Expression of SHOX in human fetal and childhood growth plate.

Abnormalities in the growth plate may lead to short stature and skeletal deformity including Leri Weil syndrome, which has been shown to result from deletions or mutations in the SHOX gene, a homeobox gene located at the pseudoautosomal region of the X and Y chromosome. We studied the expression of SHOX protein, by immunohistochemistry, in human fetal and childhood growth plates and mRNA by in situ hybridization in childhood normal and Leri Weil growth plate. SHOX protein was found in reserve, proliferative, and hypertrophic zones of fetal growth plate from 12 wk to term and childhood control and Leri Weil growth plates. The pattern of immunostaining in the proliferative zone of childhood growth plate was patchy, with more intense uniform immunostaining in the hypertrophic zone. In situ hybridization studies of childhood growth plate demonstrated SHOX mRNA expression throughout the growth plate. No difference in the pattern of SHOX protein or mRNA expression was seen between the control and Leri Weil growth plate. These findings suggest that SHOX plays a role in chondrocyte function in the growth plate.

Treatment of luteal phase inadequacy with bromocriptine.

A group of 15 women diagnosed as having luteal phase inadequacy (LPI) and a history of 1--2 years of infertility were treated with bromocriptine continuously for 4 months. Plasma levels of prolactin, progesterone, 17 alpha-hydroxy-progesterone and estradiol were measured by radioimmunoassay in a control month and each of the treatment months. There was no significant improvement in the levels of the steroids over the 4-month period, although the prolactin levels were depressed, and 2 subjects became pregnant during the study.

Expression of bone matrix protein mRNAs by primary and cloned cultures of the regenerative phenotype of human periodontal fibroblasts.

The successful regeneration of periodontal tissues is dependent, in part, on the ability of cells to reconstitute the mineralized tissues of cementum and bone. The aim of the present study was to characterize regeneration-associated cells in terms of their ability to express mineralized tissue macromolecules. Following guided tissue regeneration, cell cultures were established from regenerating tissue, periodontal ligament, and gingiva. Additionally, these cells were transfected, and single-cell-derived clones were established. Following treatment with platelet-derived growth factor-BB and insulin-derived growth factor-1, the presence of mRNA for alkaline phosphatase, osteocalcin, bone sialoprotein, osteopontin, and bone morphogenetic proteins-2 and -4 was assessed. The three cell types expressed similar mRNA levels for alkaline phosphatase, bone morphogenetic protein-2, and bone morphogenetic protein-4, whereas the expression of osteopontin, osteocalcin, and bone sialoprotein was greater in the periodontal ligament and regenerating tissue fibroblasts compared with the gingival fibroblasts. The two growth factors did not affect the expression of any of the genes. This study has identified markers that correlate with the known ability of periodontal ligament and regenerating tissue-derived fibroblasts to facilitate regeneration of the mineralized tissues of the periodontium.

Enamel matrix derivative induces matrix synthesis by cultured human periodontal fibroblast cells.

BACKGROUND: Periodontal wound healing and regeneration require that new matrix be synthesized, creating an environment into which cells can migrate. One agent which has been described as promoting periodontal regeneration is an enamel matrix protein derivative (EMD). Since no specific growth factors have been identified in EMD preparations, it is postulated that EMD acts as a matrix enhancement factor. This study was designed to investigate the effect of EMD in vitro on matrix synthesis by cultured periodontal fibroblasts. METHODS: The matrix response of the cells was evaluated by determination of the total proteoglycan synthesis, glycosaminoglycan profile, and hyaluronan synthesis by the uptake of radiolabeled precursors. The response of the individual proteoglycans, versican, decorin, and biglycan were examined at the mRNA level by Northern blot analysis. Hyaluronan synthesis was probed by identifying the isotypes of hyaluronan synthase (HAS) expressed in periodontal fibroblasts as HAS-2 and HAS-3 and the effect of EMD on the levels of mRNA for each enzyme was monitored by reverse transcription polymerase chain reaction (RTPCR). Comparisons were made between gingival fibroblast (GF) cells and periodontal ligament (PDLF) cells. RESULTS: EMD was found to significantly affect the synthesis of the mRNAs for the matrix proteoglycans versican, biglycan, and decorin, producing a response similar to, but potentially greater than, mitogenic cytokines. EMD also stimulated hyaluronan synthesis in both GF and PDLF cells. Although mRNA for HAS-2 was elevated in GF after exposure to EMD, the PDLF did not show a similar response. Therefore, the point at which the stimulation of hyaluronan becomes effective may not be at the level of stimulation of the mRNA for hyaluronan synthase, but, rather, at a later point in the pathway of regulation of hyaluronan synthesis. In all cases, GF cells appeared to be more responsive to EMD than PDLF cells in vitro. CONCLUSIONS: EMD has the potential to significantly modulate matrix synthesis in a manner consistent with early regenerative events.

Isolation and characterization of fibroblasts derived from regenerating human periodontal defects.

In an attempt to understand better the cells responsible for periodontal regeneration, cells from human gingiva, periodontal ligament and regenerating periodontal defects treated with expanded polytetrafluorethylene membranes were isolated, cultured and characterized. Guided tissue regeneration procedures were carried out on three human volunteers around molar teeth destined for extraction. After a 6-week 'healing phase', fibroblast cell cultures were established from explants of the regenerating soft connective tissue (RTF), as well as from the associated periodontal ligament (PLF) and gingiva (GF). Following stimulation with platelet-derived growth factor-beta (PDGF) and insulin-like growth factor-1 (IGF-1), [3H]thymidine-uptake and dye-binding assays were used to assess the rate of DNA synthesis and cell proliferation, respectively. Northern blotting was used to measure the expression of mRNA for the extracellular matrix proteoglycans decorin, biglycan and versican. The results show that the GF and RTF proliferated more quickly than the PLF. PDGF and IGF-1 were mitogenic for all three cell types. Decorin mRNA expression was stronger in the GF than the RTF and PLF, whereas versican mRNA expression was stronger in the GF and PLF than the RTF. Biglycan mRNA expression was strong in the PLF, moderate in the GF and weak in the RTF. The growth factors did not affect the mRNA expression for biglycan, but they upregulated versican and downregulated decorin mRNA. It can be concluded that RTF exhibits properties characteristic of a reparative phenotype. More specifically, it proliferates faster than PLF, from which it is derived, while exhibiting a unique pattern of proteoglycan mRNA expression. Therefore, this study demonstrates that fibroblasts obtained from the regenerating periodontal defects exhibit characteristics consistent with their ability to facilitate periodontal regeneration.

Effect of Emdogain on human periodontal fibroblasts in an in vitro wound-healing model.

OBJECTIVE: The aim of this study was to evaluate the influence of Emdogain (EMD) on cultured gingival fibroblasts, periodontal ligament fibroblasts and dermal fibroblasts, using an in vitro model of wound healing. BACKGROUND: Enamel matrix derivative has been demonstrated to promote periodontal regeneration. However, the precise mechanisms by which this agent acts are still unclear. METHODS: The effect of EMD on proliferation of the cells was studied using subconfluent cultures of gingival fibroblasts and periodontal ligament fibroblasts. The cells were made quiescent overnight and then stimulated with various concentrations of EMD (10, 50, 100 and 150 microg/ml) for 24 h. Negative and positive controls were cells cultured in media containing 0.2% and 10% fetal calf serum (FCS). The DNA synthesis was measured by the cellular uptake of [3H]thymidine. For in vitro wounding the cells were cultured, wounded and stimulated with 0.2% FCS, 10% FCS and EMD at a concentration of 20 microg/ml. The percentage of wound fill after treatment was measured after d 1, 4, 6, 12 and 16. The proliferation of cells was also calculated by the extent of incorporation of crystal violet. RESULTS: The results demonstrated that cells in vitro fill an empty space by a combination of proliferation and cell migration. The most rapid closure of a wound area occurred where both proliferation and migration can occur as was seen when wounded cultures were maintained in 10% FCS or at a concentration of 20 microg/ml EMD which promoted proliferation. CONCLUSIONS: Therefore, EMD appears to exert an influence on cells that is compatible with improved wound healing.

Plasma beta-endorphin concentrations prior to and during pregnancy, in labor, and after delivery.

Beta-Endorphin was measured by radioimmunoassay in peripheral plasma of nonpregnant women (58 +/- 2.4 pg/ml, n = 17, mean +/- SE), during the first trimester (47 +/- 2.4 pg/ml, n = 11), the second trimester (33 +/- 1.9, n = 11), and the third trimester (49 +/- 2.7 pg/ml, n = 10) of pregnancy, during early (202 +/- 32 pg/ml, n = 12) and advanced labor (389 +/- 78 pg/ml, n = 10), and 30 to 60 minutes post partum (177 +/- 22 pg/ml, n = 12). Mean plasma levels of beta-endorphin were significantly lower in each trimester of gestation than the levels in nonpregnant control subjects. During labor and the early postpartum period, maternal plasma levels of beta-endorphin were significantly elevated. Furthermore, peripheral plasma levels of beta-endorphin during labor fell from 189 +/- 31 to 97.6 +/- 12 pg/ml (n = 13, p = 0.015) in response to epidural anesthesia, as compared to peripheral plasma concentrations of beta-endorphin of 223 +/- 71 and 193 +/- 47 pg/ml prior to and after injection of saline solution into epidural catheters, respectively, in 10 control subjects. Mean plasma levels of beta-endorphin in patients immediately prior to elective repeat cesarean section who were not in labor (151 +/- 23 pg/ml, n = 15) were significantly higher (p less than 0.005) than the levels in third-trimester control subjects. These data indicate that the pain associated with labor and the psychological stress of anticipating an operation are potent stimuli for the pituitary release of beta-endorphin.

Cytokine regulation of syndecan-1 and -2 gene expression in human periodontal fibroblasts and osteoblasts.

Cell-surface proteoglycans participate in several biological functions including interactions with a variety of growth factors and cytokines. Regulation of syndecan-1 and -2 gene expression was investigated in human periodontal ligament fibroblasts (PDLF), osteoblasts (OB) and gingival fibroblasts (GF), in response to platelet-derived growth factor (PDGF-BB), transforming growth factor (TGF-beta 1), and interleukin (IL-1 beta) by Northern blot analyses. We also compared the effect of PDGF-BB and TGF-beta 1, separately and in combination, in the prolonged presence of IL-1 beta on the expression of both syndecan genes. The results demonstrated that the three cell lines regulated the expression of syndecan-1 and -2 in response to growth factors and cytokines in different manners. These cell lines increased syndecan-1 mRNA levels in response to either PDGF-BB or TGF-beta 1 and decreased levels in response to IL-1 beta. The effect of IL-1 beta on syndecan-1 mRNA synthesis was partially reversed after adding PDGF-BB and TGF-beta 1, separately or in combination, in the presence of IL-1 beta. In contrast, syndecan-2 mRNA level was markedly upregulated in response to either TGF-beta 1 or IL-1 beta in OB when compared with the other two cell lines. However, the stimulatory effect of TGF-beta 1 on syndecan-2 mRNA production in OB was abolished in the prolonged presence of IL-1 beta. These findings lend support to the notion that syndecan-1 and syndecan-2 have distinct functions which correlate with their source and functions within the periodontium.

Nitric oxide synthase type-II is synthesized by human gingival tissue and cultured human gingival fibroblasts.

Nitric oxide is known to be an important inflammatory mediator, and is implicated in the pathophysiology of a range of inflammatory disorders. The aim of this study was to determine the localization and distribution of endothelial NOS (NOS-II) in human gingival tissue, and to ascertain if human gingival fibroblasts express NOS-II when stimulated with interferon gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS). The distribution of NOS-II in inflamed and non-inflamed specimens of human gingivae was studied using a monoclonal antibody against nitric oxide synthase II. Cultures of fibroblasts derived from healthy human gingivae were used for the cell culture experiments. The results from immunohistochemical staining of the tissues indicated an upregulation of NOS-II expression in inflamed compared to non-inflamed gingival tissue. Fibroblasts and inflammatory cells within the inflamed connective tissue were positively stained for NOS-II. In addition, basal keratinocytes also stained strongly for NOS-II, in both healthy and inflamed tissue sections. When cultured human gingival fibroblasts were stimulated by INF-gamma and Porphyromonas gingivalis LPS, NOS-II was more strongly expressed than when the cells were exposed to LPS or IFN-gamma alone. These data suggest that, as for other inflammatory diseases, NO plays a role in the pathophysiology of periodontitis.

New overlapping gene encoded by the cucumber mosaic virus genome.

Cucumber mosaic virus (CMV) contains three genomic RNAs (RNAs 1, 2, and 3) and a subgenomic RNA (RNA 4), a shared feature of the Bromoviridae family which includes cucumoviruses, bromoviruses, alfalfa mosaic virus, and ilarviruses. We describe in this paper the molecular characterization of a novel subgenomic RNA of the Q strain of CMV (Q-CMV), RNA 4A, which was first reported in 1973 (K. W. C. Peden and R. H. Symons, Virology 53, 487-492, 1973). RNA 4A is 682 nucleotides and is identical in sequence to the 3'-terminal 682 nucleotides of RNA 2. RNA 4A encodes a small open reading frame (ORF) of 100 codons, which, in RNA 2, overlaps the C-terminal portion of the major 2a gene; thus it is likely that RNA 4A functions as the mRNA for the in vivo expression of the ORF, called ORF 2b. Polyclonal antibodies raised against a 2b fusion protein expressed in Escherichia coli specifically detected the 2b gene product in Q-CMV-infected cucumber plants by Western immunoblotting. Examination of published viral RNA sequences revealed the conservation of ORF 2b in all of the four other cucumoviruses sequenced to date; however, it is absent from the rest of the Bromoviridae. We suggest that the proposed ORF 2b may be expressed in other cucumoviruses, most likely via 4A-like subgenomic RNAs, and that the predicted gene product may have a unique functional role in the infection process of cucumoviruses.

Growth factors and cytokines modulate gene expression of cell-surface proteoglycans in human periodontal ligament cells.

Cell-surface proteoglycans have been known to be involved in many functions including interactions with components of the extracellular microenvironment, and act as co-receptors which bind and modify the action of various growth factors and cytokines. The purpose of this study was to determine the regulation by growth factors and cytokines on cell-surface proteoglycan gene expression in cultured human periodontal ligament (PDL) cells. Subconfluent, quiescent PDL cells were treated with various concentrations of serum, bFGF, PDGF-BB, TGF-beta1, IL-beta1, and IFN-gamma. RT-PCR technique was used, complemented with Northern blot for syndecan-1, to examine the effects of these agents on the mRNA expression of five cell-surface proteoglycans (syndecan-1, syndecan-2, syndecan-4, glypican and betaglycan). Syndecan-1 mRNA levels increased in response to serum, bFGF and PDGF-BB, but decreased in response to TGF-beta1, IL-1beta and IFN-gamma. In contrast, syndecan-2 mRNA levels were upregulated by TGF-beta1 and IL-1beta stimulation, but remained unchanged with the other agents. Betaglycan gene expression decreased in response to serum, but was upregulated by TGF-beta1 and unchanged by the other stimulants. Additionally, syndecan-4 and glypican were not significantly altered in response to the regulator molecules studied, with the exception that glypican is decreased in response to IFN-gamma. These data demonstrate that the gene expression of the five cell-surface proteoglycans studied is differentially regulated in PDL cells lending support to the notion of distinct functions for these cell-surface proteoglycans.

Growth factor modulation of mitogenic responses and proteoglycan synthesis by human periodontal fibroblasts.

In order to understand the relationship between specific growth factors and matrix synthesis by periodontal cells, we have investigated the effects of platelet-derived growth factor BB (PDGF-BB), insulin-like growth factor-I (IGF-1), and growth hormone on DNA and proteoglycan synthesis by cultured human gingival and periodontal ligament fibroblasts in vitro. PDGF-BB and IGF-1, but not growth hormone, were mitogenic for both periodontal ligament fibroblasts and gingival fibroblasts, although the periodontal ligament cells responded more strongly. The mitogenic response was accompanied by alterations in expression of matrix proteoglycan mRNA. For both the gingival and periodontal ligament cells, there was a decrease in mRNA for decorin and an increase in mRNA for versican following exposure to IGF-1 and PDGF-BB. Although no change was seen in response to PDGF, biglycan mRNA level was increased by IGF-1 in periodontal ligament fibroblasts. With the gingival fibroblasts, biglycan mRNA levels were unaffected by IGF-1, PDGF-BB, or growth hormone. These findings suggest variable responses of fibroblasts to growth factors depending upon anatomical site within the periodontium. Moreover, there appears to be a correlation between cell proliferation and the types of proteoglycan synthesised with decorin expression being suppressed, and versican being increased during fibroblast proliferation.

Expression of bone associated macromolecules by gingival and periodontal ligament fibroblasts.

The expression of hard tissue associated proteins may be used to identify periodontal fibroblasts with the capability to facilitate periodontal regeneration. The aim of this study was to describe, by immunohistochemistry, the distribution of osteocalcin, osteopontin, bone sialoprotein and bone morphogenic proteins-2 and -4 (BMP-2 and BMP-4) within the human periodontium. Furthermore, the expression of mRNA for the above proteins and alkaline phosphatase by gingival and periodontal ligament fibroblasts in vitro was also assessed by reverse transcriptase polymerase chain reaction (RT-PCR). Localization of osteopontin, osteocalcin, BMP-2 and BM P-4 within sections of human periodontal structures was stronger in the periodontal ligament compared to the gingiva. Bone sialoprotein was not detected in either of the soft tissues but, along with osteopontin and osteocalcin, it was localized in the cementum and bone. In vitro, both the gingival and periodontal ligament fibroblasts expressed mRNA for alkaline phosphatase, BMP-2, BMP-4 and osteopontin. Although there were no differences in the expression of alkaline phosphatase and BMP-4 mRNA between the two cell types, we noted significantly higher mRNA levels of osteopontin in the periodontal ligament and BM P-2 in the gingival fibroblasts. Osteocalcin and bone sialoprotein mRNA expression was only noted in the cultured periodontal ligament fibroblasts. From these results, it can be concluded that distinct differences exist between the two fibroblast populations in terms of the localization and mRNA expression of the majority of the hard tissue associated proteins. Furthermore, the elevated in vitro mRNA expression for osteocalcin, osteopontin and bone sialoprotein may be used to identify cells with the potential to facilitate hard tissue formation and hence periodontal regeneration.

Growth hormone regulates osteogenic marker mRNA expression in human periodontal fibroblasts and alveolar bone-derived cells.

BACKGROUND: Growth hormone (GH) is a potent regulator of bone formation. The proposed mechanism of GH action is through the stimulation of osteogenic precursor cell proliferation and, following clonal expansion of these cells, promotion of differentiation along the osteogenic lineage. OBJECTIVES: We tested this hypothesis by studying the effects of GH on primary cell populations of human periodontal ligament cells (PLC) and alveolar bone cells (ABC), which contain a spectrum of osteogenic precursors. METHODS: The cell populations were assessed for mineralization potential after long-term culture in media containing beta-glycerophosphate and ascorbic acid, by the demonstration of mineral deposition by Von Kossa staining. The proliferative response of the cells to GH was determined over a 48-h period using a crystal violet dye-binding assay. The profile of the cells in terms of osteogenic marker expression was established using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for alkaline phosphatase (ALP), osteopontin, osteocalcin, bone sialoprotein (BSP), as well as the bone morphogenetic proteins BMP-2, BMP-4 and BMP-7. RESULTS: As expected, a variety of responses were observed ranging from no mineralization in the PLC populations to dense mineralized deposition observed in one GH-treated ABC population. Over a 48-h period GH was found to be non-mitogenic for all cell populations. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) BSP mRNA expression correlated well with mineralizing potential of the cells. The change in the mRNA expression of the osteogenic markers was determined following GH treatment of the cells over a 48-h period. GH caused an increase in ALP in most cell populations, and also in BMP expression in some cell populations. However a decrease in BSP, osteocalcin and osteopontin expression in the more highly differentiated cell populations was observed in response to GH. CONCLUSION: The response of the cells indicates that while long-term treatment with GH may promote mineralization, short-term treatment does not promote proliferation of osteoblast precursors nor induce expression of late osteogenic markers.

Amniotic fluid beta-endorphin and beta-lipotropin concentrations during the second and third trimesters.

Amniotic fluid beta-endorphin (beta-EP) and beta-lipotropin (beta-LPH) were measured by radioimmunoassay after silicic acid extraction and gel chromatographic separation of the two peptides in uncomplicated second-trimester and term pregnancies, in diabetic patients at term, and in pregnancies complicated by Rh-isoimmunization, premature labor, and intrauterine growth retardation. Furthermore, the lecithin/sphingomyelin (L/S) ratios as well as the dehydroepiandrosterone sulfate (DHEA-S) and cortisol levels were determined in most of the amniotic fluid specimens. Both the mean (+/- SE) beta-EP (65.3 +/- 9.1 fmol/ml) and beta-LPH (150 +/- 15.8 fmol/ml) concentrations were significantly higher in the 20 patients with normal pregnancies of 16 to 21 weeks' duration than those found in 21 patients with uncomplicated term pregnancies of 38 weeks' gestation, averaging 42.6 +/- 6.0 and 80.1 +/- 10.7 fmol/ml, respectively. The mean amniotic fluid beta-EP and beta-LPH concentrations measured in the latter subjects were similar to those observed in 23 diabetic patients with otherwise uncomplicated term pregnancies. The mean amniotic fluid beta-EP and beta-LPH levels found in the limited number of patients with Rh-isoimmunization (N = 9), premature labor (n = 8), and intrauterine growth retardation (n = 5) with pregnancies of 24 to 36, 24 to 36, and 34 to 38 weeks' gestation, respectively, were not significantly different from the mean amniotic fluid beta-EP and beta-LPH concentrations of uncomplicated term pregnancies. In all patients but those with Rh-isoimmunization, beta-EP concentrations exhibited a positive correlation with beta-LPH levels. However, the molar beta-LPH:beta-EP ratio was significantly lower at term than during the early second trimester. Neither beta-EP nor beta-LPH correlated with the amniotic fluid L/S ratio and only beta-LPH exhibited a significant inverse correlation with amniotic fluid DHEA-S. The latter was significantly higher in uncomplicated term than second-trimester pregnancies. These results confirm that immunoassayable beta-EP is present in amniotic fluid and declines toward term. These data demonstrate that immunoassayable beta-LPH is present in amniotic fluid and show a more pronounced decrease toward the end of pregnancy than beta-EP. Neither peptide, at least on account of the amniotic fluid levels, appears to be associated with fetal maturation. The physiologic significance of amniotic fluid beta-EP and beta-LPH and their possible role as markers of fetal response to stress remain to be elucidated.