Osteopontin Deficiency Suppresses Tumor Necrosis Factor-α-Induced Apoptosis in Chondrocytes.

OBJECTIVE: Apoptosis of chondrocytes in articular cartilage has been observed in rheumatoid arthritis patients. However, molecules involved in such chondrocyte apoptosis in arthritic joints have not been fully understood. We previously observed that apoptosis of chondrocytes is enhanced in a murine arthritis model induced by injection with anti-type II collagen antibodies and lipopolysaccharide (mAbs/LPS), and osteopontin (OPN) deficiency suppresses chondrocyte apoptosis in this arthritis model in vivo. To understand how OPN deficiency renders resistance against chondrocyte apoptosis, we examined the cellular basis for this protection. DESIGN: Chondrocytes were prepared from wild-type and OPN-deficient mouse ribs, and tumor necrosis factor (TNF)-α-induced cell death was examined based on lactate dehydrogenase (LDH) release assay and TUNEL assay. RESULTS: TNF-α treatment induced LDH release in wild-type chondrocytes, while OPN deficiency suppressed such LDH release in the cultures of these cells. TNF-α-induced increase in the number of TUNEL-positive cells was observed in wild-type chondrocytes, while OPN deficiency in chondrocytes suppressed the TNF-α induction of TUNEL-positive cells. OPN deficiency suppressed TNF-α-induced increase in caspase-3 activity in chondrocytes in culture. Furthermore, OPN overexpression in chondrocytes enhanced TNF-α-induced apoptosis. CONCLUSION: These results indicated that the presence of OPN in chondrocytes is involved in the susceptibility of these cells to TNF-α-induced apoptosis.

Rapid detection of the cariogenic pathogens Streptococcus mutans and Streptococcus sobrinus using loop-mediated isothermal amplification.

INTRODUCTION: Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. In this study, we developed a rapid, sensitive method for detecting these major cariogenic pathogens using loop-mediated isothermal amplification (LAMP). The assay procedure is quite simple: the amplification is carried out in a single tube under isothermal conditions at 63 degrees C, and the result can be obtained in less than 1 h. METHODS: Initially, a set of six primers was designed by targeting S. mutans-specific and S. sobrinus-specific regions, identified using the genomic subtractive hybridization technique. We evaluated the specificities and sensitivities of these assays. Furthermore, we detected and quantified these bacteria in saliva and carious dentin from eight children. RESULTS: The sensitivities of the S. mutans-specific and S. sobrinus-specific LAMP methods, examined using agarose gel electrophoresis, were each one cell for a 30-min reaction. The detection limits using real-time turbidimetry analysis were 1 to 10(7) cells (3.28 x 10(1) to 3.28 x 10(8) fg S. mutans template DNA) per reaction tube and 1 to 10(5) cells (2.72 x 10(3) to 2.72 x 10(8) fg S. sobrinus template DNA) per reaction tube. Using these assays, we detected and quantified these cariogenic bacteria for evaluation of the LAMP assay for clinical diagnosis. CONCLUSIONS: Our results suggest that the LAMP-based assay in combination with subtractive hybridization is valuable for preparing species-specific primers for closely related species. Furthermore, the LAMP-based assay will be a useful tool for the rapid and sensitive prediction of dental caries.

Rapid detection of Actinobacillus actinomycetemcomitans using a loop-mediated isothermal amplification method.

INTRODUCTION: Actinobacillus actinomycetemcomitans has been implicated in the etiology of aggressive periodontitis. In this study, we applied a novel nucleic acid amplification method, called loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions, allowing the rapid detection of A. actinomycetemcomitans. METHODS: We designed the primers for detecting A. actinomycetemcomitans and evaluated the specificity and sensitivity of the assay. RESULTS: The LAMP primers used in this study successfully amplified serotypes a-e of A. actinomycetemcomitans, while other oral bacteria were not amplified. By measuring the precipitation of magnesium pyrophosphate, we could quantify the chromosomal DNA of A. actinomycetemcomitans. The detection limits using the real-time turbidimetry analysis were 5.8 x 10(2)-5.8 x 10(7) copies of A. actinomycetemcomitans template DNA per reaction tube. In addition, the LAMP assay was used for the rapid detection of A. actinomycetemcomitans in clinical specimens from eight individuals. The results with the LAMP method were similar to those using conventional polymerase chain reaction. CONCLUSION: Our results suggest that the LAMP-based assay is very useful for the rapid detection of A. actinomycetemcomitans.

Loop-mediated isothermal amplification method for the rapid detection of Enterococcus faecalis in infected root canals.

INTRODUCTION: Enterococcus faecalis is a major pathogen in the etiology of apical periodontitis after root canal treatment. A loop-mediated isothermal amplification method, which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand-displacement activity, was developed for the rapid detection of E. faecalis in clinical specimens from root canals. METHODS: Primers for detecting E. faecalis from the azoA gene were designed. The specificity of this assay was evaluated using various oral bacteria and the sensitivity was evaluated using serially diluted E. faecalis chromosomal DNA. In addition, loop-mediated isothermal amplification assays were applied to the rapid detection of E. faecalis from endodontic samples. RESULTS: The loop-mediated isothermal amplification products amplified with the primer set were specific for E. faecalis. To confirm the specificity of the amplicon, the amplified products were digested with the restriction endonuclease Sau3AI. The lower detection limit of the E. faecalis primer set without the loop primer set was 10 microg/tube for a 50-min loop-mediated isothermal amplification reaction. Using loop primers increased the detection sensitivity by several orders of magnitude. Furthermore, E. faecalis was detected with the loop-mediated isothermal amplification assay in four root canals from 18 individuals and the detection results were consistent with those of conventional polymerase chain reactions. CONCLUSION: These results indicate that the loop-mediated isothermal amplification assay is very useful for rapid detection of E. faecalis and diagnosis of endodontic infection.

Use of loop-mediated isothermal amplification of the IS900 sequence for rapid detection of cultured Mycobacterium avium subsp. paratuberculosis.

We evaluated the usefulness of loop-mediated isothermal amplification (LAMP) in detecting specific gene sequences of Mycobacterium avium subsp. paratuberculosis (MAP). A total of 102 primer sets for LAMP was designed to amplify the IS900, HspX, and F57 gene sequences of MAP. Using each of two primer sets (P-1 and P-2) derived from the IS900 fragment, it was possible to detect MAP in a manner similar to that used with nested PCR. The sensitivity of LAMP with P-1 was 0.5 pg/tube, which was more sensitive than nested PCR. When P-2 was used, 5 pg/tube could be detected, which was the same level of sensitivity as that for nested PCR. LAMP with P-1 was specific. Although only 2 Mycobacterium scrofulaceum strains out of 43 non-MAP mycobacterial strains were amplified, the amplification reaction for these strains was less efficient than for MAP strains, and their products could be distinguished from MAP products by restriction digestion. LAMP with P-2 resulted in very specific amplification only from MAP, the same result obtained with nested PCR. Our LAMP method was highly specific, and the white turbidity of magnesium pyrophosphate, a by-product of the LAMP reaction, allowed simple visual detection. Our method is rapid, taking only 2 h, compared with 4 h for nested PCR. In addition, the LAMP method is performed under isothermal conditions and no special apparatus is needed, which makes it more economical and practical than nested PCR or real-time PCR. These results indicate that LAMP can provide a rapid yet simple test for the detection of MAP.

Accelerated reaction by loop-mediated isothermal amplification using loop primers.

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. We have developed a method that accelerates the LAMP reaction by using additional primers, termed loop primers. Loop primers hybridize to the stem-loops, except for the loops that are hybridized by the inner primers, and prime strand displacement DNA synthesis. Although both inner and loop primers react via the loops, they do so by different mechanisms. The LAMP method presented here uses loop primers to achieve reaction times of less than half that of the original LAMP method. Since the total time of analysis including detection is less than 1h, this new method should facilitate genetic analysis, including genetic diagnosis in the clinical laboratory.

Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.

The loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that uses only one type of enzyme. One of the characteristics of the LAMP method is its ability to synthesize extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced, yielding white precipitate of magnesium pyrophosphate in the reaction mixture. Judging the presence or absence of this white precipitate allows easy distinction of whether nucleic acid was amplified by the LAMP method. Since an increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized, real-time monitoring of the LAMP reaction was achieved by real-time measurement of turbidity.

Effects of tower climbing exercise on bone mass, strength, and turnover in growing rats.

To determine the effects of tower climbing exercise on mass, strength, and local turnover of bone, 50 Sprague-Dawley rats, 10 weeks of age, were assigned to five groups: a baseline control and two groups of sedentary and exercise rats. Rats voluntarily climbed the 200-cm tower to drink water from the bottle set at the top of it. In 4 weeks, the trabecular bone formation rate (BFR/bone surface [BS]), bone volume (BV/TV), and trabecular thickness (Tb.Th) of both the lumbar vertebra and tibia and the bone mineral density (BMD) of the tibia increased, while the osteoclast surface (Oc.S) decreased. The parameter values in the midfemur, such as the total cross-sectional area, the moment of inertia, the periosteal mineralizing surface (MS/BS), mineral apposition rate (MAR), BFR/BS, and bending load increased, while the endosteal MAR decreased. In 8 weeks, the increases in the bone mineral content (BMC), BMD of the femur and tibia, and the bending load values of the femur were significant, but the climbing exercise did not increase BMC, BMD, or the compression load of the lumbar vertebra. Although the periosteal MS/BS, MAR, and BFR/BS increased, the endosteal MS/BS, MAR, and BFR/BS decreased. These results show that climbing exercise has a beneficial effect on the femoral cortex and tibia trabecular, rather than the vertebral trabecular. In the midfemur, effects on bone formation are site specific, supporting accelerated cortical drift by mechanical stimulation.

Effects of resistance exercise training on mass, strength, and turnover of bone in growing rats.

To determine the effects of resistance exercise on mass, strength and local turnover of bone, 50 Sprague Dawley rats, 8 weeks of age, were assigned to five groups: a baseline control and two groups of sedentary and exercising rats. The trunk of the rats was kept upright during electrically stimulated jumping exercise for 1 h every other day. In 4 weeks, the trabecular mineralizing surface per bone surface (MS/BS), bone formation rate per bone surface (BFR/BS) and the compression load of the lumbar body increased and the number of osteoclasts decreased, but bone mineral density (BMD) and structure did not increase. In the mid femur, the cross-sectional area, the cortical bone area, the moment of inertia, the periosteal MS/BS, BFR/BS and the bending load increased in the exercise group. In 8 weeks, the increases in BMD, structure and load values were significant in both the lumbar and mid femur. At both 4 and 8 weeks, the MS/BS for the endocortical surface of mid femur were not increased and mineral apposition rate (MAR) remained reduced. These results show that jumping exercise increases the mass and strength of the lumbar vertebrae and mid femur by stimulating bone formation and accelerates cortical drift by both increasing periosteal bone formation and reducing the endocortical MAR.

Loop-mediated isothermal amplification of DNA.

We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 10(9) copies of target in less than an hour. The final products are stem-loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.

A comparison of resistance and aerobic training for mass, strength and turnover of bone in growing rats.

To determine the effects of resistance versus aerobic exercise on the mass, strength and turnover of bone. thirty Sprague Dawley rats (4 weeks of age) were assigned to one of three experimental groups: sedentary, running or jumping. In the jumping group, the trunk was kept upright during electrically stimulated jumping exercise for 1 h every other day. The running rats ran at speeds of 24 m/min for 1 h every other day. After 4 weeks, the jumping rats exhibited increases in the mass and strength of the lumbar vertebrae and of the mid-diaphysis of the femur (mid-femur), and increases in the cross-sectional morphology of these bones: the trabecular bone volume per bone surface, the trabecular thickness, the trabecular bone formation rate per bone surface (BFR/BS). In addition, they exhibited reduced trabecular separation and the area of osteoclast surface per bone surface. The running and sedentary rats showed no such changes. With regard to the mid-femur, in both the jumping and running rats the periosteal BFR/BS was increased. However, only the jumping rats showed a reduction in the BFR/BS at the endocortical surface. These results suggest that resistance exercise accelerates cortical drift and increases the bone mass and strength by stimulating bone formation more efficiently than does aerobic exercise.

Reactivity of a dual amplified chlamydia immunoassay with different serovars of Chlamydia trachomatis.

A study was undertaken with different serovars (D, E, F, L2, MoPn) of Chlamydia trachomatis to determine the analytical sensitivity of a new dual amplified immunoassay (IDEIA PCE Chlamydia) for detecting chlamydial lipopolysaccharide. IDEIA PCE Chlamydia incorporates a polymer conjugate consisting of multiple copies of antibody and enzyme molecules to provide signal amplification. The test was also assessed with different protein A producing strains of Staphylococcus aureus in order to assess whether the use of a multiple antibody conjugate increased nonspecific binding. The detection limits varied for each serovar with a detection limit of 38 IFU/ml obtained with serovar F and 237 IFU/ml obtained with serovar D. The incorporation of the polymer conjugate resulted in a 2-5 fold increase in analytical sensitivity compared to an earlier version of the test using a conventional conjugate. No increase in cross reactivity with protein A producing strains of S. aureus was obtained. The new dual amplified test format offers potential as a sensitive low-cost screening assay for C. trachomatis infections.

Minimum inhibitory and minimal lethal concentration against Chlamydia trachomatis dependent on the time of addition and the duration of the presence of antibiotics.

The purpose of this study was to investigate the properties of several antimicrobial agents found to be effective against Chlamydia trachomatis and to verify the eradication therapy schedule. The in vitro activities of two quinolones (sparfloxacin, ofloxacin), of three macrolides (azithromycin, erythromycin, clarithromycin) and of a tetracycline (doxycycline) against C. trachomatis were evaluated by several methods for the determination of the minimum inhibitory concentration (MIC) and minimal lethal concentration (MLC). MLC of azithromycin was only 2 times higher than that of MIC. On the other hand, MLCs of other antibiotics were 4-16 times higher than their respective MICs. When all antimicrobial agents were added to the infected culture at different times, we found that the quinolones even at a concentration of 64 microg/ml could not inhibit the formation of inclusion if they were added after 20 h from the start of infection. The corresponding period for macrolides and doxycycline was 24 h. When the antibiotics were removed at 8 h after the start of the infection, all antibiotics except azithromycin and clarithromycin were needed at a concentration much higher than their MLCs to inhibit the formation of inclusion. We consider macrolides, especially azithromycin, to be an excellent anti-C. trachomatis drug because of its lower MICs and MLCs values which were also closer together.

The inhibitory effect of phosphate on the ligase chain reaction used for detecting Chlamydia trachomatis.

AIMS: To examine the detection limit of the ligase chain reaction kit for Chlamydia trachomatis, to study the inhibitory effect of phosphate on the ligase chain reaction, and to clarify the mechanism of inhibition. METHODS: Three reference serovars of C trachomatis--D/UW-3/Cx, F/UW-6/Cx, and L2/434/Bu--were used to test the sensitivity of the chlamydia ligase chain reaction. Comparison was made of the inhibition by phosphate before and after DNA amplification. Phosphate in up to 2.4 mM concentration was added to specimens of C trachomatis serovar D (1 to 50 inclusion forming units (IFU)/reaction) before DNA amplification to examine the concentration dependency of phosphate inhibition of the ligase chain reaction. RESULTS: The detection limits were 0.6 IFU/reaction for serovar D/UW-3/Cx and F/UW-6/Cx, and 0.4 IFU/reaction for L2/434/Bu. Phosphate inhibited the ligase chain reaction only when it was added before the amplification stage. The specimens containing chlamydia at 1 to 50 IFU/reaction were negative when the concentration of phosphate added at the prethermocycle stage was more than 1.2 mM. CONCLUSIONS: Ligase chain reaction analysis is a reliable method of diagnosing C trachomatis infection because of its high sensitivity. It would be clearly superior to the currently used methods if the problem of inhibitors could be eliminated. The mechanism of inhibition of the ligase chain reaction by phosphate was thought to be blockade of the amplification of the target DNA. The efficacy of the ligase chain reaction could be inhibited by phosphate in the urine, so duplicate dilution analysis of some negative specimens should be useful.

[Basic evaluation of Chlamydia antigen detection by EIA using a dual amplification enhanced immunoassay method].

Chlamydia trachomatis is one of the important pathogens of STD in our country. Therefore, rapid accurate, reliable and convenient tests for its detection are required. So far, IDEIA Chlamydia has been employed as a useful diagnostic kit. Now, IDEIA PCE Chlamydia, applied as a dual amplification EIA method, has been developed. In our present studies, the sensitivity, reproducibility, cross reactivity, and reliability of IDEIA PCE Chlamydia were investigated and compared with those of IDEIA Chlamydia and LCR Chlamydia. The sensitivity of IDEIA PCE Chlamydia showed 2.4 x 10(2) IFU/ml for C. trachomatis D, 1.2 x 10(2) IFU/ml for C. trachomatis E, 3.8 x 10 IFU/ml for C. trachomatis F, and 1.25 x 10(2) IFU/ml for C. trachomatis L2. With regard to reproducibility, more than 2.4 x 10(2) IFU/ml of all strains of C. trachomatis and negative samples gave highly reproducible values. Though no cross reactivity was recognized among three strains of Staphylococcus aureus with concentrations of more than 10(9) IFU/ml, non-heated samples of over 10(6) CFU/ml showed cross reactivity. In our observations, phosphate, Mg2+, Ca2+, and Fe3+ inhibited the efficacy of both IDEIA and IDEIA PCE Chlamydia. Ca2+ per se could be an inhibitor in the case of urine samples analyzed by IDEIA and IDEIA PCE Chlamydia. These results indicate that IDEIA PCE Chlamydia kit for detection of C. trachomatis may be clinically useful because of its improved sensitivity over IDEIA Chlamydia and its invariable specificity and reliability.

Cretinism with combined hormone deficiency caused by a mutation in the PIT1 gene.

Cretinism is marked by irreversible mental and growth retardation. We describe here an entirely new case of cretinism showing combined pituitary hormone deficiencies of thyrotropin, growth hormone and prolactin that appears to be caused by homozygosity for a nonsense mutation in the gene for the pituitary specific transcription activator, Pit-1/GHF-1 (designated PIT1 in humans for pituitary specific factor 1). This is the first report in humans of a defect in a transcription activator causing deficiency of multiple target genes.

Nucleotide sequence of the complementary DNA for human Pit-1/GHF-1.

Human cDNA clones encoding Pit-1/GHF-1, a pituitary-specific DNA binding factor, were obtained by PCR following reverse transcription of human pituitary RNA. It is approx. 1.3 kb in size with 0.1 kb 5' non-coding region, 0.9 kb protein-coding region and 0.3 kb 3' non-coding region. The predicted human Pit-1/GHF-1 peptide structure has 291 amino acids and is highly conserved among mouse, rat and bovine. In addition, the 5' non-coding region is highly conserved with rat pit-1/GHF-1 sequence to the transcription start site.

Clinical significance of gene amplification studied in human neuroblastoma xenografts: relationship with tumor growth rate, chemotherapeutic sensitivities and levels of neuron-specific enolase.

Tumor doubling time, sensitivity to chemotherapeutic agents and concentrations of neuron-specific enolase were studied in nine human neuroblastoma xenografts, in which amplifications of N-myc, clones 8 and G21 were known; N-myc was amplified in eight, clone 8 in five and clone G21 in four of these nine xenografts. Tumor doubling time was longest in one xenograft, TNB10, which lacks the amplification of either N-myc or clone 8 or G21, and shortest in TNB1 in which all three DNA sequences are amplified with a DNA rearrangement in clone 8. No correlations were found between genomic amplification of N-myc, clones 8 and G21 and effectiveness of five chemotherapeutic drugs tested, except for cis-platinum. cis-Platinum was found to be effective on all but the one xenograft, TNB10, with the longest tumor doubling time. Concentration of neuron-specific enolase in tumor extract was lowest in TNB1 and correlated with the length of the tumor doubling time.

Radioimmunoassay development for human neuron-specific enolase: with some clinical results in lung cancers and neuroblastoma.

A double-antibody radioimmunoassay for human neuron-specific enolase (NSE) was developed, using rabbit antiserum against the gamma subunit of enolase purified from human brain. Intra-assay variance was 3.8-5.1% and inter-assay variance 4.3-7.3%, and recovery of NSE added to normal serum was 100.2% on average. Normal serum NSE levels for 451 adults ranged from 3.6 to 10.8 ng/ml (mean 6.6 ng/ml). Antibodies raised against the gamma gamma enolase isozyme did not cross-react with the alpha alpha and beta beta isozymes at concentrations of 1,000 ng/ml, but showed a cross-reactivity of 41.5% (theoretically 50%) with the alpha gamma isozyme. It was also shown that hemolysis of 160 mg/dl hemoglobin can add 5.73 ng/ml of NSE to the true level. The coefficient of correlation between the radioimmunoassay and the sandwich enzyme immunoassay [1] was 0.99 (n = 21), and values determined by the RIA were about twice those obtained by the EIA. Serum NSE was abnormally high in 42 of 52 patients (80.8%) with small cell lung carcinoma, and in all 38 children with neuroblastoma.