Carin Sahlberg

Cloning of a novel bacteria-binding receptor structurally related to scavenger receptors and expressed in a subset of macrophages.

A novel murine plasma membrane protein has been identified in subpopulations of macrophages. It has an intracellular N-terminal domain, a transmembrane domain, and an extracellular region with a short spacer, an 89 Gly-Xaa-Yaa repeat-containing collagenous domain, and a C-terminal cysteine-rich domain. In situ hybridization and immunohistochemical staining have localized the protein to a subset of macrophages in the marginal zone of the spleen and the medullary cord of lymph nodes. No expression was observed in macrophages of liver or lung. Transfected COS cells synthesized a native trimeric plasma membrane protein that bound labeled bacteria and acetylated LDL, but not yeast or Ficoll. The results suggest that the novel protein is a macrophage-specific membrane receptor with a role in host defense, as it shows postnatal expression in macrophages, which are considered responsible for the binding of bacterial antigens and phagocytosis.

Expression of syndecan gene is induced early, is transient, and correlates with changes in mesenchymal cell proliferation during tooth organogenesis

Syndecan is an integral cell surface proteoglycan which contains an extracellular matrix-binding domain and a cytoskeleton-associated domain and may therefore transfer changes in the extracellular environment to cellular behavior. Changes in syndecan gene expression during embryonic and early postnatal mouse tooth development were analyzed by in situ hybridization and compared with the distribution of syndecan core protein and cell proliferation studied by immunohistochemistry. Syndecan RNA became accumulated in the condensing mesenchymal cells around the invaginating epithelial tooth bud during early development, and this accumulation became more intense when morphogenesis advanced to the cap stage. During the bell stage, when the cuspal pattern of the tooth is established, syndecan transcripts were lost, and RNA was not detected in the terminally differentiated or postmitotic odontoblasts. In the epithelium, syndecan RNA was intensely expressed in the invaginating epithelial bud, but the expression was reduced during the cap and bell stages. However, local stimulation in syndecan gene expression was observed in the epithelial preameloblasts immediately preceding their terminal differentiation into ameloblasts, which was accompanied by a complete loss of transcripts. There was a close correlation between the changes in syndecan transcripts and the distribution of syndecan core protein. Furthermore, analysis of cell proliferation by immunohistochemical detection of BrdU incorporation revealed that in the mesenchyme, but not in the epithelium, syndecan was intensely expressed by proliferating cells. The analysis of mRNA by Northern blot indicated that the transcripts in mesenchymal and epithelial cells were of similar size. In the slot-blot analysis the changes in syndecan transcripts correlated with the overall changes observed in the in situ hybridization analysis. The role of tissue interactions in the regulation of the syndecan gene was studied by using tissue recombination cultures of separated epithelial and mesenchymal components of the early tooth germ. The in situ hybridization and Northern blot analysis of these explants showed that the expression was increased in the mesenchyme cultured in contact with the epithelium. Our results indicate that syndecan gene expression in the embryonic tooth mesenchyme is induced by epithelial-mesenchymal interactions and thereafter expressed stage-dependently and transiently by the differentiating cells during organogenesis. The association of syndecan expression with mesenchymal cell proliferation raises the possibility that, in addition to behaving as a matrix receptor, syndecan may have a role in controlling growth and that syndecan may have different functions in epithelial and mesenchymal cells.

Amoxicillin May Cause Molar Incisor Hypomineralization

The etiology of molar incisor hypomineralization (MIH) is unclear. Our hypothesis was that certain antibiotics cause MIH. We examined 141 schoolchildren for MIH and, from their medical files, recorded the use of antibiotics under the age of 4 yrs. MIH was found in 16.3% of children. MIH was more common among those children who had taken, during the first year of life, amoxicillin (OR = 2.06; 95% CI, 1.01–4.17) or the rarely prescribed erythromycin (OR = 4.14; 95% CI, 1.05–16.4), compared with children who had not received treatment. Mouse E18 teeth were cultured for 10 days with/without amoxicillin at concentrations of 100 μg/mL–4 mg/mL. Amoxicillin increased enamel but not dentin thickness. An altered pattern of amelogenesis may have interfered with mineralization. We conclude that the early use of amoxicillin is among the causative factors of MIH.

Primary Structure, Developmental Expression, and Immunolocalization of the Murine Laminin α4 Chain

The complete primary structure of the mouse laminin α4 chain was derived from cDNA clones. The translation product contains a 24-residue signal peptide preceding the mature α4 chain of 1,792 residues. Northern analysis on whole mouse embryos revealed that the expression was weak at day 7, but it later increased and peaked at day 15. In adult tissues the strongest expression was observed in lung and cardiac and skeletal muscles. Weak expression was also seen in other adult tissues such as brain, spleen, liver, kidney, and testis. By in situ hybridization of fetal and newborn tissues, expression of the laminin α4 chain was mainly localized to mesenchymal cells. Strong expression was seen in the villi and submucosa of the developing intestine, the mesenchymal stroma surrounding the branching lung epithelia, and the external root sheath of vibrissae follicles, as well as in cardiac and skeletal muscle fibers. In the developing kidney, intense but transient expression was associated with the differentiation of epithelial kidney tubules from the nephrogenic mesenchyme. Immunohistologic staining with affinity-purified IgG localized the laminin α4 chain primarily to lung septa, heart, and skeletal muscle, capillaries, and perineurium.

Association between the expression of murine 72 kDa type IV collagenase by odontoblasts and basement membrane degradation during mouse tooth development

In situ hybridization was used to study the expression of the 72 kDa type IV collagenase gene and its association with morphogenesis and cell differentiation during advancing mouse tooth development. The epithelia were completely negative during all developmental stages. The dental mesenchyme was uniformly positive during the early stages of tooth morphogenesis, and no association of type IV collagenase with morphogenetic events was observed. However, at the bell stage the expression increased in differentiating preodontoblasts. Expression was intense in the odontoblasts during secretion of the first predentine matrix. The expression was, however, transient; it decreased around the time when mineralization of dentine started until it completely ceased. Transcripts for 72 kDa type IV collagenase also gradually disappeared from the dental pulp. The expression of 72 kDa type IV collagenase was also strong in the osteoblastic cell lineage. The preosteoblasts at the beginning of the formation of mandibular bone as well as the osteoblasts of the alveolar bone expressed more 72 kDa type IV collagenase than did other mesenchymal cells. The increased gene expression in the odontoblasts correlates with the disappearance of the dental basement membrane as shown by immunolabelling with antibodies against type IV collagen. The onset of increased expression in the odontoblasts preceded the disappearance of the basement membrane and at the time when type IV collagenase transcripts were lost from all odontoblasts the basement membrane was completely removed. It can be speculated that during early stages of tooth development the 72 kDa type IV collagenase acts as a gelatinase whereas during later stages, when odontoblasts and ameloblasts differentiate and the deposition of predentine and enamel matrix is initiated, the enzyme may act as a type IV collagenase and contribute to the degradation of the dental basement membrane.

Lactational Exposure of Han/Wistar Rats to 2,3,7,8-Tetrachlorodibenzo-p-dioxin Interferes with Enamel Maturation and Retards Dentin Mineralization:

Exposure to environmental dioxins via mother’s milk may be one causative factor of mineralization defects in children’s teeth. A prerequisite for the completion of enamel mineralization is the removal of enamel matrix. To test the hypothesis that dioxins interfere with enamel maturation, we administered lactating Han/Wistar rats a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 50 or 1000 μg/kg) on the day after delivery and analyzed tissue sections of the pup heads at post-natal days (Pn) 9 and 22. By Pn22, the first and second molars of the exposed pups, but not controls, showed retention of enamel matrix. Predentin was thicker than normal. Immunostaining for the aryl hydrocarbon/dioxin receptor (AhR) and cytochrome P4501A1 (CYP1A1) in ameloblasts and odontoblasts was reduced, suggesting that TCDD interferes with tooth mineralization via AhR. Extinction of AhR may lead to abolition of CYP1A1 expression as a sign of impaired dental cell function.

Combined effect of amoxicillin and sodium fluoride on the structure of developing mouse enamel in vitro.

OBJECTIVE Excess fluoride intake during tooth development is known to cause dental fluorosis. It has also been suggested that amoxicillin use in early childhood is associated with enamel hypomineralization. The aim was to investigate separate and combined effects of sodium fluoride (NaF) and amoxicillin on enamel formation in vitro. DESIGN Mandibular molar tooth germs of E18 mouse embryos were cultured for 10 days in a medium containing NaF (10, 12 or 15μM) and/or amoxicillin (0.5, 1, 2 or 3.6mg/mL) or sodium clavulanate (0.07mg/mL) alone or in combination with 0.5mg/mL of amoxicillin. Morphological changes were studied from the whole tooth photographs and histological tissue sections with light microscope. RESULTS Only with the highest concentrations of NaF or amoxicillin alone the extent of enamel in the first molars measured as the vertical enamel height/crown height ratio was reduced (p<0.01, p<0.001, respectively). At lower concentrations, combination of NaF (12μM) and amoxicillin (2mg/mL) significantly reduced enamel extent compared with the controls (p<0.001). Histologically, the ameloblasts were still columnar but poorly organized and the nascent enamel was often non-homogeneous. Enamel formation was not seen in any second molars exposed to 12μM NaF and 2mg/mL of amoxicillin (or higher concentrations) compared with the presence of enamel in half of the controls (p<0.001). CONCLUSIONS Amoxicillin and NaF dose dependently affect developing enamel of mouse molars in vitro and the effects are potentiative. The clinical significance of the results remains to be studied.

Dioxin Alters Gene Expression in Mouse Embryonic Tooth Explants

Dioxins are ubiquitous environmental poisons that cause disturbances in developing organs, including the teeth. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at the cap stage leads to reduced tooth size and deformation of cuspal morphology. Our hypothesis was that TCDD affects the expression of genes specific for tooth development, which leads to these aberrations. Mouse embryonic E14 tooth germs were cultured for 24 hrs with/without 1 μM TCDD. Analysis of total RNA on Affymetrix arrays showed that TCDD altered the expression of 31 known genes by a fold factor of at least 2. Genes implied in tooth development expressed only slight changes. Genes active at the cap stage were selected for quantitative PCR analysis. Of these, the most highly up-regulated were Follistatin and Runx2, while TGFβ1 and p21 were the most down-regulated genes. Incomplete tooth morphogenesis caused by TCDD may thus result from modified expression of developmentally regulated genes.

Tributyltin Alters Osteocalcin, Matrix Metalloproteinase 20 and Dentin Sialophosphoprotein Gene Expression in Mineralizing Mouse Embryonic Tooth in vitro

We showed in a previous in vitro study that tributyltin (TBT) arrests dentin mineralization and enamel formation in developing mouse tooth. The present aim was to investigate the effect of TBT on the expression of genes associated with mineralization of dental hard tissues. Embryonic day 18 mouse mandibular first molars were cultured for 3, 5 or 7 days and exposed to 1.0 µM TBT and studied by real-time quantitative polymerase chain reaction (RT-QPCR) for the expressions of osteocalcin (Ocn), alkaline phosphatase (Alpl), dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp) and matrix metalloproteinase 20 (Mmp-20).Ocn, Mmp-20 and Dspp, whose expressions showed changes in RT- QPCR, were further analyzed by in situ hybridization of tissue sections. In situ hybridization showed that TBT decreased Ocn expression in odontoblasts but increased the expression in the epithelial tooth compartment. In QPCR assays, the net effect in the whole tooth was increased expression. TBT also reduced Mmp-20 expression in ameloblasts and odontoblasts. Dspp expression varied but both QPCR assays and in situ hybridization showed a decreasing trend. TBT exposure had no clear effect on Alpl and Dmp1 expressions. Increased Ocn expression by epithelial enamel organ may inhibit dentin mineralization and enamel formation. Decreased Ocn, Mmp-20 and Dspp expressions in odontoblasts may indicate delayed cell differentiation, or TBT may specifically decrease the expression of genes involved in dentin mineralization. While decreased Mmp-20 expression by TBT in ameloblasts may impair enamel mineralization, the coincident reduction in Mmp-20 and Dspp expressions in odontoblasts may potentiate the delay of dentin mineralization.

Tributyltin Impairs Dentin Mineralization and Enamel Formation in Cultured Mouse Embryonic Molar Teeth

Tributyltin (TBT), earlier used as an antifouling agent in marine paints, causes damage to the aquatic ecosystem, for example, impaired shell calcification in oysters. TBT affects hard tissue mineralization even in mammals: delayed bone mineralization has been observed in rodents exposed to TBT in utero. To see if TBT interferes with tooth development, especially dental hard tissue formation, we exposed mouse E18 mandibular first and second molars to 0.1, 0.5, 1.0, and 2.0 microM TBT chloride in organ culture for 7-12 days. The amount of enamel was assessed and the sizes of the first molars were measured from photographs taken after the culture. TBT concentration dependently impaired enamel formation (p < 0.001) and reduced tooth size (p < 0.001). Histological analysis showed slight arrest of dentin mineralization and enamel formation in first molars exposed to 0.1 microM TBT. At the concentration of 1.0 microM the effect was overt. The differentiation of ameloblasts in the mesial cusps was retarded but TBT had no effect on odontoblast morphology. The dental epithelium showed enhanced apoptosis. The failure of ameloblasts to form enamel was likely to be secondary to the effect of TBT on dentin mineralization. In the second molars, where predentin deposition had not started, ameloblasts and odontoblasts were nonpolarized and proliferative. The results showed that TBT concentration dependently impairs dental hard tissue formation and reduces tooth size in cultured mouse embryonic molars. The effects depend on the stage of tooth development at the start of exposure and may involve epithelial-mesenchymal interactions.