: We suggest that increase in cell proliferation enables growth of enamel organ, while its selective decrease leads to disintegration of some tooth parts. Drop of proliferation coincided with initiation of ameloblast and odontoblast differentiation. Additionally, cell differentiation was accompanied by increased expression of Oct-4 and probably by signalling via primary cilia, both regulating processes of cell proliferation and differentiation.