The Lack of Association between FCN2 Gene Promoter Region Polymorphisms and Dental Caries in Polish Children.

The aim of this study was to examine the association of single-nucleotide polymorphisms (SNPs) in the gene encoding ficolin-2 protein (FCN2 gene) at positions -986 (rs17514136), -602 (rs3124953), and -4 (rs3124952) with dental caries in Polish children. Two hundred and sixty Polish Caucasian children aged 15 years were enrolled in this study: 82 with "higher" caries experience (DMFT >5) and 178 with "lower" caries experience (DMFT ≤5). In addition, subjects with caries experience (DMFT ≥1) and caries-free subjects (DMFT = 0) were compared. FCN2 SNPs were genotyped with PCR-RFLP methods. There were no significant differences in the genotype, allele, or haplotype distributions in 3 analyzed SNPs of the FCN2 gene between children with "higher" and those with "lower" caries experience as well as between children with caries experience and caries-free children. In conclusion, we did not find any association of FCN2 promoter polymorphisms at positions -986, -602, and -4 with dental caries in Polish children.

MBL2, MASP2, AMELX, and ENAM gene polymorphisms and dental caries in Polish children.

OBJECTIVE: The aim of the study was to examine whether the MBL2 C(-290)G and G161A, MASP2 A359G, AMELX C287T and C522T, and ENAM C2452T polymorphisms are associated with dental caries. SUBJECTS AND METHODS: Genomic DNA of 95 Polish children with 'higher caries experience' (HC) and 84 subjects with 'lower caries experience' (LC) belonging to two age-groups (5 and 13 years old) was extracted from the buccal mucosa. SNPs were genotyped with PCR-RFLP methods. RESULTS: Among 5-year-old children, we found significantly higher percentage of subjects carrying MBL2 (-290)G allele in HC group compared with LC group (43.2%vs 17.6%, P = 0.023). MBL2 C(-290)G-G161A C-G haplotype was overrepresented in LC group in 5-year-olds (P = 0.01), while the opposite association was observed in 13-year-olds, where C-G was overrepresented in HC group (P = 0.028). In 5-year-old children, the frequency of MBL2 G-G haplotype was higher in HC group compared with LC subjects (P = 0.045), while the opposite association (with borderline significance) was observed in 13-year-old children (P = 0.057). SNPs in MASP2, AMELX, and ENAM were not associated with dental caries. CONCLUSION: MBL2 gene polymorphism is associated with caries experience in Polish children, but the direction of this association seems to be opposite in primary and permanent dentition.

Therapy of osteoporosis in patients with Crohn's disease: a randomized study comparing sodium fluoride and ibandronate.

BACKGROUND: Osteoporosis is a frequent complication in Crohn's disease. Although the efficacy of both sodium fluoride and aminobisphosphonates in postmenopausal osteoporosis has been investigated in long-term therapy studies, no long-term results are available regarding the effect of these agents in the management of osteoporosis in patients with Crohn's disease. METHODS: Eighty-four patients with Crohn's disease and pathological bone mineral density findings were randomized to receive either vitamin D3 (1000 IU) and calcium citrate (800 mg) daily (group A) or sodium fluoride (25 mg b.d., group B) or intravenous ibandronate (1 mg every 3 months, group C) in addition to daily calcium/vitamin D substitution. On admission to the study and after 12 and 27 months, patients underwent dual-energy X-ray absorptiometry and radiological examination of the spine. RESULTS: Sixty-eight patients completed the 1-year observation period and were available for the intention-to-treat analysis. No new vertebral fractures were diagnosed. In group A, lumbar bone density increased by 2.6% (P = 0.066, N.S.), in group B by 5.7% (P = 0.003) and in group C by 5.4% (P = 0.003). Therapy with sodium fluoride was associated with an increase in osteocalcin (N.S.), whereas administration of ibandronate was associated with a decrease in the resorption parameter, carboxy-terminal cross-linked type-I collagen telopeptide (P < 0.05). Both sodium fluoride and ibandronate resulted in significant decreases in the serum concentration of osteoprotegerin after 9 months (P < 0.001). CONCLUSIONS: The findings of the present study show that both sodium fluoride and ibandronate are effective in combination with calcium and vitamin D substitution in the management of osteopenia and osteoporosis in patients with Crohn's disease. Both agents are safe and well tolerated, and induce continuous increases in lumbar bone density.

Quantitation of Helicobacter pylori in dental plaque samples by competitive polymerase chain reaction.

AIM: To establish a competitive PCR (cPCR) assay for quantitation of H pylori organisms in dental plaque samples. METHODS: The cPCR co-amplified target H pylori DNA and a known amount of internal standard template in the same tube with the same primers directed to 0.86 kb DNA of H pylori. The internal standard was a synthesised DNA bearing the same primer recognition sites at two ends and a non-homologous core sequence as the target DNA fragment. Quantitation was based on determination of the relative, not absolute, amounts of the differently sized and [32P]-dCTP labelled products derived from H pylori DNA and the competitive internal standard after gel electrophoresis separation. RESULTS: A significant correlation between known amounts of H pylori added to dental plaque samples and the results of the cPCR was found, and a standard line was developed which allowed quantitation of H pylori in the plaque samples. cPCR was performed on supragingival plaque samples from 10 adult patients with H pylori infection in the stomach, and from five adults and six children without H pylori infection in the stomach. The ranges of H pylori numbers were 1-213 (median 25), 6-76 (10), and 4-94 (14) cells/mg of dental plaque in the three groups, respectively. CONCLUSIONS: cPCR is useful for quantitation of H pylori in supragingival dental plaque samples; however, the number of the organisms in dental plaque samples seems very low.

Characteristic distribution pattern of Helicobacter pylori in dental plaque and saliva detected with nested PCR.

The precise mode of transmission and the natural reservoir for Helicobacter pylori are unknown. PCR assays have proved to be highly sensitive and specific and are regarded as the method of choice for detecting H. pylori DNA in the oral cavity. The aim of this study was to investigate the prevalence and distribution of H. pylori in the oral cavity. Forty-two patients undergoing gastroscopy were investigated for the presence of H. pylori in dental plaque and saliva by nested PCR, and in the stomach by the 13C-urea breath test. Samples tested comprised dental plaque from molars, premolars and incisors and saliva. Two sets of primers homologous to the 860-bp fragment of H. pylori DNA, which have been shown previously to be highly sensitive and specific, were used for nested PCR. Eleven patients (26.2%) were infected with H. pylori in the stomach. H. pylori DNA was identified in dental plaque samples from 41 patients (97%) and in 23 saliva samples (55%). The prevalence in dental plaque from molars, premolars and incisors was 82%, 64% and 59%, with an odds ratio of 3.18, 1.24 and 1 (reference), respectively. In conclusion, H. pylori was present in the oral cavity of 97% of tested patients, with a characteristic distribution that was independent of the infection status of the stomach. Thus H. pylori may belong to the normal oral microflora.

Role of cholecystokinin in cholestyramine-induced changes of the exocrine pancreas.

This study was an investigation of the role of cholecystokinin (CCK) in the stimulatory action of cholestyramine on rat exocrine pancreas. Postprandial CCK release was significantly enhanced by acute administration of cholestyramine (12.7 +/- 1.8 vs 3.7 +/- 0.5 pmol/L in controls). Over four weeks, rats were fed either regular diet or diet containing 6% cholestyramine, and were treated with the specific CCK receptor antagonist L-364,718 (2 x 0.5 mg/kg body weight/day s.c.) or DMSO (vehicle for the antagonist). Cholestyramine significantly increased pancreatic weight and trypsin and chymotrypsin contents. L-364,718 abolished these effects. Concomitant administration of antagonist and cholestyramine elevated amylase content, compared to controls. CCK levels in fasted animals did not differ between the four groups. The effect of the same dose of L-364,718 on pancreatic enzyme depletion, induced by the protease inhibitor camostate, was studied in a control experiment. A single dose of camostate (200 mg/kg) caused a 44-68% decrease in enzyme content. L-364,718 reversed this effect for all enzymes. We conclude that CCK is the mediator of cholestyramine-induced pancreatic hypertrophy and increase in content of proteases. After long-term administration, the CCK receptor antagonist, in combination with cholestyramine revealed an agonistic effect on individual, pancreatic enzyme content.

Sequential quantitative radionuclide evaluation of mandibular bone graft repair.

We have developed a noninvasive method to derive quantitative radionuclide data from images of grafted beagle dog mandibles. This method includes an accurate means for localizing the graft in sequential radionuclide images. The resultant data permit quantitative confirmation of the progress of osseous repair in the grafted bones. With this method, it is possible to compare bone repair activity between experimental subjects and also between selected zones within individual bones and thus objectively define the pattern of repair that occurred in various anatomic regions of the grafted mandible.

Comparisons of electron beam dose measurements in water and polystyrene using various dosimeters.

A comparison has been made of central axis percent depth dose and absorbed dose in electron beams of 7.8 and 10.2 MeV, measured with devices of differing geometry and construction. Flat and cylindrical ionization chambers have been used as well as thin thermoluminescent dosimeters. The ionization chambers had walls of air equivalent or tissue equivalent plastic. Results indicate that central axis depth dose measurements are independent of measuring device. No significant difference was found among the various ionization chambers with air equivalent walls in the determination of absorbed dose. The dose determined by the tissue-equivalent wall chamber was about 3% higher than the dose determined by the other ionization chambers. Dose maximum on the central axis in water is about 4% greater than when this same quantity is calculated from data measured in polystyrene.

Increase of bone mineral density with sodium fluoride in patients with Crohn's disease.

BACKGROUND AND AIMS: Low bone density with an increased risk of vertebral fractures is a frequent complication in inflammatory bowel disease. Since the aetiology of osteopathia in these patients is different compared to postmenopausal or steroid-induced osteoporosis, no treatment strategy is established. Supplementation of calcium and vitamin D has been shown to prevent further bone loss, but no data are available showing the anabolic effect of sodium fluoride in Crohn's disease. METHODS: We carried out a one-year prospective clinical trial in 33 patients with chronic active Crohn's disease who were randomly assigned to receive either calcium (500 mg b.i.d.) and 1000 IU vitamin D3 only, or retarded-release sodium fluoride (25 mg t.i.d.) additionally. The diagnosis of Crohn's disease had been made at least two years ago, and all patients had received systemic high-dose steroid therapy during the previous year. Eleven of 15 patients who received calcium/vitamin D and 15 of 18 patients who additionally received sodium fluoride completed the study. The primary endpoint of the study was the increase of bone mineral density, measured by dual energy X-ray absorptiometry (DXA) after one year of treatment. Bone-specific alkaline phosphatase and osteocalcin were used as markers for bone turnover. RESULTS: In the calcium/vitamin D only group, bone density was not significantly changed after one year of treatment, whereas in the calcium/vitamin D/fluoride group, bone density of the lumbar spine increased from -1.39+/-0.3 (Z-score, mean +/- SEM) to -0.65+/-0.3 (P<0.05) after one year of treatment. Increase of bone density was positively correlated to the osteoblastic markers bone-specific alkaline phosphatase (r = 0.53) and osteocalcin (r = 0.43). CONCLUSIONS: Sodium fluoride in combination with vitamin D and calcium is an effective, well-tolerated and inexpensive treatment to increase lumbar bone density in patients with chronic active Crohn's disease and osteoporosis.

Topical anesthesia and topical toxicity of bupivacaine combined with hydroxypolyethoxydodecane.

The biological effects of adding hydroxypolyethoxydodecane (HPED) to N-butylpipecolic acid 2,6-xyiidide (bupivacaine) has been studied in animals and in man. The topical toxicity of bupivacaine was somewhat aggravated by HPED 0.25-1.0%, and the topical anesthetic effect was strongly potentiated both in animals and in man. It was concluded that HPED increased the absorption of bupivacaine after topical application. It was also found that D(+)-bupivacaine was more potent as a topical anesthetic than L(-)-bupivacaine.

Direct gene transfer into the colon using a double-balloon catheter.

BACKGROUND AND STUDY AIMS: In spite of the many advances that have been made in understanding the molecular basis for diseases, a major obstacle to the treatment of human disorders remains the inability to express genes at specific sites in vivo. Recent progress in gene transfer technology has provided access to a variety of recombinant gene products that can be applied in clinical medicine for therapeutic purposes. MATERIALS AND METHODS: In an animal model, we describe here the way in which a marker gene can be introduced into the colon using a double-balloon catheter. Cationic liposomes were used as vehicles to introduce DNA into the living organism. RSV-LacZ plasmid coding for the enzyme beta-galactosidase was used as a marker gene. Cells expressing beta-galactosidase can be stained using the chromogen X-gal. Positive cells show a blue coloration in the cytoplasm. RESULTS: Both absorptive cells and goblet cells were successfully transduced with the marker gene. No evidence of similar staining was observed in control animals receiving a control plasmid or liposomes alone. CONCLUSIONS: The method used is a simple, safe, and nontoxic way of delivering genes of interest to specific sites in the colon. Gene transfer may offer fresh potential for endoscopic interventions in colonic disease.

Colon epithelium can be transiently transfected with liposomes, calcium phosphate precipitation and DEAE dextran in vivo.

BACKGROUND: The introduction of recombinant DNA into cells is the initial step toward the development of gene therapy. It has been shown that cationic liposomes are useful vehicles to introduce DNA into colon epithelial cells in vivo. METHODS: In the present study we compared the efficacy of different nonviral transfection methods into the colon wall. In anesthetized rats, a double balloon catheter was advanced into the colon and a chloramphenicol acetyltransferase (CAT) reporter plasmid complexed to liposomes, mixed with DEAE dextran, or precipitated with calcium phosphate was instilled. Following 2 days CAT activity was determined in the transfected colon segments. RESULTS: DEAE dextran and liposomes were more effective than calcium phosphate, whereas naked DNA was not taken up by the colon epithelial cells. Reporter gene expression was dose-dependent. Expressing cell types did not differ utilizing the various transfection methods as judged by X-gal staining of colon sections after transfection with a LacZ reporter plasmid. CONCLUSION: These data indicate that in addition to liposomes, plasmid DNA mixed with DEAE dextran can be taken up by colon epithelial cells. This transfection techniques may prove useful in the development of gene therapy approaches for colon disease.

Helicobacter pylori in the oral cavity: high prevalence and great DNA diversity.

To test the hypothesis that Helicobacter pylori may be transmitted by the oral-oral route, we applied nested PCR and DNA sequencing to detect and analyze H. pylori DNA in the oral cavity of 20 adult patients undergoing endoscopy. Dental plaques of molars, premolars, and incisors and saliva were collected. Additional paraffin-embedded gastric biopsies were analyzed in four patients. Two sets of highly sensitive and specific primers, EHC-U/EHC-L and ET5-U/ET-5L directed to a 860-bp fragment of H. pylori DNA, were used in the nested PCR. Eight patients had an active infection in the stomach determined with the [13C]urea breath test and the other 12 were negative. Nested PCR showed that all 20 subjects (100%) were positive for H. pylori in the oral cavity. DNA sequencing demonstrated that all tested PCR products of the expected size from the oral samples have more than 97% identity with that from H. pylori type strain ATCC 43629. However, sequences differed in oral samples from different subjects as well as between different oral locations and gastric biopsies within the same individuals. In conclusion, the oral cavity may be a permanent reservoir for H. pylori and can harbor multiple H. pylori strains at the same time.

Liposome mediated gene transfer into the rat oesophagus.

BACKGROUND: Cancer of the oesophagus has so far eluded every attempt at pharmacological treatment. The recent advent of somatic gene therapy offers a new therapeutic approach to malignant tumours. AIM: To investigate whether and how gene transfer into the oesophagus can be achieved. METHODS: A LacZ reporter gene was used as marker and transferred into the oesophagus of rats using cationic liposomes. Gene transfer was achieved by either luminal instillation into a closed segment using a double balloon catheter, or by intramural injection through a needle. Expression of beta-galactosidase was monitored in the oesophagus and various control tissues by histochemistry, polymerase chain reaction (PCR), reverse transcriptase PCR, and Southern blotting. RESULTS: Up to 100 days after in vivo gene transfer beta-galactosidase activity could be demonstrated in the oesophagus. Following luminal application, the transgene was expressed in epithelial cells whereas intramural injection induced preferential expression in fibroblasts. CONCLUSION: In vivo gene transfer into the esophagus is feasible and safe, and the route of administration largely determines cell type specificity. This novel approach will enable in vivo studies of growth, differentiation, and malignant transformation in the oesophagus, and may open new avenues to the confinement of oesophageal malignancies.

Helicobacter pylori in dental plaque: a comparison of different PCR primer sets.

This study was designed to compare different primer sets for PCR analysis of H. pylori in the same series of 40 dental plaque samples. Three pairs of primers, HPU1/HPU2, HP1/HP2, and EHC-U/EHC-L, directed to the urease A gene, 16S rRNA gene, or 860-bp DNA of H. pylori, respectively, were used. Our results demonstrate that EHC-L/EHC-U were more specific and sensitive for H. pylori added to saliva or dental plaque than HPU1/HPU2 and HP1/HP2. The detection rates for H. pylori DNA in dental plaque samples from randomly selected adult patients from the Dental Clinic of the University of Ulm were 26.5% (9/34) for HPU1/HPU2, 78.9% (30/38) for HP1/HP2, and 100% (40/40) for EHC-U/EHC-L (P < 0.001). Nested PCR using primers directed to the 860-bp DNA of H. pylori further confirmed the presence of H. pylori DNA (40/40) in all these samples. Our results indicate that primers EHC-U/EHC-L are to be recommended for PCR detection of H. pylori in the oral cavity.

Technetium-99m radionuclide bone imaging for evaluating mandibular osseous allografts.

Sequential interpretation of osseous repair, more sensitive than with conventional radiography, is possible with a noninvasive, nondestructive radio-nuclide method. The method was used in the evaluation of the progress of osteogenic activity in mandibular bone grafts in 24 beagle dogs.

Direct gene transfer into the rat pancreas using DNA-liposomes.

BACKGROUND: Pancreatic cancer represents a malignancy with very poor clinical prognosis and limited therapeutic potential. Recent developments of gene transfer technology offer new therapeutic avenues by delivering recombinant genes directly into normal or neoplastic tissue in vivo. METHODS: Here we show that the LacZ marker gene, complexed to cationic liposomes, can be introduced into the pancreas by either intraductal or intra-arterial injection. Expression of the beta-galactosidase gene product was monitored by polymerase chain reaction and histochemistry. RESULTS: Up to 28 days after in vivo gene transfer, beta-galactosidase activity could be demonstrated in the pancreas. Intraductal application induced gene expression in lining duct cells preferentially. Twenty-four hours after intraductal injection of liposomes, a dose-dependent, transient increase in serum amylase levels was detected. Nevertheless, no histological signs of pancreatitis were evident. Intra-arterial injection resulted in beta-galactosidase expression in endothelial cells of intrapancreatic arteries, as well as in the spleen, lymph nodes and liver, but not in ductal cells of the pancreas. Only occasionally were acinar cells positive for blue staining by either type of treatment. CONCLUSION: These experiments demonstrate that in vivo gene transfer into the pancreas is feasible using DNA-liposome complexes. Furthermore, the route of administration largely determines cell type specificity and side-effects. This technique might have an impact for the development of gene therapy strategies for pancreatic diseases.

Liposome mediated in vivo gene transfer into different tissues of the gastrointestinal tract.

The possibility to transfer and express genetic material in mammalian cells represents a new approach to the treatment of genetic and acquired disorders. So far, most studies use in vitro techniques to introduce foreign DNA into cultured cells, followed by reintroduction of these genetically altered cells into living organisms. In the present study we demonstrate that the LacZ marker gene can be selectively delivered, by in vivo techniques, to various locations of the gastrointestinal tract. Genetic material was targeted to the stomach, the colon, the liver and the pancreas using cationic liposomes. For transfer into the stomach and colon an intraluminal application, in the liver a portal access and in the pancreas an intraductal infusion was chosen. 48 hours after administration, the LacZ gene product beta-galactosidase could be localized in these tissues by cytochemistry. These experiments suggest a new approach to study gastrointestinal physiology and may offer novel aspects for the treatment of gastrointestinal diseases.