Immunohistochemical Characterization of Sarcomas in Trp53+/- Haploinsufficient Mice.

The use of immunohistochemical (IHC) staining in determining and/or confirming the cellular origin of poorly differentiated sarcomas was evaluated in this study. Sarcomatous neoplasms were evaluated in a research study conducted in 2 strains of p53+/- haploinsufficient mice. The most common neoplasms were undifferentiated sarcomas, followed by osteosarcomas and rhabdomyosarcomas (RMSs). The RMSs were poorly differentiated and appeared similar to the pleomorphic, or adult type, RMS of humans. All sarcomas stained positive by IHC for the mesenchymal cell intermediate filament vimentin. The RMSs were identified by positive IHC staining for myogenin, a transcription factor specific to skeletal muscle. Osteosarcomas were easily identifiable on hematoxylin and eosin-stained slides; no generally accepted IHC stain specific for bone is presently available. Some of the undifferentiated sarcomas contained numerous macrophages that stained positive for F4/80, a macrophage marker; the positive-staining cells were considered to be infiltrating macrophages. One-third of the neoplasms observed in this study were associated with subcutaneous implanted electronic microchips used for animal identification. Based upon histopathologic evaluation and IHC staining, it was not possible to distinguish neoplasms associated with subcutaneous microchips from neoplasms not associated with microchips.

Extended release of adenovirus from silica implants in vitro and in vivo.

Despite promising preclinical results, the clinical benefits of cancer gene therapy have been modest heretofore. The main obstacle continues to be the level and persistence of gene delivery to sufficiently large areas of the tumor. One approach for overcoming this might entail extended local virus release. We studied the utility of silica gel monoliths for delivery of adenovirus to advanced orthotopic gastric and pancreatic cancer tumors. Initially, the biochemical properties of the silica-virus matrix were studied and nearly linear release as a function of time was detected. Virus stayed infective for weeks at +37 degrees C and months at +4 degrees C, which may facilitate storage and distribution. In vivo, extended release of functional replication deficient and also replication-competent, capsid-modified oncolytic viruses was seen. Treatment of mice with pancreatic cancer doubled their survival (P<0.001). Also, silica gel-based delivery slowed the development of antiadenovirus antibodies.

Inhibition of beta(2) integrin-mediated leukocyte cell adhesion by leucine-leucine-glycine motif-containing peptides.

Many integrins mediate cell attachment to the extracellular matrix by recognizing short tripeptide sequences such as arginine-glycine-aspartic acid and leucine-aspartate-valine. Using phage display, we have now found that the leukocyte-specific beta(2) integrins bind sequences containing a leucine-leucine-glycine (LLG) tripeptide motif. An LLG motif is present on intercellular adhesion molecule (ICAM)-1, the major beta(2) integrin ligand, but also on several matrix proteins, including von Willebrand factor. We developed a novel beta(2) integrin antagonist peptide CPCFLLGCC (called LLG-C4), the structure of which was determined by nuclear magnetic resonance. The LLG-C4 peptide inhibited leukocyte adhesion to ICAM-1, and, interestingly, also to von Willebrand factor. When immobilized on plastic, the LLG-C4 sequence supported the beta(2) integrin-mediated leukocyte adhesion, but not beta(1) or beta(3) integrin-mediated cell adhesion. These results suggest that LLG sequences exposed on ICAM-1 and on von Willebrand factor at sites of vascular injury play a role in the binding of leukocytes, and LLG-C4 and peptidomimetics derived from it could provide a therapeutic approach to inflammatory reactions.

Comparison between sol-gel-derived anatase- and rutile-structured TiO2 coatings in soft-tissue environment.

The bioactivity of the surface reactive TiO(2) coatings for medical implants can be locally modified by CO(2) laser processing to match with the properties of surrounding tissues. The TiO(2) coatings heat-treated at 500 degrees C exhibit in vitro bioactivity. With further CO(2) laser treatment they exhibit enhanced in vitro bioactivity. The aim of this in vivo study was to compare the performance of heat-treated anatase-structured TiO(2) coatings with preheat-treated and CO(2) laser-treated rutile-structured coatings in terms of their ability to attach soft connective tissues. The coatings were characterized with TF-XRD and AFM. TiO(2)-coated discs were implanted in rats. The samples were analyzed with routine histology, SEM-EDS, and TEM. In both groups, already at 3 days, soft connective tissues were in immediate contact with the surface. No thick crystalline CaP layer was detected by SEM-EDS, but a thin amorphous CaP layer was detected by XPS. No gap between the cell membrane and the coating could be observed in TEM pictures. No differences were observed between the anatase- and rutile-structured coatings in terms of tissue responses. Further studies are needed to verify if the tissues are adherent to the surface of the implant.

Phospholipid composition and external labeling of aminophospholipids of human En(a--) erythrocyte membranes which lack the major sialoglycorprotein (glycophorin A).

Erythrocytes of the rare human blood group En(a--) lack the major sialoglycoprotein, glycophorin A, and the cell population heterozygous for the En(a) antigen contain half the normal amount of glycophorin A. With such cells we have studied whether glycophorin A influences the phospholipid composition and the availability of aminophospholipids to external labeling reagents. We here demonstrate that the amounts of all phospholipids are closely similar in normal and variant membranes. However, using the amino-reactive reagent trinitrobenzenesulfonate, we show that phosphatidylethanolamine is more easily labeled in intact En(a--) cells as compared to normal cells, whereas phosphatidylethanolamine shows an intermediate labeling in En(a) heterozygous cells.

Effect of rifampin and tobacco smoking on the pharmacokinetics of ropivacaine.

OBJECTIVE: Our objective was to assess the effect of rifampin (INN, rifampicin) and tobacco smoking on the pharmacokinetics of ropivacaine. METHODS: A randomized, 2-phase, crossover study was performed in both a group of 10 healthy nonsmokers and a group of 8 healthy smokers. In both groups each subject ingested daily for 5 days either placebo or 600 mg rifampin. On day 6 each subject received intravenously over 30 minutes a single dose of 0.6 mg/kg ropivacaine. Ropivacaine, 3-hydroxyropivacaine (3-OH-ropivacaine), and (S) -2',6'-pipecoloxylidide (PPX) in venous plasma and urine were measured for up to 12 hours and 24 hours, respectively. Pharmacokinetic parameters were calculated with noncompartmental methods, and t tests were used for comparisons between the phases and between the smokers and nonsmokers. The electrocardiogram was monitored for 3 hours. RESULTS: There were no statistically significant differences in the area under the plasma concentration-time curve (AUC), plasma clearance (CL), or half-life (t(1/2)) of ropivacaine between the smokers and nonsmokers. However, smokers excreted in urine 31% more 3-OH-ropivacaine and 62% less PPX than nonsmokers did. Rifampin decreased the AUC of ropivacaine in nonsmokers by 52% and in smokers by 38%. In nonsmokers rifampin increased the CL of ropivacaine by 93% and shortened its t(1/2) by 25%. In smokers rifampin increased the CL of ropivacaine by 47% and shortened its t(1/2) by 20%. Rifampin decreased the urinary excretion of 3-OH-ropivacaine in nonsmokers by 74% and in smokers by 68%, and it increased the excretion of PPX by 97% and 158%, respectively. No clinically significant differences in the QTc times were found between the groups or treatments. CONCLUSIONS: Tobacco smoking increases the excretion of 3-OH-ropivacaine in urine, probably because of the increased cytochrome P450 (CYP) 1A2-mediated metabolism of ropivacaine, and decreases the excretion of CYP3A4-formed PPX in urine. Rifampin considerably increases the metabolism of ropivacaine to PPX and decreases the metabolism to 3-OH-ropivacaine in both nonsmokers and smokers.

Inhalation of tetranitromethane causes nasal passage irritation and pulmonary carcinogenesis in rodents.

Fischer 344 rats and B6C3F1 mice were exposed for 2 years to vapors of tetranitromethane at concentrations below (0.5 ppm) and slightly above (2 or 5 ppm) the current U.S. recommended occupational exposure limit. Under the conditions of exposure of 6 h/day, 5 days/week, tetranitromethane was found to cause mild irritation and hyperplastic lesions in the nasal passages, but not nasal cavity neoplasms were observed. In contrast, nearly all animals exposed to the higher TNM concentrations, and the majority of animals exposed to the lower concentrations developed alveolar/bronchiolar adenoma or carcinoma; squamous cell neoplasms of the lung also occurred in exposed rats. The extent of the lung tumor response, and the low concentrations of tetranitromethane required for this response, are unprecedented in National Toxicology Program (NTP) studies.

Summary of the National Toxicology Program benzidine dye initiative.

The benzidine dye initiative is a research program established by the National Toxicology Program to generate an integrated body of scientific information regarding the potential health risks associated with exposure to benzidine- and benzidine-congener-derived dyes. Because an in-depth evaluation of each of the hundreds of benzidine-congener-derived dyes was considered impractical, the research program was designed to study the metabolism and disposition, genetic toxicity, and in vivo toxicity and carcinogenicity of two primary benzidine congeners, 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine, and a select group of prototypical dyes derived from those amines. It was anticipated that by applying the basic information generated in these extensive studies, it would be possible to make regulatory decisions about other dyes after conducting only a minimal number of experiments such as studies of disposition and metabolism, and in vitro mutagenicity. This paper summarizes the results of studies conducted to evaluate the metabolism, disposition, mutagenicity, toxicity, and carcinogenicity of representative benzidine congeners and derived dyes.

Influence of sol and stage of spinnability on in vitro bioactivity and dissolution of sol-gel-derived SiO2 fibers.

The ability of the sol-gel-derived green state silica fibers to induce the formation of bone-like calcium phosphate (HCA) on their surfaces has not been studied earlier. Bioactive silica fibers provide alternatives for the design of novel products, e.g., as implants used in tissue guiding or bone repairs. In this study, dry spinning was used to prepare the sol-gel fibers. Different fibers with different bulk structures were prepared by changing the composition and controlling the stage of spinnability. Additionally, the influence of the aging time of the fibers on the bulk structure of the samples was investigated. Furthermore, the ability to form calcium phosphate was investigated in vitro in the simulated body fluid (SBF). Transmission electron microscopy was used to illustrate the bulk structure of the green state fibers and scanning electron microscopy to illustrate the formed calcium phosphate layer on the fibers. The fibers were additionally characterized by measuring the dissolution of the silica in the SBF. In vitro bioactive silica fibers were successfully prepared. The calcium phosphate layer was formed within 1-5 days in the best case. The structural stability and the in vitro bioactivity varied with the aging time expect in one case where practically stable fibers could be prepared. The concentration of silica released in the SBF had no direct connection with the HCA formation. The silica-rich gel layer was not observed on the fibers, but the structure of the fibers was suggested to have an important role in the HCA formation.

Surface properties of in vitro bioactive and non-bioactive sol-gel derived materials.

The acid-base properties of several in vitro bioactive (able to form bone mineral-like calcium phosphate on their surfaces) and non-bioactive sol-gel processed oxides are studied. The amount of Lewis acid sites was calculated from the pyridine adsorption using the Langmuir adsorption model. The Henry adsorption model was used in cases where no specific affinity between the adsorbent and the probe molecule was observed. The results were used to calculate the specific amounts of acidic and basic sites on SiO2- and TiO2-based materials. The zeta potential was measured for dip-coated TiO2 films, calcium- and phosphate-doped TiO2 films and for a non-bioactive Al2O3 film. Also, the calcium phosphate formation in simulated body fluid on in vitro bioactive TiO2 film was studied with zeta potential measurements. The results showed dependence on the negative surface charge and the important role of calcium adsorption in the beginning of the calcium phosphate formation. Surface topography of the films was investigated with atomic force microscopy, including a detailed analysis of the peak heights and distribution over cross sections. It was observed that in vitro bioactivity was strongly dependent on the nanoscale dimensions. Consequently, the in vitro calcium phosphate formation seems to be due to both the chemical interactions and the surface structure.

In vitro Ca-P precipitation on biodegradable thermoplastic composite of poly(epsilon-caprolactone-co-DL-lactide) and bioactive glass (S53P4).

Bioactive properties of composites containing poly(epsilon-caprolactone-co-DL-lactide) with molar ratio 96/4 and bioactive glass (BAG), S53P4, were tested in vitro. The glass content in the tested materials was 40, 60 or 70 wt%, and two granule size ranges (<45 and 90-315 microm) were used. The composites were analysed for their apatite-forming ability. This was determined as a function of time by the dissolution pattern of Si and Ca ions and structural changes on the specimen surfaces. Composite specimens were immersed in simulated body fluid at 37 degrees C for up to 6 months. The changes in Si and Ca concentrations of the immersion medium were determined with UV-Vis and atomic absorption spectrophotometry. The calcium phosphate precipitation and apatite formation were evaluated by scanning electron microscopy (SEM) and infra-red spectroscopy (IR) using the attenuated total reflectance (ATR) system. The SEM and SEM-EDX analysis of the depositions formed on the composite surfaces was in line with the changes in ion concentrations. The clearest results with IR were seen in the material containing 60 wt% small glass particles. The results indicate that composites containing over 40 wt% BAG granules are bioactive, and that a higher BAG surface area/volume ratio favors the apatite formation in vitro.

Toxicity of monochloroacetic acid administered by gavage to F344 rats and B6C3F1 mice for up to 13 weeks.

Groups of 20 rats and 20 mice of each sex were administered monochloroacetic acid (MCAA) once daily, 5 days per week, in water by gavage for up to 13 weeks. Doses used were 0, 30, 60, 90, 120, or 150 mg/kg for rats and 0, 25, 50, 100, 150, or 200 mg/kg for mice. Compound-related deaths occurred at the four highest dose levels in rats and at the highest dose level in mice. Mean body weights of treated groups of rats and mice surviving until the end of the study were similar to those of the controls. A dose-related increase in blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase, as well as a dose-related increase in the relative liver and kidney weights was observed in rats but not in mice. A dose-related increase in the incidence and severity of cardiomyopathy occurred in rats. This lesion may be related to the inhibition of heart mitochondrial aconitase activity. No compound-related lesions were observed in mice. The results of this study indicate that F344 rats are more sensitive than B6C3F1 mice; sexes within the species were equally sensitive. The no-observable-effect level was estimated as 30 mg MCAA/kg body weight for rats and 100 mg MCAA/kg body weight for mice.

Local induction of calcium phosphate formation on TiO2 coatings on titanium via surface treatment with a CO2 laser.

Sol-gel-derived TiO(2) coatings are known to promote bonelike hydroxyapatite formation on their surfaces in vitro and in vivo. Hydroxyapatite integrates into bone tissue. In some clinical applications, the surface of an implant is simultaneously interfaced with soft and hard tissues, so it should match the properties of both. A new method is introduced for treating the coatings locally in a controlled manner. The local densification of sol-gel-derived titania coatings on titanium substrates with a CO(2) laser was studied in terms of the in vitro calcium phosphate-inducting properties. CO(2)-laser-treated multilayer coatings were compared with furnace-fired coatings prepared with the same recipe and previously shown to be bioactive. Additionally, local areas of furnace-fired multilayer coatings (previously shown to be bioactive in vitro) were further laser-treated to achieve various properties in the same implant. Topological surface properties were examined with atomic force microscopy. The formation of hydroxyapatite was studied with Fourier transform infrared and scanning electron microscopy energy-dispersive X-ray analysis. The results show that calcium phosphate formation can be adjusted locally by laser treatment. Calcium phosphate is a bonelike hydroxyapatite. The local treatment of sol-gel-derived coatings with a CO(2) laser is a promising technique for creating implants with various properties to interface different tissues and a possible way of coating implants that do not tolerate furnace firing.

Bilateral hydronephrosis attributable to bilateral ureteral fibrosis in a cat.

A 4-year-old castrated male Burmese cat was evaluated because of nonregenerative anemia (PCV, 20%) and was found to have renal failure. Renal ultrasonography revealed bilateral hydronephrosis. Antegrade pyelography of the right kidney failed to indicate obstructive disease. Necropsy and histologic examination of the ureters revealed a markedly stenotic lumen and massive fibrosis of the mucosa. An etiologic agent could not be found.

Association between dental health and acute myocardial infarction.

Known risk factors for coronary heart disease do not explain all of the clinical and epidemiological features of the disease. To examine the role of chronic bacterial infections as risk factors for the disease the association between poor dental health and acute myocardial infarction was investigated in two separate case-control studies of a total of 100 patients with acute myocardial infarction and 102 controls selected from the community at random. Dental health was graded by using two indexes, one of which was assessed blind. Based on these indexes dental health was significantly worse in patients with acute myocardial infarction than in controls. The association remained valid after adjustment for age, social class, smoking, serum lipid concentrations, and the presence of diabetes. Further prospective studies are required in different populations to confirm the association and to elucidate its nature.

Biomimetic mineralization of partially bioresorbable glass fiber reinforced composite.

The aim of this study was to investigate the biomimetic mineralization on the surface of a glass fiber reinforced composite with partially resorbable biopolymer matrix. The E-glass fibers were preimpregnated with a novel biopolymer of poly(hydroxyproline) amide, and further impregnated in the monomer system of bis-phenyl glycidyl dimethacrylate (Bis-GMA)--triethylene glycol dimethacrylate (TEGDMA), which formed interpenetrating polymer networks (IPN) with the preimpregnation polymer. After light-initiated polymerization of the monomer system, the rhombic test specimens (n = 6) were immersed in the simulated body fluid (SBF) with the bioactive glass for 24 h, and then the apatite nuclei were allowed to grow for 1, 3, 5 and 7 days in the SBF. The control test specimens (n = 3) were immersed in SBF without the bioactive glass. According to the scanning electron microscope (SEM), a mineral layer was formed on the surface of all the specimens, which were immersed with bioactive glass. The layer was thickened by the prolonged immersion time to a uniform layer. The Ca/P atomic ratio of the mineral varied between 1.30 and 1.54 as analyzed by the energy dispersive X-ray analysis (EDXA). The Fourier transform infrared spectroscopy (FT-IR) spectra gave signals for the mineral, which are characteristic of both bone-like apatite and orthocalciumphosphate. In conclusion, the mineral layer was formed on the surfaces of the specimens by biomimetic mineralization, the mineral being a mixture of bone-like apatite, orthocalciumphosphate and other calcium phosphates.

Characterization of Glycophorin A and band 3 from Tn polyagglutinable erythrocytes.

The 2 types of erythrocytes from a person with persistent mixed-field polyagglutinability (Tn abnormality) were separated from each other by preparative cell electrophoresis. Surface labelling using the galactose oxidase/NaB3H4 technique followed by polyacrylamide gel electrophoresis showed a strong labelling in the glycophorin A region of Tn positive erythrocytes indicating exposed galactosyl N-acetyl/galactosaminyl residues. Tn positive cell membranes were labelled by the galactose oxidase/NaB3H4 technique and solubilized in non-ionic detergent. After chromatography on Helix pomatia lectin-linked Sepharose, glycophorin A was immunoprecipitated from the sugar eluate using specific antiserum. Glycophorin A from Tn negative cells and normal red blood cells did not bind to Helix pomatia lectin but to Lens culinaris lectin-Sepharose. Glycophorin A and band 3 were isolated by preparative gel electrophoresis from normal cells and the two red cell populations of the Tn individual. Pronase treatment of labelled glycophorin A followed by gel filtration revealed a more efficient proteolysis in molecules isolated from Tn positive cells. Mild alkaline treatment of galactose oxidase/NaB3H4 or periodate/NaB3H4 labelled glycophorin A liberated 3 different oligosaccharides from Tn positive cells. No significant difference was found between the oligosaccharides of band 3 protein from normal and Tn positive cells and the amounts of glycophorin A were identical in both cell types when determined by radioimmunoassay.