Effectiveness of an oral health education seminar for paediatric and family medicine residents.

AIM: To assess the immediate effect of a 60-minute oral health educational seminar for paediatric and family medicine residents in improving their knowledge, attitude, likelihoodtowards incorporating oral health preventive practice in their current practices to well-child visits, and confidence in identifying and referring patients with dental trauma. MATERIALS AND METHODS: Baseline pre- and post-test design was used to evaluate the immediate effect of a 60-minute PowerPoint oral health educational seminar given to the paediatric and family medicine residents. STATISTICS: Multiple-choice items were used and the pre- and post-test data were analysed with McNemar and Wilcoxon signed-rank tests. A p-value <0.05 was considered statistically significant. RESULTS: Sixty-eight residents participated in the oral health educational seminar and completed the questionnaire. The mean age of participants was 29.9 years old (SD ±4.8 yrs.). Immediately following a 60-minute oral health educational seminar, there was an overall significant increase in participants' knowledge, attitudes and likelihood towards incorporating oral health preventive practice in their current practices to well-child visits (p<0.05). More confidence in identifying and referring patients with dental trauma was reported by 100% of participants. CONCLUSIONS: A 60-minute oral health educational seminar was effective in improving paediatric and family medicine residents' immediate knowledge, attitude, and likelihood towards incorporating oral health preventive practice in their current practices to well-child visits. Significantly more residents felt more confident in identifying and referring patients with dental trauma. Key messages: an oral health educational seminar can be effective in improving paediatric and family medicine residents' immediate knowledge, attitude, and likelihood towards incorporating oral health preventive practice in their current practices to well-child visits.

Composite Replacement of Amalgam Restoration Versus Freshly Cut Dentin: An In Vitro Microleakage Comparison.

OBJECTIVE: The aim of this study was to evaluate the microleakage of the composite restorations when bonded to tooth structure previously restored with amalgam material compared with that of freshly cut dentin. METHODS AND MATERIALS: Thirty intact, extracted intact human molars were mounted in autopolymerizing acrylic resin. Class II box preparations were prepared on the occluso-proximal surfaces of each tooth (4-mm bucco-lingual width and 2-mm mesio-distal depth) with the gingival cavosurface margin 1 mm above the CEJ. Each cavity was then restored using high copper amalgam restoration (Disperalloy, Dentsply) and then thermocycled for 10,000 thermal cycles. Twenty-five of the amalgam restorations were then carefully removed and replaced with Filtek Supreme Ultra Universal (3M ESPE); the remaining five were used for scanning electron microscopy and energy dispersive x-ray spectroscopy analysis. A preparation of the same dimensions was performed on the opposite surface of the tooth and restored with composite resin and thermocycled for 5000 thermal cycles. Twenty samples were randomly selected for dye penetration testing using silver nitrate staining to detect the microleakage. The specimens were analyzed with a stereomicroscope at a magnification of 20×. All of the measurements were done in micrometers; two readings were taken for each cavity at the occlusal and proximal margins. Two measurements were taken using a 0-3 scale and the percentage measurements. RESULTS: Corrosion products were not detected in either group (fresh cut dentin and teeth previously restored with amalgam). No statistically significant difference was found between the microleakage of the two groups using a 0-3 scale at the occlusal margins (McNemar test, p=0.727) or proximal margins (Wilcoxon signed-rank test, p=0.174). No significance difference was found between the two groups using the percentage measurements and a Wilcoxon signed-rank test at either the occlusal (p=0.675) or proximal (p=0.513) margins. However, marginal microleakage was statistically significant between the proximal and occlusal margins (p<0.001). CONCLUSION: Within the limitations of this in vitro study, no significant difference was found between the microleakage of nondiscolored dentin in teeth that were previously restored with amalgam compared with freshly cut dentin. However, marginal microleakage in the proximal surface was higher than that in the occlusal surface.

Gingival crevicular fluid levels of interferon-γ, but not interleukin-4 or -33 or thymic stromal lymphopoietin, are increased in inflamed sites in patients with periodontal disease.

OBJECTIVE: To investigate the hypothesis that levels of interferon (IFN)-γ and interleukin (IL)-4, as well as the newer cytokines IL-33 and thymic stromal lymphopoietin (TSLP), in gingival crevicular fluid (GCF) samples differ from sites of patients at various clinical stages of periodontal disease and controls. BACKGROUND: Periodontal diseases result from the complex interplay between pathogenic bacteria and the host's immune responses. Several inflammatory mediators, such as IFN-γ and IL-4, have been detected in GCF samples in patients with periodontitis, but the results are mostly contradicting due to the lack of uniformity and collection of sites and methods of analysis. MATERIAL AND METHODS: GCF samples were collected from sites with different clinical characteristics (healthy, gingivitis and periodontitis sites) from periodontally healthy ( n = 14), plaque-induced gingivitis (n = 17) and chronic periodontitis (n = 11) subjects. The GCF samples were analyzed for the frequency of detection and levels of IFN-γ, IL-4, IL-33 and TSLP using a multiplex bead immunoassay. RESULTS: Inflamed sites in both patients with plaque-induced gingivitis and chronic periodontitis showed statistically significantly higher volume of GCF compared to non-inflamed sites in all patients. IFN-γ could be detected in about 50-70% of the samples analyzed and at significantly higher levels in sites with periodontitis compared to healthy sites in patients with chronic periodontitis (p = 0.035). We also show a statistically significant decrease of IFN-? in healthy sites of patients with chronic periodontitis as compared to gingivitis sites in patients with plaque-induced gingivitis (p = 0.047). Only some of the GCF samples showed detectable levels for IL-4 and TSLP, while IL-33 was below the detection level in all samples collected. CONCLUSIONS: These results suggest that IFN-γ levels in GCF depend on the clinical stage of the site and not on the disease stage of the patient, but need to be expanded to a greater number of subjects and additional analysis of corresponding gingival tissue biopsies for cytokine gene expression.

Prospective study in critically ill non-neutropenic patients: diagnostic potential of (1,3)-ß-D-glucan assay and circulating galactomannan for the diagnosis of invasive fungal disease.

Diagnosis of invasive fungal disease (IFD) in patients under intensive care is challenging. Circulating biomarkers, (1,3)-ß-D-glucan (BG) and galactomannan (GM), were prospectively assessed in 98 critically ill patients at risk of IFD. There were 11 cases of invasive aspergillosis (IA; 4 proven and 7 probable), 9 cases of proven invasive candidiasis (IC), 1 case of mixed proven IC and probable IA, 1 case of proven zygomycosis, and 1 case of mixed mycelial proven IFD. In all IA cases there was no significant difference when the area under the receiver operating characteristic curve (AUC) of GM (0.873 [95%CI, 0.75-0.99]) and BG (0.856 [95% CI, 0.71-0.99]) were compared (p = 0.871). The AUC for BG in IC and for the rest of the IFD cases was 0.605 (95% CI, 0.39-0.82) and 0.768 (95% CI, 0.63-0.90) respectively. Positive BG (40%) predated blood culture (n = 3) and abdominal pus (n = 1) a mean of 3.25 days before Candida was grown. In patients with IFD caused by molds, BG appeared a mean of 5.65 days before culture results. For the diagnosis of patients at risk of IC, BG has shown a high NPV (94.5%), with positive results also predating blood cultures in 30% of patients. In conclusion, early BG results permit a timely initiation of antifungal therapy in patients at risk of IFD.

Confirming nasal airway dimensions observed on panoramic and posterior-anterior cephalometric radiographs using an acoustic rhinometer.

AIM: This study aimed to confirm the nasal airway dimensions observed in standard dental radiographs through analysis of the anterior nasal cross-sectional area and nasal volume with acoustic rhinometry. METHOD: Participants were chosen from the patient population under consideration for interceptive orthodontic treatment at Tufts University's Pediatric Dentistry Clinic. A total of 55 Caucasian subjects, of both sexes (males=28, females =27), and ranging from age 6-9 years (mean= 7.33) were enrolled. Utilizing the ImageJ program, the subjects' panoramic and PA cephalometric radiographs were analyzed for radiolucent anterior nasal cross-sectional area. Subjects were then examined with the Eccovision Acoustic Rhinometer, which measured anterior nasal cross-sectional area and nasal volume. Data were grouped together, and areas and volume gathered from the radiographs and rhinometer analyzed for any correlation. Statistical analysis was performed using the SPSS program. Data was further divided into right and left nasal side, and a Pearson Correlation Matrix was created for 8 predictor variables (right and left panoramic area, right and left cephalometric area, right and left rhinometer area, and right and left rhinometer volume). RESULTS: Values in this correlation matrix ranged from 0.712 to 0.988; all were statistically significant at the <0.01 level (2-tailed test). CONCLUSION: A very strong correlation was found between the anterior nasal cross-sectional area calculated from the radiographs, and the anterior nasal cross-sectional area and nasal volume from the rhinometer.

Prostatic secretory protein (PSP94) expression in human female reproductive tissues, breast and in endometrial cancer cell lines.

PSP94 (beta microseminoprotein, beta MSP) is one of the three major proteins secreted by the normal human prostate gland. Using reverse transcriptase polymerase chain reaction (RT-PCR) and Southern blotting, PSP94 transcripts were shown in human endometrium, myometrium, ovary, breast, placenta and in the human endometrial cancer cell lines KLE and AN3 CA. Primers used in these studies were specific for human prostate PSP94, and were derived from its flanking non-coding regions. The results were confirmed by sequence analysis of two independently derived clones from normal human breast tissues and the other two from KLE cells respectively. The sequences were identical with the coding sequence of human prostate PSP94 cDNA. Using RNA from the endometrial tissues, two different transcripts of approximately 487 bp, equivalent to prostate PSP94 and approximately 381 bp, corresponding to prostate PSP57, its alternately spliced form, were amplified by RT-PCR. Human ovary, breast, placenta and endometrial cancer cell lines (KLE, AN3 CA), however, showed only the full length, approximately 487 bp, PSP94 transcript. We further demonstrated by in situ hybridization that PSP94 mRNA is expressed specifically in the glandular epithelial cells, and not in the stroma of both the human endometrial and breast tissues. Further, using image analysis of in situ hybridization data, the levels of PSP94 mRNA in the cycling endometrial tissues and in breast confirmed the differential levels of expression in the cycling endometrium (P<0.005). This study distinctly demonstrated significant expression of PSP94 mRNA in human uterine, breast and other female reproductive tissues as well in the endometrial cancer cell lines, suggesting that it may have a role in these tissues as a local autocrine paracrine factor.

Prostate secretory protein (PSP94) suppresses the growth of androgen-independent prostate cancer cell line (PC3) and xenografts by inducing apoptosis.

BACKGROUND: PSP94 (prostate secretory protein of 94 aa; also called PIP), one of the predominant proteins secreted into the seminal fluid, was proposed as an independent diagnostic/prognostic marker for prostate cancers. It was also shown to inhibit rat prostate cancer growth. In this study, we investigated the effect of purified PSP94 on the growth of androgen-independent human prostate cancer cells (PC3) and its potential mechanism of action. METHODS AND RESULTS: PSP94, in a dose- and time-dependent manner, inhibited the growth of PC3 cells. The protein demonstrated a stronger inhibitory effect on the colony-forming ability of PC3 cells in soft agar. A daily injection of PSP94 at 5 microg/kg/body weight resulted in a 50-60% inhibition in the growth of PC3 xenografts in athymic mice. PC3 cell growth inhibition by PSP94 resulted from cell death characteristic of morphological apoptosis, which was confirmed by dual fluorescence microscopy, electron microscopy, and DNA fragmentation assays. Mechanistic studies indicated that PSP94 enhanced the expression of proapoptotic protein Bax without affecting Bcl-2 levels. CONCLUSIONS: This study suggests that PSP94 may represent a novel, apoptosis-based, antitumor agent applicable to the treatment of hormone-refractory human prostate cancers.

Detection of PSP94 and its specific binding sites in the prostate adenocarcinoma cell line LNCaP.

PURPOSE: To evaluate the expression of prostate secretory protein of 94 amino acids (PSP94) and PSP94 binding proteins in the LNCaP cell line. MATERIALS AND METHODS: The reverse-transcription polymerase chain reaction (RT-PCR) and Southern blot hybridization were employed to assay the expression of PSP94. Immunoprecipitation with specific polyclonal antibodies was used to detect PSP94 secreted by the LNCaP cells. The binding proteins were assayed by equilibrium binding assays. RESULTS: PSP94 was expressed and secreted in the LNCaP cells. As well as, LNCaP cells expressed surface membrane proteins capable of binding PSP94 in a specific and saturable manner. Exposure of LNCaP cells to exogenous PSP94 resulted in the up-regulation of PSP94 binding sites, indicating functional interactions for PSP94 and its receptor in this cell line. CONCLUSION: The expression of PSP94 and its receptors may be partially regulated by an autocrine pathway in the LNCaP cell line.

Identification of binding proteins for PSP94 in human prostate adenocarcinoma cell lines LNCaP and PC-3.

BACKGROUND: Prostatic secretory protein of 94 amino acids (PSP94) is one of the predominant proteins found in human seminal fluid. Limited information is available regarding a physiological function for PSP94. An important step in the elucidation of this function is the determination of the mechanism of interaction of PSP94 with potential cellular targets. METHODS: Equilibrium binding assay was employed to demonstrate specific binding of biotinylated-PSP94 to the LNCaP and PC-3 cell lines. Binding proteins were partially purified by PSP94 affinity-chromatography from LNCaP, PC-3 cells, and prostate tissues. RESULTS: Binding of biotinylated-PSP94 to LNCaP and PC-3 cells was saturable and time and temperature dependent. The binding could be specifically competitively inhibited by unlabelled PSP94. Two types of PSP94 binding sites with distinct affinity (Kd) and density (Bmax) were determined by Scatchard analysis for each of the two cell lines. For the LNCaP cells, these values were Kd 1 = 0.75 nM and Bmax1 = 300 fmol/mg protein and Kd 2 = 4.5 nM, Bmax2 = 780 fmol/mg protein, respectively. Similar affinity and density results were obtained for PC-3 cells: Kd 1 = 0.83 nM, Bmax1 = 250 fmol/mg protein, and Kd 2 = 5.0 nM, Bmax2 = 700 fmol/mg. The binding of biotinylated-PSP94 to the LNCaP cells was competitively inhibited by the partially purified proteins. Analysis of these proteins SDS-PAGE showed three main bands and the molecular weights of these three bands were approximately 180, 100 and 60 kD, respectively. CONCLUSIONS: The data showed the presence of specific binding proteins to the PSP94 in LNCaP, PC-3 cells, and prostate tissue.

A new scalable purification procedure for prostatic secretory protein (PSP94) from human seminal plasma.

A simple three-step procedure for the purification of native prostate secretory protein (PSP94) from human seminal plasma is described. The steps are ammonium sulfate fractionation followed by a Macro-Prep S support (cation) flowthrough process and the final Macro-Prep high Q support (anion exchange) chromatography using two step-gradient elution. The benefits of this procedure lie in its simplicity, speed, and relatively inexpensive materials, thus providing advantages over the previously reported schemes. The purity of the product as judged by single band on SDS-polyacrylaminde gel electrophoresis was equivalent to that attained by analytical HPLC anion exchange procedure. Yields were in the range of 18-25 mg highly pure PSP94 per 50 ml of seminal plasma. The desalted, lyophilized, purified PSP94 sample was characterized by SDS-PAGE, Western blot, and N-terminal sequencing. All parameters tested confirm its identity. Preliminary data show that this procedure is suitable for a large-scale production. The direct quantitation of PSP94 by SDS-PAGE and densitometric image analysis at various purification steps for evaluating the recovery of PSP94 is described. The results obtained show that this is an efficient strategy for preparation of highly purified native PSP94.

Alternative splicing of PSP94 (prostatic secretory protein of 94 amino acids) mRNA in prostate tissue.

While performing reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of total mRNA from prostate cancer specimens, two forms of PSP94 cDNA were detected. RT-PCR products were analysed by Southern blotting and probing with exon-specific oligonucleotides. In the short form of PSP94 mRNA, designated as PSP57, exon III was found to be deleted. The two mRNA forms were confirmed by cloning and sequencing of the RT-PCR products and were found to result from alternative splicing. The alternatively spliced form, PSP57, was characterized by sequence analysis. PSP94 and PSP57 possess identical exons I and II, including identical secretion signal peptide and the 5' untranslated sequences. PSP57 has a frame-shifted exon IV and encodes a putative 57 amino acid protein with a novel, highly basic C-terminus of 41 amino acids. PSP57 mRNA was detected in other urogenital tissues (kidney, bladder) and in most tumor cell lines tested, but was not detectable in other tissues such as breast and lung. In prostate tumor cell lines, PSP57 mRNA was aberrantly spliced and localized in the nuclear fraction of the cell. Our results suggest the possible existence of a novel PSP protein that originates from alternative splicing of PSP94 mRNA in urogenital tissues.

Construction, binding properties, metabolism, and tumor targeting of a single-chain Fv derived from the pancarcinoma monoclonal antibody CC49.

CC49 is a "second generation" monoclonal antibody to B72.3, which reacts with the pancarcinoma antigen TAG-72. CC49 has been shown to efficiently target human colon carcinoma xenografts and is currently being evaluated in both diagnostic and therapeutic clinical trials. We describe here the construction and characterization of a recombinant single-chain Fv (sFv) of CC49. The sFv was shown to be a Mr 27,000 homogeneous entity which could be efficiently radiolabeled with 125I or 131I. Comparative direct binding studies and competition radioimmunoassays using CC49 intact IgG, F(ab')2, Fab', and sFv revealed that the monomeric CC49 Fab' and sFv had relative binding affinities 8-fold lower than the dimeric F(ab')2 and intact IgG. Nonetheless, the 131I-labeled sFv was shown to bind biopsies of TAG-72-expressing tumors. Metabolism studies in mice, using radiolabeled CC49 IgG, F(ab')2, Fab', and sFv, demonstrated an extremely rapid plasma and whole body clearance for the sFv. CC49 sFv plasma pharmacokinetic studies in rhesus monkeys also showed a very rapid plasma clearance (T1/2 alpha of 3.9 min and T1/2 beta of 4.2 h). Tumor targeting studies with all four radiolabeled Ig CC49 forms, using the LS-174T human colon carcinoma xenograft model, revealed a much lower percentage injected dose/g tumor binding for the CC49 monomeric sFv and Fab' as compared to the dimeric F(ab')2 and intact IgG. However, tumor:normal tissue ratios (radiolocalization indices) for the sFv were comparable to or greater than those of the other Ig forms. High kidney uptake with 125I-labeled Fab' and F(ab')2 was not seen with 125I-sFv. Gamma scanning studies also showed that 131I-CC49 sFv could efficiently localize tumors. The CC49 sFv may thus have utility in diagnostic and perhaps therapeutic applications for a range of human carcinomas.

Construction and characterization of a single-chain catalytic antibody.

The antigen-binding (Fab) fragment of the catalytic monoclonal antibody NPN43C9 has recently been cloned by using bacteriophage lambda. By inserting the variable regions of this Fab coding sequence into a (NH2)-VL-linker-VH-(COOH) construct (where VL and VH represent the heavy and light chain variable regions), we have assembled a recombinant gene encoding a catalytic single-chain antigen-binding protein. This protein has been expressed in Escherichia coli and exhibits the same catalytic parameters as the parent monoclonal antibody NPN43C9. Single-chain forms of catalytic antibodies may prove valuable for structural and site-directed mutagenesis studies as well as for large-scale applications of catalytic antibodies.

Pullulan Elaboration by Aureobasidium pullulans Protoplasts.

Protoplasts of Aureobasidium pullulans are capable of producing pullulan. Biosynthesis of the polymer pullulan required induction with kinetics similar to those of whole cells. The protoplasts also produced a heteropolysaccharide component containing mannose, glucose, and galactose. The relative proportions of the pullulan and heteropolysaccharide fractions were a function of glucose concentration, with the pullulan content of the total polysaccharide rising from 20% at 2.5 mM glucose to 45% at 20 mM glucose. Elaboration of pullulan by both cells and protoplasts was sensitive to 0.6 M KCl, which was present as the osmotic stabilizer in protoplast experiments. The presence of KCl resulted in a shift in the pH optimum to a more acidic value. The molecular weight of the protoplast-derived pullulan was sharply reduced from the molecular weight of the whole-cell-derived product. Exposure of the protoplasts to proteolytic enzymes had no effect on polysaccharide elaboration.

Simplified microassay for pullulan synthesis.

A simple radiometric microassay for extracellular polysaccharide elaboration by yeast-like cells of Aureobasidium pullulans was developed, based upon a procedure originally described by Catley (FEBS Lett. 20:174-176, 1972). Incorporation of [C]glucose into pullulan was linear with respect to time and cell dose. The pH and temperature optima for elaboration were 5.3 and 30 degrees C, respectively. Polysaccharide elaboration declined linearly with culture age.

New Method of Producing Protoplasts of Aureobasidium pullulans.

A rapid and relatively inexpensive method for producing protoplasts of the black yeast Aureobasidium pullulans is described. The procedure involves anaerobic incubation with the lytic preparation Driselase.