Comparison of the Effect of two Denture Cleansers on Tensile bond Strength of a Denture Liner.

STATEMENT OF PROBLEM: One of the most clinical challenging issues in prosthodontics is debonding of soft liners from the denture base. PURPOSE: The aim of this study was to evaluate and compare tensile bond strength between soft liner and heat-cured acrylic resin when immersed in two different types of denture cleanser and distilled water, at different period of times. MATERIALS AND METHOD: In this experimental in vivo study, 238 heat-cured acrylic blocks were made. A soft liner was embedded between the acrylic blocks. Samples were divided into four groups: 17 samples were in the control group and were not soaked in any solution .The remaining samples were divided into 3 groups (Distilled water, Calgon and Fittydent). Each group was then subdivided into two subcategories, regarding the immersion time variable; 15 and 45 minutes. All samples were placed in tension force and tensile bond strength was recorded with the testing machine. One- way ANOVA and Tucky HSD post-hoc test were adopted to analyze the yielded data (α> 0.05). RESULTS: Specimens which were immersed in two denture cleansers (Fittydent and Calgon) and in distilled water showed significant difference (p= 0.001) in bonding strength when compared to the control group. The subjects immersed in denture cleanser solutions and distilled water did not reveal any significant difference (p= 0.90). For all groups; most of the bonding failures (72%) were cohesive type. CONCLUSION: The effect of the denture cleansers and distilled water on the bond strength was not statistically different; however, the difference was significant between the immersed groups with the non-immersed group. Moreover, type of the denture cleanser did not show any effect on the tensile strength. The tensile strength increases with time of immersion.

Evaluation of cellular toxicity of three denture base acrylic resins.

OBJECTIVE: This study aimed to evaluate the cellular toxicity of two newly-released acrylic resins (Futura Gen and GC Reline Hard) in comparison with the conventional heat-cure resin (Meliodent). MATERIALS AND METHODS: Sample discs from each acrylic resin were placed in 24-well culture plates along with L929 mouse fibroblast cell line. A mixture of the RPMI 1640 medium, antibiotics and 10% FBS was added to the plates and the specimens were incubated in a CO(2) incubator. The amount of light absorption by each plate was determined after 1 hour, 24 hours and 1 week by the MTT assay and ELISA. The cytotoxic effect of the resins was compared among groups using the two-way ANOVA and further paired comparison was performed using the post-hoc Tukey's test. RESULTS: After 1 hour, Futura Gen displayed a significantly lower level of light absorption in comparison with Meliodent (P=0.03). After 24 hours, GC Reline Hard rendered a significantly lower level of light absorption compared to Meliodent (P=0.02). After one-week of incubation, the mean absorption rates for GC Reline Hard, Meliodent and Futura Gen were relatively similar (P>0.05). The lowest and highest level of cytotoxicity among all resins were observed after one week and 1 hour of immersion in water, respectively. CONCLUSION: All the tested resins induced some degree of cytotoxicity. Cytotoxicity of Futura Gen, GC Reline Hard and Meliodent resins failed to show any significant reduction from 24 hours to one week. Thus, it is recommended to immerse the dentures in water for 24 hours prior to delivery to the patient.

Serum immunoglobulin response and protection from homologous challenge by Proteus mirabilis in a mouse model of ascending urinary tract infection.

We tested the hypothesis that experimental Proteus mirabilis urinary tract infection in mice would protect against homologous bladder rechallenge. Despite production of serum immunoglobulin G (IgG) and IgM (median titers of 1:320 and 1:80, respectively), vaccinated (infected and antibiotic-cured) mice did not show a decrease in mortality upon rechallenge; the survivors experienced only modest protection from infection (mean log(10) number of CFU of P. mirabilis Nal(r) HI4320 per milliliter or gram in vaccinated mice versus sham-vaccinated mice: urine, 100-fold less [3.5 versus 5.5; P = 0.13]; bladder, 100-fold less [3.1 versus 5.1; P = 0.066]; kidneys, 40-fold less [2.7 versus 4.3; P = 0.016]). Western blots using protein from the wild-type strain and isogenic mutants demonstrated antibody responses to MR/P and PMF fimbriae and flagella. There was no correlation between serum IgG or IgM levels and protection from mortality or infection. There was a trend toward elevated serum IgA titers and protection from subsequent challenge (P >/= 0.09), although only a few mice developed significant serum IgA levels. We conclude that prior infection with P. mirabilis does not protect significantly against homologous challenge.

Secretion of Ipa proteins by Shigella flexneri: inducer molecules and kinetics of activation.

The type III Mxi-Spa secretion machinery of Shigella flexneri is responsible for secretion of Ipa proteins, which are involved in the entry of bacteria into epithelial cells. Ipa proteins accumulate within bacteria growing in laboratory media, and their secretion is activated upon contact of bacteria with eukaryotic cells. In this study, we have identified a group of chemical compounds, including Congo red, Evans blue, and direct orange, which are able to induce secretion of Ipa proteins by bacteria suspended in phosphate-buffered saline. Parameters of kinetics of activation of Ipa secretion by Congo red were determined by measuring by enzyme-linked immunosorbent assay the amount of IpaC secreted and by investigating the increase in susceptibility of Ipa proteins to proteinase K degradation. Ipa secretion occurred at 37 degrees C, was obtained with 5 to 10 microM Congo red, and was complete within 30 min. In addition, activation of Ipa secretion by Congo red was observed with bacteria harvested throughout the exponential phase of growth but not with bacteria in the stationary phase. The interactions of Congo red and Congo red-related compounds with the Mxi-Spa secretion apparatus might be specific hydrophobic interactions similar to those involved in binding of Congo red to amyloid proteins.

Proteus mirabilis mannose-resistant, Proteus-like fimbriae: MrpG is located at the fimbrial tip and is required for fimbrial assembly.

The mannose-resistant, Proteus-like (MR/P) fimbria, responsible for mannose-resistant hemagglutination, is a virulence factor for uropathogenic Proteus mirabilis. Based on known fimbrial gene organization, we postulated that MrpG, a putative minor subunit of the MR/P fimbria, functions as an adhesin responsible for hemagglutination, while MrpA serves as the major structural subunit for the filamentous structure. To test this hypothesis, an mrpG mutant was constructed by allelic-exchange mutagenesis and verified by PCR and Southern blotting. The mrpG mutant was found to be negative for hemagglutination, while wild-type strain H14320 and the complemented mutant were positive. Western blots with antiserum raised against an overexpressed MrpG'-His6 fusion protein showed that MrpG was present in the fimbrial preparations of both the wild-type strain and the complemented mutant but absent in that of the mrpG mutant. The mrpG mutant was significantly less virulent in a CBA mouse model of ascending urinary tract infection. Western blots with antiserum to whole MR/P fimbriae showed that MrpA protein was also missing from the fimbrial preparation of the mrpG mutant. Using immunogold electron microscopy, we found that the normal MR/P-fimbrial structure was absent in the mrpG mutant, suggesting that MrpG is essential for initiation of normal fimbrial formation. In the wild-type strain, MrpG protein was localized to the tips of the fimbriae or at the surface of the cell when antiserum raised against overexpressed MrpG was used. Given the tip localization, MrpG may be required for initiation of assembly of MR/P fimbriae but does not appear to be the fimbrial adhesin.

Secretion of Shigella flexneri Ipa invasins on contact with epithelial cells and subsequent entry of the bacterium into cells are growth stage dependent.

Upon contact with the surface of epithelial cells, Shigella flexneri secretes Ipa proteins through the Mxi-Spa type III secretion apparatus. Among the Ipa proteins, IpaB and IpaC form a soluble complex in the bacterial supernatant which appears to be sufficient to initiate the cellular rearrangements necessary to achieve bacterial entry. Here, we provide the first evidence that efficiency of bacterial entry into cells depends on the stage of bacterial growth. Bacteria in the early phase of exponential growth are six times more invasive than those in the stationary phase. The entry efficiency of the bacteria present on the cell surface appears to correlate with the percentage of those that are able to secrete their invasins. This suggests that the capacity to activate the Mxi-Spa apparatus is a major factor in the regulation of bacterial entry efficiency. Consistent with these observations, we have further shown that bacteria which have reached the stage of division secrete Ipa proteins more often than those that have not. Also, initial secretion occurs essentially in the area of the septation furrow. The Ipa proteins, secreted in the vicinity of the septation furrow, seem to initiate the early stages of reorganization of the host cell cytoskeleton.

Construction of an MR/P fimbrial mutant of Proteus mirabilis: role in virulence in a mouse model of ascending urinary tract infection.

Proteus mirabilis, a cause of acute pyelonephritis, produces at least four types of fimbriae, including MR/P (mannose-resistant/Proteus-like) fimbriae. To investigate the contribution of MR/P fimbriae to colonization of the urinary tract, we constructed an MR/P fimbrial mutant by allelic exchange. A 4.2-kb BamHI fragment carrying the mrpA gene was subcloned into a mobilizable plasmid, pSUP202. A 1.3-kb Kanr cassette was inserted into the mrpA open reading frame, and the construct was transferred to the parent P. mirabilis strain by conjugation. Following passage on nonselective medium, 1 of 500 transconjugants screened was found to have undergone allelic exchange as demonstrated by Southern blot. Colony immunoblot, Western immunoblot, and immunogold labeling with a monoclonal antibody to MR/P fimbriae revealed that MrpA was not expressed. Complementation with cloned mrpA restored MR/P expression as shown by hemagglutination, Western blot, and immunogold electron microscopy. To assess virulence, we challenged 40 CBA mice transurethrally with 10(7) CFU of wild-type or mutant strains. After 1 week, geometric means of log10 CFU per milliliter of urine or per gram of bladder or kidney for the wild-type and mutant strains were as follows: urine, 7.79 (wild type) versus 7.02 (mutant) (P = 0.035); bladder, 6.22 versus 4.78 (P = 0.019); left kidney, 5.02 versus 3.31 (P = 0.009); and right kidney, 5.28 versus 4.46 (P = 0.039). Mice challenged with the wild-type strain showed significantly more severe renal damage than did mice challenged with the MR/P-negative mutant (P = 0.007). We conclude that MR/P fimbriae contribute significantly to colonization of the urinary tract and increase the risk of development of acute pyelonephritis.

Proteus mirabilis MR/P fimbrial operon: genetic organization, nucleotide sequence, and conditions for expression.

Proteus mirabilis, an agent of urinary tract infection, expresses at least four fimbrial types. Among these are the MR/P (mannose-resistant/Proteus-like) fimbriae. MrpA, the structural subunit, is optimally expressed at 37 degrees C in Luria broth cultured statically for 48 h by each of seven strains examined. Genes encoding this fimbria were isolated, and the complete nucleotide sequence was determined. The mrp gene cluster encoded by 7,293 bp predicts eight polypeptides: MrpI (22,133 Da), MrpA (17,909 Da), MrpB (19,632 Da), MrpC (96,823 Da), MrpD (27,886 Da), MrpE (19,470 Da), MrpF (17,363 Da), and MrpG (13,169 Da). mrpI is upstream of the gene encoding the major structural subunit gene mrpA and is transcribed in the direction opposite to that of the rest of the operon. All predicted polypeptides share > or = 25% amino acid identity with at least one other enteric fimbrial gene product encoded by the pap, fim, smf, fan, or mrk gene clusters.

Proteus mirabilis fimbriae: identification, isolation, and characterization of a new ambient-temperature fimbria.

Urinary tract infections involving Proteus mirabilis may lead to complications including bladder and kidney stones, acute pyelonephritis, and bacteremia. This bacterium produces a number of fimbriae, two of which, MR/P fimbria and P. mirabilis fimbria, have been shown to contribute to the ability of this pathogen to colonize the bladder and kidney. We have now purified and characterized a previously undescribed fimbria of P. mirabilis, named ambient-temperature fimbria (ATF). Electron microscopy of a pure preparation and immunogold labeling of cells demonstrated that ATF was fimbrial in nature. The major fimbrial subunit of ATF has an apparent molecular weight of 24,000. The N-terminal amino acid sequence, E-X-T-G-T-P-A-P-T-E-V-T-V-D-G-G-T-I-D-F, did not show significant similarity to that of any previously described fimbrial protein. ATF was expressed by all eight P. mirabilis strains examined. Culture conditions affected expression of ATF, with optimal expression observed in static broth cultures at 23 degrees C. This fimbria was not produced by cells grown at 42 degrees C or on solid medium. Expression of ATF did not correlate with mannose-resistant/Proteus-like (MR/P) or mannose-resistant/Klebsiella-like (MR/K) hemagglutination and represents a novel fimbria of P. mirabilis.

Proteus mirabilis fimbriae: N-terminal amino acid sequence of a major fimbrial subunit and nucleotide sequences of the genes from two strains.

Proteus mirabilis, a common cause of urinary tract infection in hospitalized and catheterized patients, produces mannose-resistant/klebsiella-like (MR/K) and mannose-resistant/proteus-like (MR/P) hemagglutinins. The gene encoding the major structural subunit of a fimbria, possibly MR/K, was identified in two strains. A degenerate oligonucleotide probe based on the N terminus of the Proteus uroepithelial cell adhesin and antiserum raised against the denatured polypeptide were used to screen a cosmid gene bank of strain HU1069. A cosmid clone that reacted with the probe and antiserum was identified, and a fimbria-like open reading frame was determined by nucleotide sequencing. The predicted N-terminal amino acid sequence of the processed polypeptide, ENETPAPKVSSTKGEIQLKG (residues 23 to 42), did not match the uroepithelial cell adhesin N terminus but, rather, matched exactly the N-terminal amino acid sequence of a polypeptide with an apparent molecular size of 19.5 kDa isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a fimbrial preparation from strain HI4320 expressing MR/K hemagglutinin. By using an oligonucleotide from the HU1069 open reading frame, the fimbrial gene was isolated and sequenced from a cosmid gene bank clone of strain HI4320. A 552-bp open reading frame predicts a 184-amino-acid polypeptide including a 22-amino-acid hydrophobic leader sequence. The unprocessed polypeptide is predicted to be 18,921 Da; the processed polypeptide is predicted to be 16,749 Da. The predicted amino acid sequence of the polypeptide encoded by the gene, designated pmfA, displayed 36% exact matches with the mannose-resistant fimbrial subunit encoded by smfA of Serratia marcescens but only 15% exact matches with the predicted sequence encoded by mrkA of Klebsiella pneumoniae.

Proteus mirabilis MR/P fimbriae: molecular cloning, expression, and nucleotide sequence of the major fimbrial subunit gene.

Proteus mirabilis, a cause of serious urinary tract infection and acute pyelonephritis, produces several putative virulence determinants, among them, fimbriae. Principally, two fimbrial types are produced by this species: mannose-resistant/Proteus-like (MR/P) fimbriae and mannose-resistant/Klebsiella-like (MR/K) fimbriae. To isolate MR/P fimbrial gene sequences, a P. mirabilis cosmid library was screened by immunoblotting and by hybridization with an oligonucleotide probe based on the N-terminal amino acid sequence of the isolated fimbrial polypeptide, ADQGHGTVKFVGSIIDAPCS. One clone, pMRP101, reacted strongly with a monoclonal antibody specific for MR/P fimbriae and with the DNA probe. This clone hemagglutinated both tannic acid-treated and untreated chicken erythrocytes with or without 50 mM D-mannose and was shown to be fimbriated by transmission electron microscopy. A 525-bp open reading frame, designated mrpA, predicted a 175-amino-acid polypeptide including a 23-amino-acid hydrophobic leader peptide. The unprocessed and processed polypeptides are predicted to be 17,909 and 15,689 Da, respectively. The N-terminal amino acid sequence of the processed fimbrial subunit exactly matched amino acid residues 24 to 43 predicted by the mrpA nucleotide sequence. The MrpA polypeptide shares 57% amino acid sequence identity with SmfA, the major fimbrial subunit of Serratia marcescens mannose-resistant fimbriae.

Proteus mirabilis flagella and MR/P fimbriae: isolation, purification, N-terminal analysis, and serum antibody response following experimental urinary tract infection.

Urinary tract infection with Proteus mirabilis may lead to serious complications, including cystitis, acute pyelonephritis, fever, bacteremia, and death. In addition to the production of hemolysin and the enzyme urease, fimbriae and flagellum-mediated motility have been postulated as virulence factors for this species. We purified mannose-resistant/proteuslike (MR/P) fimbriae and flagella from strains CFT322 and HU2450, respectively. Electron microscopy revealed highly concentrated preparations of fimbriae and flagella. Fimbrial and flagellar structural subunits were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 18.5 and 41 kDa, respectively. N-terminal sequencing revealed that 10 of the first 20 amino acids of the major MR/P subunit matched the sequence of the P. mirabilis uroepithelial cell adhesin N terminus and 11 of 20 amino acids matched the predicted amino acid sequence of the Escherichia coli P fimbriae structural subunit, PapA. In addition, 90 and 80% homologies were found between the first 20 amino acids of P. mirabilis flagellin and those of Salmonella typhimurium phase-1 flagellin and the E. coli hag gene product, respectively. An enzyme-linked immunosorbent assay using purified antigens showed a strong reaction between the MR/P fimbriae or flagella and sera of CBA mice challenged transurethrally with P. mirabilis. A possible role for MR/P fimbriae in the pathogenesis of urinary tract infection is supported by (i) a strong immune response to the antigen in experimentally infected animals, (ii) amino acid sequence similarity to other enteric surface structure, and (iii) our previously reported observation that MR/P fimbriae are expressed preferentially as the sole fimbrial type in human pyelonephritis isolates.