TNFα promotes osteosarcoma progression by maintaining tumor cells in an undifferentiated state.

Chronic inflammation is frequently associated with tumorigenesis in elderly people. By contrast, young people without chronic inflammation often develop tumors considered independent of chronic inflammation but driven instead by mutations. Thus, whether inflammation has a significant role in tumor progression in tumors driven by mutations remains largely unknown. Here we show that TNFα is required for the tumorigenesis of osteosarcoma, the most common tumor in children and adolescents. We show that transplantation of AX osteosarcoma cells, which harbor mutations driving c-Myc overexpression and Ink4a-deficiency, in wild-type mice promotes lethal tumorigenesis accompanied by ectopic bone formation and multiple metastases, phenotypes seen in osteosarcoma patients. Such tumorigenesis was completely abrogated in TNFα-deficient mice. AX cells have the capacity to undergo osteoblastic differentiation; however, that activity was significantly inhibited by TNFα treatment, suggesting that TNFα maintains AX cells in an undifferentiated state. TNFα inhibition of AX cell osteoblastic differentiation occurred through ERK activation, and a pharmacological TNFα inhibitor effectively inhibited both AX cell tumorigenesis and increased osteoblastic gene expression and increased survival of tumor-bearing mice. Lethal tumorigenesis of AX cells was also abrogated in IL-1α/IL-1ß doubly deficient mice. We found that both TNFα and IL-1 maintained AX cells in an undifferentiated state via ERK activation. Thus, inflammatory cytokines are required to promote tumorigenesis even in mutation-induced tumors, and TNFα/IL-1 and ERK may represent therapeutic targets for osteosarcoma.

Relationship between attitudes toward oral health at initial office visit and compliance with supportive periodontal treatment.

OBJECTIVES: The objectives of the present study include investigation of the relationship between attitudes and desires with respect to oral health at initial office visit and compliance with supportive periodontal treatment (SPT) and identification of prognostic factors with respect to low-compliance with SPT. MATERIALS AND METHODS: Four hundred thirty-one patients were evaluated. Subjects completed a questionnaire concerning attitude and desire with respect to oral health and subjective symptoms prior to periodontal treatment. Survival probabilities of SPT were estimated by the Kaplan-Meier method and compared between answers for each item of the questionnaire via the Cox-Mantel test. Finally, a multivariate Cox proportional hazards regression model was constructed, which included age and gender. RESULTS: Greater than 95% of participants desired toothbrushing proficiency and lifelong retention of teeth at the initial office visit; however, the overall survival probabilities of SPT were only 52.7% after about 5 years. Patients exhibiting unfavourable attitudes toward oral health at the initial office visit, in comparison with those displaying favourable attitudes, exhibited greater tendency to abandon SPT. A Cox regression model revealed that lack of brushing on the gingival margin, non-use of an inter-dental brush or dental floss, non-use of fluoride toothpaste and frequent consumption of sugar-containing drinks were significant independent prognostic factors for low-compliance with SPT (p<0.05; Hazard ratios=2.27, 2.00, 2.56 and 2.06, respectively). CONCLUSIONS: Desire for satisfactory oral health is not related consistently to continuation of SPT. Unfavourable attitudes toward oral health were correlated to low-compliance with SPT. Clinicians may wish to establish methods for improvement of patient compliance employing behavioural approaches applicable to the attitudes of potential low-compliance individuals.

Manipulated mouse embryos as bioassay system for water quality control.

Mouse pronuclear stage embryos with intact slit zona pellucida (manipulated) were cultured in vitro until the hatched blastocyst stage in simplex optimized medium with higher K+ concentration (KSOM) prepared with three different water types: tap, deionized reverse osmosis (D-O) water and Milli-Q system (M-Q) water. The culture media were supplemented with or without protein and ethylenediaminetetraacetic acid (EDTA, disodium salt). The rates of hatched blastocysts were significantly affected (p < 0.01) by micromanipulation, protein supplement and water source. The water source has no influence (p > 0.05) on development in EDTA-supplemented protein-free culture media, whereas in EDTA- and protein-free culture media, the water quality significantly (p < 0.001) affected the rates of development, with higher rates in media prepared with M-Q water. The micromanipulated embryos showed higher sensitivity to the water quality (p < 0.01). It worth mentioning that the rates of hatched blastocysts in protein-free culture media were very low (0-7.5%). Furthermore, the three different water types were analysed by measuring the electrical conductivity, inorganic ions, total organic carbon and endotoxins to evaluate the purity. M-Q water showed the lowest levels of inorganic ion, total organic carbon and endotoxin concentrations. We concluded that manipulated mouse embryos are good system to evaluate the quality of water used in biological system.

Expression pattern of a newly recognized protein, LECT2, in hepatocellular carcinoma and its premalignant lesion.

Leukocyte cell-derived chemotoxin 2 (LECT2) is a recently isolated protein and has been shown to be synthesized by human hepatocytes. All hepatocytes show diffuse immunostaining for LECT2 within the cytoplasm. In the present study, an attempt was made to clarify the expression pattern of LECT2 in nine cases of low-grade malignant hepatocellular carcinoma (LGM-HCC) and five cases of advanced HCC and 19 cases of premalignant lesion, termed atypical hyperplasia (AH), using the indirect immunoperoxidase technique. Variable spotty to coarsely diffuse staining in the majority of cells, a mixture of positively staining and negatively staining areas, and essentially negative staining was observed within the cellular cytoplasm of AH, LGM-HCC and advanced HCC, respectively. The expression of LECT2 became weaker with the progression of multistep hepatocarcinogenesis. The data clearly demonstrate that LECT2 becomes essentially negative in full-blown HCC cells and that the histological distinction between AH and LGM-HCC is valid. It also seems likely that LECT2 is related to hepatocyte growth.

Influences of retrieval stages and glutathione addition on post-thaw viability of quick frozen mouse morula during in vitro culture.

Effects of the embryo retrieval stages and addition of glutathione (GSH) on post-thaw development of mouse morula were evaluated in 2 consecutive experiments. In the first experiment, 1-, 2-, 3- to 4- and 5- to 8-cell stage embryos were collected and cultured to the morula stage in Whitten's medium containing 0.1 mM ethylenediaminetetraacetic acid (EDTA). The development rate of 1-cell embryos to the morula stage was lower than that of the other stages (P<0.01). The post-thaw development rate of the morulae obtained from in vitro culture of 1-, 2-, 3- to 4-, and 5- to 8-cell embryos and from in vivo embryos (control) to the blastocyst stage was 55.5, 84.9, 87.4, 90.1 and 90.8%, respectively. The post-thaw development rate of morula obtained from in vitro produced 1-cell embryos was significantly lower than from the other stages or from the in vivo counterparts (P<0.0001). In Experiment 2, the impact of GSH supplementation of the culture medium in the presence or absence of EDTA was evaluated for embryo development to the morula stage and post-thaw survival, using in the 2 x 2 factorial design. Although EDTA supplementation increased development rates to the morulae (P<0.01) stage, GSH did not have an influence on morula development. However, the presence of either GSH or EDTA in the culture medium supported development to the blastocyst stage (P<0.01) of in vitro produced morulae. These data demonstrate that 1-cell embryos from a blocking-strain mouse cultured in vitro to the morula stage have a lower development rate following freezing and thawing than embryos collected at the 2-cell or later stages. Addition of EDTA or GSH, individually or in combination, to the culture medium may improve the development rate of morula to blastocyst stage following cryopreservation.

Electroejaculation and semen characteristics of the captive Hokkaido brown bear (Ursus arctos yesoensis).

An electroejaculation technique was applied to the Hokkaido brown bear (Ursus arctos yesoensis) for semen collection and characterization of their seminal traits. Ten captive sexually mature bears were anesthetized and subjected to 21 electroejaculation trials during their mating season in 1995 and 1996. Spermic electroejaculates were recovered from 6 of the 10 bears (14 of 21 trials). The semen was characterized by serous fluid of semitransparent white color and a neutral pH. The mean values of ejaculate volume, sperm concentration, percentage of sperm motility, percentage of live spermatozoa, and percentage of pleiomorphic forms were 2.7 ml, 471.6 x 10(6) cells/ml, 80.2%, 89.7% and 21.8%, respectively. Although there was considerable variation among the seminal traits of the individual bears, the electroejaculation technique was effective in obtaining ejaculates from captive bears.

A delayed-implantation-associated protein prevents resumption of DNA synthesis by the trophectoderm of delayed-implanting mouse blastocysts in vitro.

Objective of this study was to determine the ability of a delayed-implantation-associated protein (MW 170,000, DIAP170K) to inhibit DNA synthesis by mouse blastocysts. Mice were ovariectomized on day 3 of pregnancy and treated with daily injections with 1 mg progesterone till day 7 to induce delayed implantation. Blastocysts were collected on day 8 with or without a single injection of 25 ng estradiol-17 beta on day 7 that activates blastocyst metabolisms (activated blastocysts and delayed-implanting blastocysts respectively). DNA synthesis was determined by measuring [3H]thymidine incorporation by blastocysts. DIAP170K at 10 micrograms/m/ suppressed resumption of DNA synthesis by delayed-implanting blastocysts and suppression was maximal at 50 micrograms/m/. However, DIAP170K did not affect DNA synthesis by blastocysts obtained on day 5 of pregnancy (normal blastocysts) and activated blastocysts. Resumption of DNA synthesis in the inner cell mass (ICM) and trophectoderm from delayed-implanting blastocysts was then separately assessed. DNA synthesis resumed in the trophectoderm of intact blastocysts during 24-hr culture but not in the trophectoderm cultured apart from the ICM. DIAP170K inhibited the resumption of DNA synthesis by the trophectoderm of intact delayed-implanting blastocysts but did not affect DNA synthesis by the ICM. In conclusion, DIAP170K inhibits resumption of DNA synthesis by trophectoderm of delayed-implanting blastocysts. This action of DIAP170K may play a central role in maintaining, but not achieving, dormancy of DNA synthesis by delayed-implanting blastocysts in mice.

In vitro viability of mouse oocytes vitrified in an ethylene glycol-based solution.

Ovulated mouse oocytes denuded of their cumulus cells, were vitrified in a solution containing 7 M ethylene glycol as the sole cryoprotectant using one or two steps of exposure before vitrification and were diluted in 1 M sucrose solution in 5 or 10 min after warming. The results proved that the viability of oocytes are detrimentally affected by exposure to the vitrification solution even without vitrification. At 5 min dilution time, the two-step exposure was superior to the one-step in terms of the post-warming recovery rate of vitrified oocytes with normal morphology and their subsequent development to the blastocyst stage (p < 0.01) after fertilization in vitro. At 10 min dilution time, no significant difference between one- or two-step exposure was found. The effect of the addition of 0.5 M sucrose to the vitrification solution was also determined and did not result in a significant improvement in the viability of oocytes vitrified in one-step and diluted for 10 min. In conclusion, the results in this study indicate that oocytes can be vitrified with 7 M ethylene glycol as the sole cryoprotectant in the vitrification solution, and that the recovery of normal oocytes after one-step exposure in the vitrification solution can be improved by 10 min dilution time. However, the improvement in the recovery rate of oocytes with normal morphology and their subsequent developmental in vitro was not improved by the addition of 0.5 M sucrose to the vitrification solution.

Differentiative potential of a mouse parthenogenetic embryonic stem cell line revealed by embryoid body formation in vitro.

The in vitro differentiative potential of mouse parthenogenetic (PG) embryonic stem (PGES) cells were investigated in the formation of embryoid bodies (EBs). EBs derived from PGES cells retarded in growth and showed restricted differentiation compared to their fertilized counterpart. In chimeric EBs from the aggregation of PGES and fertilized ES cells, morphological examination revealed that PGES cells were reduced in their population and distributed in endodermal layer as culture periods proceeded. These findings were comparable to those in aggregation chimeras of fertilized and PG embryos, and suggest that the differentiation of PGES cells in vitro is restricted in the formation of EBs.

Trisomy 8 does not affect differentiative potential in a murine parthenogenetic embryonic stem cell line.

Murine parthenogenetic embryonic stem (ES) cell lines expressing lac Z reporter gene were isolated after co-transfection with lac Z reporter gene (pENL) and neo gene (pSTneo) to TMA-48P cell line of 129/Sv origin. Karyotype analyses showed that all of four transfected cell lines examined contained 41 chromosomes with trisomy 8. Bacterial neo transgene required for G418 selection were integrated into several chromosomes including chromosome 8. Histological studies of teratomas formed in syngenic mice and embryoid bodies grown in vitro showed that the differentiative potential remained almost identical in chromosomally normal parental cell line and its derivative cell lines trisomic for chromosome 8.

Effects of glutamine, glycine and taurine on the development of in vitro fertilized bovine zygotes in a chemically defined medium.

The effects of glutamine, glycine and taurine on the development of bovine zygotes derived from IVM/IVF were determined using a protein-free chemically defined medium. After the cumulus cells were removed at 18 hr post-insemination, the presumptive zygotes were cultured for 4 or 6 days (about 104 or 154 hr) under a gas atmosphere of 5% O2: 5% CO2: 90% N2. A modified synthetic oviduct fluid medium supplemented with 20 amino acids (1 mM glutamine, essential amino acids for basal medium Eagle and non-essential amino acids for minimum essential medium), insulin, and PVA was used as a basic medium (mSOFai). Omitting 1 mM glutamine from mSOFai did not affect the embryonic development after 4 and 6 days of culture. After 4 days of culture, no significant effects of glycine and taurine on the development of zygotes to the morula stage were observed. However, supplementation with glycine or taurine significantly (P < 0.05) affected, with no interaction, the embryonic development to blastocysts after 6 days of culture. Addition of 5 mM glycine and 2 or 10 mM taurine significantly (P < 0.05) increased the percentage of blastocysts. The mean cell number in the blastocysts was affected by the glycine level, and was increased by the addition of 10 mM glycine (P < 0.001). These results demonstrate that glycine and taurine in a chemically defined medium containing a group of essential and non-essential amino acids improve the development of bovine zygotes to the blastocyst stage under 5% O2.

Effect of oxygen concentration in the gas atmosphere during in vitro insemination of bovine oocytes on the subsequent embryonic development in vitro.

In vitro matured bovine oocytes were co-incubated with sperm for 18 hr in a droplet of fertilization medium under a gas atmosphere of 5% CO2 with 5 or 20% O2. After removing the cumulus cells, they were fixed to examine their fertilization rate, or cultured for another 154 hr in a chemically defined medium under 5% O2 to determine their development to the blastocyst stage. There was no difference between the 5 and 20% O2 groups in the fertilization rate. However, the percentage of inseminated oocytes which developed to the blastocyst stage was higher when in vitro insemination was conducted under 5% O2 compared with that under 20% O2 (34.4 vs. 24.7%, P < 0.05).

Current status of vitrification of embryos and oocytes in domestic animals: ethylene glycol as an emerging cryoprotectant of choice.

The cryopreservation of mammalian embryos has become an integral part of methods to control animal reproduction. Numerous vitrification solutions have been formulated with ethylene glycol in combination with macromolecules, sugars and other cryoprotective agents. These indicate that a study of ethylene glycol as a cryoprotectant of choice in vitrification studies would be promising. To understand the cryobiology of ethylene glycol, several factors have to be studied. These are: cryoprotectant toxicity, osmotic stress and temperature at exposure. Understanding these factors could lead to the formulation of vitrification protocols that would lead to higher viability rates after cooling. First, ethylene glycol must be used as the sole cryoprotectant in a solution without macromolecules and sugars. Second, partial dehydration and permeation prior to cooling to subzero temperatures must be studied to achieve accurate exposure and a one-step dilution method. Third, the toxic effects of ethylene glycol must be overcome without sacrificing its vitrification properties by combining step-wise exposure at appropriate temperatures, low concentration and decreased volume. Fourth, the long-term effects of ethylene glycol on exposed or vitrified embryos must be determined. Lastly, the influence of culture on the viability of vitrified embryos must be studied to improve viability rates after warming.

In vitro viability of mouse zygotes vitrified in ethylene glycol.

A study was made to determine if mouse zygotes can be effectively vitrified in 7 M ethylene glycol in modified Dulbecco's phosphate buffered saline (PB1) and to find out if the development of vitrified-warmed zygotes in vitro can be improved by renewing the culture medium. The results showed that without medium change, vitrification reduced the development of zygotes to the expanded blastocyst stage (p < 0.01). With medium change, the development rate of vitrified-warmed zygotes exposed in 7 M ethylene glycol for 1 or 2 min was similar to that of unvitrified zygotes. However, prolonged exposure (5 min) markedly reduced the development rates of vitrified-warmed zygotes to the expanded blastocyst stage (p < 0.05). When the zygotes were vitrified in 7 M ethylene glycol and diluted at 18 degrees C to 22 degrees C, a slower efflux of ethylene glycol from the cell might have occurred, leading to a toxic effect of ethylene glycol in culture. The development rates of vitrified embryos cultured with medium change at 24 hr did not significantly differ from the untreated control (89.0% vs 96.5%). In conclusion, this study showed that mouse zygotes can be vitrified in 7 M ethylene glycol in PB1 and that changing the culture medium can improve the in vitro development rates of vitrified-warmed zygotes to the expanded blastocyst stage.

Functional enucleation of mouse metaphase II oocytes with etoposide.

Mouse metaphase II (M II) oocytes were exposed to 50 micrograms/microliters etoposide (ETO) before and after parthenogenetic activation with 7% ethanol and they were washed with 0.75 M sucrose. The ETO treated parthenogenetically activated oocytes were cultured or fused to single blastomeres of late 2-cell stage mouse embryo to test their ability to support development in vitro. In parallel untreated parthenogenetically activated oocytes were cultured to serve as control. None of ETO treated oocytes developed beyond the 2-cell stage, whereas 4% of the reconstituted embryos and 35% of control developed to blastocysts. It is concluded that mouse M II oocytes can be functionally enucleated by ETO treatment and can be used for nuclear transfer experiments.

Hepatitis C virus is frequently coinfected with serum marker-negative hepatitis B virus: probable replication promotion of the former by the latter as demonstrated by in vitro cotransfection.

Patients with hepatitis C have been reported occasionally to be coinfected with serum marker-negative (silent) hepatitis B virus (HBV). The frequency and significance of such coinfection were investigated. Thirty patients with hepatitis C virus (HCV) infections (10 acute, 10 chronic, 10 cirrhotic) were selected randomly; the acute cases were without serum hepatitis B surface antigen (HBsAg) and anti-hepatitis B core IgM, and the chronic cases were without HBsAg. A nested polymerase chain reaction for the X open reading frame was used to amplify HBV DNA in serum, and immunoperoxidase staining was carried out on liver biopsy specimens. Nucleotide sequencing was carried out to characterize the amplified HBV DNAs. In order to clarify the possibility that the silent HBV mutant promotes HCV replication in the liver, the full-length HCV RNA and the cloned silent HBV DNA dimer were cotransfected into an established cell line, HuH-7, and the amount of secreted HCV RNA was quantified serially. The target HBV DNA was amplified in 26 (86.7%) of the 30 patients. Subsequent direct nucleotide sequencing in 9 selected patients revealed an 8-nucleotide deletion, characteristic of a silent HBV mutant. Immunostaining revealed hepatitis B surface antigen in 15 (50.0%). Cotransfected silent HBV DNA augmented the secretion of HCV RNA by up to 5-fold in comparison with HCV RNA transfection alone. In conclusion, HCV is coinfected frequently with the silent HBV mutant and the latter probably promotes the replication of the former in the liver.

In vitro viability of mouse 8-cell embryos vitrified in a simple solution of ethylene glycol.

A study was made to determine if ethylene glycol (EG) can be used in a simple solution to vitrify mouse 8-cell embryos and to determine the critical factors that affect its success. Mouse 8-cell embryos were vitrified after exposure to 2M and 7M EG prepared in Dulbecco's phosphate buffered saline (PBS) with 10% heat-inactivated calf serum (CS). Mouse 8-cell embryos exposed to 2M EG for 2, 5 and 10 min, and to 7M EG for 2 and 5 min had survival rates similar to the untreated controls (93.3-100%). No significant difference in their survival rates in vitro was observed. Higher room temperatures (> 24 degrees C) at exposure before cooling resulted in poor development rates to the blastocyst stage. The survival rates of embryos vitrified after 2 min in 7M EG at 18-22 degrees C room temperature did not differ significantly from the control, but embryos vitrified after 5 min had significantly lower survival rates (p < 0.0001). In conclusion, effective vitrification of mouse 8-cell embryos can be achieved by initial exposure to 2M EG for 2-10 min followed by equilibration in 7M EG for 2 min at 18-22 degrees C room temperature. This study has shown that 7M EG in PBS with 10% CS is sufficient to provide cryoprotection of vitrified mouse 8-cell embryos and that exposure of the embryos to the vitrification solution at temperatures over 24 degrees C is critical to their subsequent development in vitro.

Effect of encapsulation on development of mouse pronuclear stage embryos in vitro.

Pronuclear stage embryos with intact (ZI), slit (ZS) or completely removed (ZF) zona pellucida were encapsulated with an artificial zona pellucida (AZP) made of 1.5% sodium alginate. Embryos were cultured in KSOM medium with or without protein and their development in vitro to the blastocyst stage was recorded. AZP significantly (P < 0.05) improved the development of embryos to the blastocyst stage regardless of the presence of the natural zona pellucida. The encapsulated embryos developed at a higher rate (P < 0.05) in the absence of protein as compared with non-encapsulated embryos. Furthermore, the cell contacts at the 4-cell stage were significantly improved (P < 0.05) with encapsulation. AZP improved (P < 0.05) the development of pronuclear stage embryos with a slit zona pellucida to morula and blastocyst stages as compared with ZS embryos. It is concluded that AZP improves the in vitro development of pronuclear stage embryos with intact or completely removed zona pellucida as well as micromanipulated embryos to the blastocyst stage.

Developmental ability of mouse late 2-cell stage blastomeres fused to chemically enucleated oocytes in vitro.

The effects of different concentrations of etoposide and cycloheximide (ETO-CHXM), used for chemical enucleation of mouse oocytes, on polar body extrusion and chromatin expulsion were tested. The developmental ability of blastomeres of late 2-cell stage embryos fused to chemically enucleated oocytes of different ages or cytoplasts from different sources was also examined in vitro. Metaphase I oocytes cultured in different concentrations of ETO-CHXM (10-50 micrograms/ml/each) extruded polar bodies at rates similar to those cultured without ETO-CHXM (58.5-65.9% and 64.6%, respectively). However, low percent of the oocytes (1.7-6.2%) expressed signs of meiotic perturbation, which was manifested by blebbing of the cytoplasmic membrane and extrusion of two or more polar body-like fragments. Twenty-three percent of the chemically enucleated oocytes cultured in ETO-CHXM-free medium spontaneously fused to their polar bodies. The rates of total chromatin expulsion were similar when ETO-CHXM concentrations were 36 and 50 micrograms/ml (93.5 and 98%, respectively). The results also showed that the cleavage rates of reconstituted embryos were significantly (P < 0.001) affected by the age of the chemically enucleated oocytes. Cytoplasts of bisected oocytes that matured in vivo supported the development of 31.7% of the reconstituted embryos to the blastocyst stage. However, both cytoplasts of chemically enucleated oocytes and in vitro matured oocytes did not support the development to the blastocyst stage. A high percentage (85.5%) of the reconstituted embryos with chemically enucleated recipients displayed abnormality of the metaphase plate. These results suggest that concentrations of etoposide between 36 and 50 micrograms/ml are optimum for enucleation of mouse oocytes. Furthermore, increasing the age or reducing the cytoplasmic volume of the chemically enucleated oocytes did not improve the development of the reconstituted embryos to the blastocyst stage.

Stimulation of the development of bovine embryos by insulin and insulin-like growth factor-I (IGF-I) is mediated through the IGF-I receptor.

To study the effects of insulin and insulin-like growth factor-I (IGF-I) on the development of bovine embryos, fertilized bovine embryos in vitro were cultured in a chemically defined, protein-free medium: modified synthetic oviduct fluid (mSOF) supplemented with 1 mg/ml polyvinyl alcohol. Dose-response studies showed that insulin (0.5 to 10 microg/ml) and IGF-I (2 to 200 ng/ml) stimulated the development of bovine embryos to the morula stage 5 d after in vitro fertilization. The addition of 0.5 microg/ml insulin or 2 ng/ml IGF-I to the mSOF had beneficial effects on embryonic development to the morula stage in the presence of amino acids, but insulin and IGF-I did not affect the development of bovine embryos to the morula stage in the absence of amino acids. The antiIGF-I receptor antibody (alphaIR-3) completely blocked the stimulation of development to the morula stage by insulin and IGF-I. These findings suggest that the stimulation of embryonic development by insulin and IGF-I is mediated through the IGF-I receptor.