Evaluation of the BAX system for detection of Listeria monocytogenes in foods: collaborative study.

A multilaboratory study was conducted to compare the automated BAX system and the standard cultural methods for detection of Listeria monocytogenes in foods. Six food types (frankfurters, soft cheese, smoked salmon, raw, ground beef, fresh radishes, and frozen peas) were analyzed by each method. For each food type, 3 inoculation levels were tested: high (average of 2 CFU/g), low (average of 0.2 CFU/g) and uninoculated controls. A total of 25 laboratories representing government and industry participated. Of the 2335 samples analyzed, 1109 were positive by the BAX system and 1115 were positive by the standard method. A Chi square analysis of each of the 6 food types, at the 3 inoculation levels tested, was performed. For all foods, except radishes, the BAX system performed as well as or better than the standard reference methods based on the Chi square results.

Insulin-like growth factor binding protein-3 does not mediate the interferon-dependent suppression of human ectocervical epithelial cell proliferation.

Interferon is a potential therapeutic agent for the treatment of cervical cancer. In the present study we examine the role of IFNgamma as a regulator of proliferation and production of IGFBP-3 expression in ectocervical epithelial cells. ECE16-1 cells are a model for studying early human papillomavirus-dependent cervical disease. IFNgamma produces a concentration-dependent inhibition of ECE16-1 cell proliferation that is associated with an increase in insulin-like growth factor binding protein-3 level. Growth suppression and IGFBP-3 increase is maximal at concentrations of IFNgamma >/=0.75 ng/ml. The increased IGFBP-3 expression is mediated via an increase in IGFBP-3 encoding mRNA. In contrast, IFNgamma inhibits proliferation of CaSki and SiHa cells, but IGFBP-3 is barely detectable and levels are not regulated by IFNgamma. These results suggest that the IFNgamma-dependent suppression of CaSki and SiHa cell proliferation is not mediated by secreted IGFBP-3. This result was confirmed when vector-mediated overexpression of immunoreactive IGFBP-3 in SiHa and CaSki cells did not consistently result in reduced cell proliferation rate.

Cultured porcine myogenic cells produce insulin-like growth factor binding protein-3 (IGFBP-3) and transforming growth factor beta-1 stimulates IGFBP-3 production.

Insulin-like growth factor binding proteins (IGFBP) may act locally as autocrine or paracrine regulators of insulin-like growth factor activity in specific tissues such as muscle. Although secretion of IGFBP by cultured myogenic cell lines has been examined, little is known about secretion of IGFBP by primary myogenic cell cultures. This may be because primary myogenic cultures contain non-muscle cells (fibroblasts) that complicate interpretation of IGFBP determinations. We have circumvented this problem by subculturing nonfusing cells from extensively fused porcine myogenic cultures and comparing the IGFBP production of these nonfusing, porcine muscle-derived cells with that of primary porcine myogenic cell cultures. Immunoprecipitation with specific antibodies and 125I-IGF-I ligand blot analysis showed that myogenic cultures secreted IGFBP-3 (doublet band, 43 kDa and 39 kDa), IGFBP-2 (34 kDa), IGFBP-4 (30 and 24 kDa), and IGFBP-5 (30 and 28 kDa). Muscle-derived fibroblasts secreted no detectable IGFBP-3 but approximately 10 times more IGFBP-2 than did myogenic cell cultures. Treatment of myogenic cultures for 24 h with transforming growth factor (TGF) beta-1 caused a concentration-dependent increase in IGFBP-3 secretion with a maximum 1.5-fold increase occurring at .5 ng of TGF beta-1/mL. In contrast, TGF beta-1 treatment did not stimulate detectable IGFBP-3 secretion by muscle-derived fibroblast cultures. Northern analysis of total RNA using a porcine IGFBP-3 probe revealed that TGF beta-1 treatment resulted in a fourfold increase in the steady-state level of IGFBP-3 mRNA in myogenic cultures. Insulin-like growth factor binding protein-3 mRNA was not detectable in fibroblast cultures either before or after TGF beta-1 treatment. This is the first report of IGFBP-3 secretion by cultured myogenic cells.

Regulation of human dermal papilla cell production of insulin-like growth factor binding protein-3 by retinoic acid, glucocorticoids, and insulin-like growth factor-1.

Insulin-like growth factor-1 (IGF1) has been reported to stimulate hair elongation and to facilitate maintenance of the hair follicle in anagen phase. However, little is known about IGF1 signaling in the hair follicle. In this study we investigate the effects of IGF1, glucocorticoids, and retinoids on dermal papilla (DP) cell production of insulin-like growth factor binding proteins (IGFBPs). IGFBPs comprise a family of IGF binding proteins that are produced and released by most cell types. They bind to IGFs to either enhance or inhibit IGF activity. In the present report we identify IGFBP-3 as being produced and released by cultured human dermal papilla (DP) cells. IGFBP-3 levels are increased fivefold by retinoic acid, eightfold by dexamethasone, and tenfold by IGF1. DP cells are known to produce IGF1, and so the observed stimulation of DP cell IGFBP-3 production by IGF1 is consistent with the idea that DP cells possess the IGF transmembrane receptor kinase and are autoregulated by IGFs. The level of another IGFBP, tentatively identified as IGFBP-2, is, in contrast, not regulated by these agents. IGFBP-3 has been shown to inhibit the activity of IGFs in a variety of systems. Our results are consistent with a model in which retinoids and glucocorticoids inhibit IGF action on DP cells and surrounding matrix cells by stimulating increased DP cell production of IGFBP-3. The IGFBP-3, in turn, forms a complex with free IGF1 to reduce the concentration of IGF1 available to stimulate hair elongation and maintenance of anagen phase.

Retinoid X receptor-specific retinoids inhibit the ability of retinoic acid receptor-specific retinoids to increase the level of insulin-like growth factor binding protein-3 in human ectocervical epithelial cells.

The hormones derived from vitamin A and related synthetic ligands (retinoids) are important regulators of differentiation and development and have been shown to be therapeutically useful in the treatment of cervical cancer. All-trans-retinoic acid exerts its effects by activation of retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers. These heterodimers bind to the retinoic acid response elements of target genes to regulate gene expression. RXR ligands act through RXR homodimers to regulate gene expression. In the present study, we describe the effects of RAR- and RXR-specific ligands on the regulation of insulin-like growth factor binding protein-3 (IGFBP-3) production and cell proliferation in human ectocervical epithelial (ECE) cell lines. Treatment of ECE16-1 cells with a RAR-specific ligand (TTNPB) or a ligand that interacts with both RAR and RXR receptors (9-cis-retinoic acid) increases IGFBP-3 levels and suppresses cell proliferation. In contrast, RXR-specific ligands (AGN191701, SR11217, and SR11237) do not regulate proliferation and slightly suppress the IGFBP-3 level. Cotreatment with increasing concentrations (0.01-1000nm) of RXR-specific ligand antagonizes the growth suppressive and IGFB-3-increasing effects of 1000 nM TTNPB. Similar results are observed in two other ECE cell lines, ECE16-D1 and ECE16-D2. These results indicate that RXR-specific ligands can antagonize RAR responses in these cell lines and suggest that a RAR-specific retinoid may be superior to one with mixed RAR/RXR binding activity for inhibiting cervical cancer cell proliferation. Moreover, the antagonism of RAR-dependent responses by RXR-specific ligands is consistent with a squelching model in which the RXR-specific ligand drives formation of RXR/RXR homodimers at the expense of the more active RAR/RXR heterodimers.

Microleakage of new dentin bonding systems using human and bovine teeth.

The objective of this study was twofold: to evaluate the microleakage behavior of three dentin bonding systems and to determine if bovine teeth are comparable substrates to human teeth when studying the microleakage of various materials. The materials evaluated were Scotchbond Multi-Purpose Adhesive, Prisma Universal Bond 3, and All-Bond 2. All three bonding systems were used in combination with Prisma APH hybrid composite for comparison of microleakage behavior. Sixty class 5 preparations were cut at the cementoenamel junction for groups containing 30 human and 30 bovine teeth. A 1 mm 45 degree bevel was placed at the enamel margin. Teeth were grouped according to the dentin bonding system used and then restored according to the manufacturer's directions. After restoration, the teeth from each group were stored in distilled water at 37 degrees C for 3 days. The teeth were then thermocycled between 4 degrees C and 58 degrees C for 100 cycles and returned to distilled water at 37 degrees C for an additional 4 days. The teeth were then sealed with nail polish up to 1 mm from the margins of the restoration and placed in 45Ca isotope for 2 hours. The teeth were then sectioned and placed on x-ray film to produce autoradiographs. Microleakage was evaluated for the enamel and dentin margins separately using the following scale: 0 = no leakage, 1 = penetration of isotope to less than 1/2 the distance to the axial wall, 2 = penetration of isotope greater than 1/2 of the distance to the axial wall but short of the axial wall, and 3 = penetration of isotope to the axial wall or beyond. The materials were compared to each other using the Mann-Whitney U and Kruskal-Wallis tests. The gingival margins were compared to the incisal margins for all materials. No statistically significant differences in microleakage were revealed between the incisal and gingival location for human substrates, but there was statistically significant greater gingival microleakage for bovine substrates. All-Bond 2 leaked significantly more than Scotchbond Multi-Purpose for human substrates at the incisal margin. All-Bond 2 had significantly more microleakage than Prisma Universal Bond 3 at both dentin and enamel margins for the bovine substrate. There were no statistically significant differences in microleakage among the bonding systems for the human substrate. No statistically significant differences between the microleakage behavior of human and bovine substrates were found. These results support the use of bovine teeth for in vitro microleakage studies.

Microleakage and thermal properties of hybrid ionomer restoratives.

The objective of this study was to compare the microleakage and thermal properties of two recently introduced hybrid materials to those of a conventional glass-ionomer cement. Class V preparations were prepared at the cementoenamel junctions in freshly extracted bovine incisor and evaluated for microleakage with a 45Ca radioisotope method. Thermal properties were evaluated with thermal mechanical analysis and differential scanning calorimetry. The degree of microleakage and the coefficient of thermal expansion of the conventional glass-ionomer cement were found to be significantly less than those of either hybrid.

Microleakage of a new dental adhesive comparing microfilled and hybrid resin composites.

The objective of this study was to evaluate the in vitro microleakage of a new adhesive system, Syntac. Class V preparations were cut at the cementoenamel junction in 20 extracted bovine incisors and 20 extracted human incisors. One group of 10 human and 10 bovine teeth was restored with Syntac and a microfilled posterior resin composite; the other group was restored with Syntac and a hybrid resin composite. Microleakage was evaluated at 1 week with a 45Ca radioisotope method. No statistically significant differences between human and bovine substrates were found with either resin composite. The Syntac/hybrid resin restorations leaked less than did the Syntac/microfilled resin restorations at the gingival (dentin) margin in the bovine teeth; however, this difference was not statistically significant.

Comparisons of fit of CAD-CAM restorations using three imaging surfaces.

One of the factors that influences the overall fit of a Cerec restoration is the thickness of the optical powder. The objective of this study was to compare the measurement of the fit of glass inlays produced with the Cerec instrument after each preparation had been coated, in separate trials, with imaging powder applied with an aerosol; imaging powder applied with air from a dental unit; and a water-soluble paint applied with a brush. Ten inlay preparations were made in extracted teeth. Each preparation was coated with one of the three imaging media and an inlay was made. This was repeated until each preparation received each imaging surface. Each inlay was measured at eight different points by using an image analysis system interfaced to a stereoscopic measuring microscope. The inlays fabricated on the two powder surfaces were not significantly different, but the painted surfaces were found to result in a significantly better-fitting inlay.

Human cervical cancer. Retinoids, interferon and human papillomavirus.

Our studies highlight the importance of dietary vitamin A (retinol) and other retinoids in maintaining normal cervical cell function and in inhibiting the growth of cervical tumors. Based on our results we conclude that 1) HPV 16-immortalization enhances cervical cell sensitivity to retinoids, 2) cytokeratin expression may be useful as a marker for evaluating the success of retinoid therapy in vivo, 3) retinoids do not necessarily act to inhibit proliferation of HPV-immortalized cervical cells via effects on HPV E6 and E7 RNA levels and 4) retinoids may act to inhibit cervical proliferation by "suppressing" the activity of the EGF and IGF signalling pathways. Based on these and other results, it is worth considering the possibility that vitamin A or related retinoids could be administered therapeutically, early in the neoplastic process (either systemically or locally), to inhibit the progress of the disease. These results also suggest that combined interferon/retinoid therapy may provide an enhanced beneficial effect to reduce cervical tumor size due to the fact that each agent is inhibiting cervical cell proliferation via distinct, but reinforcing, pathways (i.e., IFN gamma reduces E6/E7 expression, RA inhibits the function of the EGF and IGF1 signalling pathways).

Transforming growth factor beta-1 facilitates establishing clonal populations of ovine muscle satellite cells.

Myogenic cells isolated from lamb fetuses (approximately mid-gestation) exhibited a concentration-dependent decrease in myogenic cell proliferation in response to transforming growth factor (TGF) beta-1 (P < .001). Half-maximal inhibition of proliferation occurred at approximately .05 ng of TGF beta-1/mL and maximal inhibition of proliferation occurred at approximately .1 ng of TGF beta-1/mL. The specificity of this inhibition was confirmed by neutralization of the activity following exposure to a TGF beta antibody. The TGF beta-1 also suppressed proliferation of ovine satellite cells isolated from 5-d-old lambs (P < .0035), but to a lesser extent than observed for embryonic cells. In contrast, TGF beta-1 did not significantly suppress serum-stimulated proliferation of ovine satellite cells isolated from 30- or 150-d-old lambs. Similarly, TGF beta-1 did not suppress proliferation of skeletal muscle fibroblast-like cells isolated from either fetal lambs or 150-d-old lambs. In fact, proliferation of fibroblast-like cells derived from embryonic ovine muscle was enhanced by exposure to TGF beta-1 at all levels tested; however, a concentration-dependent response was not observed. Media transfer experiments showed that conditioning of culture media by postnatally derived cells did not render TGF beta-1 inactive. The studies described in this manuscript suggest that sensitivity of ovine myogenic cells to the antiproliferative effect of TGF-beta may vary with the stage of development.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of insulin-like growth factor 1 binding protein 3 levels by epidermal growth factor and retinoic acid in cervical epithelial cells.

Insulin-like growth factors (IGFs) are important regulators of epithelial cell growth. The mitogenic activity of these factors is influenced by the levels of extracellular IGF binding proteins, including insulin-like growth factor binding protein 3 (IGFBP-3). In the present report we study the effects of epidermal growth factor (EGF) and all-trans-retinoic acid (RA) on IGFBP-3 RNA and protein levels in human papillomavirus-immortalized cervical epithelial cells. Treatment of ECE16-1 cells with 3-20 ng/ml EGF causes a marked reduction in IGFBP-3 levels. In contrast, 1 microM RA increases IGFBP-3 mRNA and protein levels in the presence or absence of 20 ng/ml EGF. The response is concentration dependent with a half-maximal increase observed at 1 nM RA. RA is able to reverse the EGF suppression when added simultaneously or 3 days after initiation of EGF treatment. Conversely, when cells are treated with RA, IGFBP-3 levels increase within 24 h and subsequent addition of EGF is without effect. Thus, the RA-dependent increase in IGFBP-3 levels is dominant over the EGF suppression. The increased IGFBP-3 levels are correlated with RA suppression of proliferation. Similar RA effects on IGFBP-3 mRNA levels were observed in other cervical epithelial cell lines (i.e., ECE16-D1, ECE16-D2, and CaSki). These results suggest that RA may act to inhibit cervical cell growth by increasing IGFBP-3 levels and reducing the extracellular concentration of free insulin-like growth factor I (IGFI) and/or alternatively, IGFBP-3 may inhibit cell growth by direct effects on the cell, independent of IGFI.

Epidermal growth factor suppresses insulin-like growth factor binding protein 3 levels in human papillomavirus type 16-immortalized cervical epithelial cells and thereby potentiates the effects of insulin-like growth factor 1.

Human ectocervical epithelial cells are a primary target for infection by oncogenic papillomaviruses, which are strongly implicated as causative agents in the genesis of cervical cancer. Growth factors have been implicated as agents that stimulate proliferation and enhance the possibility of malignant transformation. In the present study we utilize several human papillomavirus (HPV) type 16-immortalized ectocervical epithelial cell lines to investigate the effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on cell proliferation and the production of IGF binding proteins (IGFBPs). ECE16-1 cells, an HPV16-immortalized/nontumorigenic cell line, maintained in defined medium, produce and release high levels of IGFBP-3 (38/42 kDa) as well as smaller amounts of a 24-kDa IGFBP. Supplementation of defined medium with EGF causes a dose-dependent increase in cell growth and a concomitant decrease in the levels of IGFBP-3 released into the culture medium. EGF suppression of IGFBP-3 is maintained even when EGF-stimulated cell growth is suppressed 67% due to the simultaneous presence of 3 ng/ml of TGF beta 1, indicating that EGF suppression of IGFBP-3 levels is independent of EGF effects on cell growth. EGF suppression of IGFBP-3 production is correlated with a reduction in IGFBP-3 mRNA level. In the presence of EGF, the growth response of the cells to ng amounts of IGF-I is significantly enhanced. Moreover, the simultaneous presence of both EGF and IGF-I reduces the level of IGFBP-3 more efficiently than EGF alone. We also observe that the IGFBP-3 level is decreased and the 24-kDa IGFBP level is increased in HPV16-positive tumorigenic versus nontumorigenic cell lines. This is the first report of EGF acting as a positive regulator of IGF-I action via the IGFBPs. On the basis of these findings, we propose that EGF stimulates ECE16-1 cell growth via a dual-action mechanism by (a) stimulating growth directly via the EGF mitogenic pathway and (b) stimulating growth indirectly by reducing the levels of inhibitory IGFBPs and thereby potentiating the effects of IGF-I. In addition, the observation that more highly transformed cell types produce lower levels of IGFBP-3 and higher levels of 24-kDa IGFBP suggests that tumor cells in more advanced cervical cancers may have an altered response to IGF-I.

Interferon and retinoic acid suppress the growth of human papillomavirus type 16 immortalized cervical epithelial cells, but only interferon suppresses the level of the human papillomavirus transforming oncogenes.

In the present study, we examine the effects of all-trans-retinoic acid (RA) and interferons-alpha and -gamma (IFN-alpha and IFN-gamma) on the growth of HPV16-immortalized cell lines, ECE16-1 and CaSki. Treating proliferating ECE16-1 cells with RA causes a concentration-dependent decrease in cell number. At 1 microM RA, cell growth is suppressed by 65% and the level of mRNA encoding cytokeratin K5, a biochemical marker of retinoid action, is also suppressed. In contrast, the level of transcript encoding the HPV16 oncogenes, E6 and E7, is reduced by only 5 to 10%. IFN-alpha at 1000 IU/ml or IFN-gamma at 200 IU/ml suppresses growth by 70%. This growth suppression by IFN-gamma is correlated with a > 90% reduction in E6/E7 mRNA levels. Additional growth suppression is observed upon simultaneous treatment with retinoid and interferon. Optimal suppression is observed in the presence of 200 IU/ml IFN-gamma and 1 microM RA. The rank order of effectiveness is IFN-gamma/RA > IFN-alpha/RA = IFN-gamma > RA > IFN-alpha. In contrast to the suppression of ECE16-1 cell growth, RA causes a concentration-dependent increase in CaSki cell number (50-60%) which is optimal at 1 microM RA. Cytokeratin K5 mRNA levels are markedly suppressed, and E6/E7 mRNA levels increased by 5% under these conditions. IFN-alpha at 1000 IU/ml or IFN-gamma at 200 IU/ml decreases CaSki cell growth by 20 and 45%, respectively, and 200 IU/ml of IFN-gamma reduce E6/E7 expression to undetectable levels. Addition of RA (1 microM) partially counters the IFN-dependent suppression of growth and E6/E7 mRNA levels. Our results suggest that retinoid-dependent changes in human papillomavirus-immortalized cervical cell proliferation are not always correlated with changes in E6/E7 transcript levels.

Transforming growth factor beta 1 regulation of metalloproteinase production in cultured human cervical epithelial cells.

Collagenase levels are regulated in a cell type-specific manner by a variety of growth factors and cytokines, and increased type IV collagenase activity in tumor cells has been linked to metastatic growth. In this study we compare the effects of epidermal growth factor (EGF) and transforming growth factor beta 1 (TGF beta 1) on gelatinase production in cervical epithelial cell lines. EGF is a strong mitogen for cervical epithelial cells and TGF beta 1 suppresses growth. Metalloproteinase zymograms of conditioned medium from normal human ectocervical cells reveal two major bands of metalloproteinase activity at 72 and 92 Kd. In contrast, the level of the 92-Kd activity is greatly reduced in the human papillomavirus type 16-positive ECE16-1 and CaSki cells. EGF treatment produces minimal changes in metalloproteinase levels. Treatment of CaSki cells with 20 ng/ml of EGF reduces by 30 to 50% the level of both activities. In ECE16-1 cells, EGF decreases the 72-Kd activity by 50% and the 92-Kd activity slightly. TGF beta 1 treatment, in contrast, increases the 72-Kd activity 3- to 10-fold and the 92-Kd activity by > or = 25-fold in each cell type. In CaSki and ECE16-1 cells, the changes in metalloproteinase level are mediated by changes in level of the corresponding mRNAs. In each case, the metalloproteinases are secreted as inactive proenzymes which can be activated by in vitro treatment with organomercurials. Tests of a series of additional cervical cell lines reveal that metalloproteinase levels are generally higher in normal cervical cells and in cells immortalized by transfection with HPV16, whereas lower levels are observed in cells derived from human tumors. Moreover, a higher percentage of cell lines derived from human tumors do not respond to TGF beta 1 regulation of metalloproteinase levels. Parallel studies indicate that the TGF beta 1-stimulated increase in the 72- and 92-Kd activities is correlated with enhanced chemotactic and chemoinvasive behavior in both ECE16-1 and CaSki cells.

Evaluation of a new dentinal bonding system.

The purpose of this study was to compare the in vitro microleakage of a new dentinal bonding system, not yet released in the United States, to the microleakage of two first-generation bonding agents. Simulated Class V cavities were prepared in freshly extracted bovine incisors and restored with the appropriate composite resin-bonding agent combination. Restorations were thermocycled and examined for microleakage at 24 hours and 60 days. Comparison of the materials revealed no statistically significant difference in microleakage scores at the gingival or incisal margins after 24 hours. However, the first-generation bonding agents exhibited significantly more microleakage at the gingival margin than did the new bonding system after 60 days. No significant increase in microleakage was measured at the incisal margin for any of the materials after 60 days. In contrast, all materials exhibited a significant increase in leakage at the gingival margin after 60 days. No correlation between microleakage and polymerization shrinkage or coefficients of thermal expansion of the composite resins could be established.