Efficacy and safety of weekly alendronate plus vitamin D3 5600 IU versus weekly alendronate alone in Korean osteoporotic women: 16-week randomized trial.

Vitamin D (vit-D) is essential for bone health, although many osteoporosis patients have low levels of 25-hydroxy-vit-D [25(OH)D]. This randomized, open-label study compared the effects of once weekly alendronate 70 mg containing 5600 IU vit-D3 (ALN/D5600) to alendronate 70 mg without additional vit-D (ALN) on the percent of patients with vit-D insufficiency [25(OH)D <15 ng/mL, primary endpoint] and serum parathyroid hormone (PTH, secondary endpoint) levels in postmenopausal, osteoporotic Korean women. Neuromuscular function was also measured. A total of 268 subjects were randomized. Overall, 35% of patients had vit-D insufficiency at baseline. After 16-weeks, there were fewer patients with vit-D insufficiency in the ALN/D5600 group (1.47%) than in the ALN group (41.67%) (p<0.001). Patients receiving ALN/D5600 compared with ALN were at a significantly decreased risk of vit-D insufficiency [odds ratio=0.02, 95% confidence interval (CI) 0.00-0.08]. In the ALN/D5600 group, significant increases in serum 25(OH)D were observed at weeks 8 (9.60 ng/mL) and 16 (11.41 ng/mL), where as a significant decrease was recorded in the ALN group at week 16 (-1.61 ng/mL). By multiple regression analysis, major determinants of increases in serum 25(OH)D were ALN/D5600 administration, seasonal variation, and baseline 25(OH)D. The least squares mean percent change from baseline in serum PTH in the ALN/D5600 group (8.17%) was lower than that in the ALN group (29.98%) (p=0.0091). There was no significant difference between treatment groups in neuromuscular function. Overall safety was similar between groups. In conclusion, the administration of 5600 IU vit-D in the ALN/D5600 group improved vit-D status and reduced the magnitude of PTH increase without significant side-effects after 16 weeks in Korean osteoporotic patients.

Vascular expression of the chemokine CX3CL1 promotes osteoclast recruitment and exacerbates bone resorption in an irradiated murine model.

Circulating osteoclast precursor cells highly express CX3C chemokine receptor 1 (CX3CR1), which is the only receptor for the unique CX3C membrane-anchored chemokine, fractalkine (CX3CL1). An irradiated murine model was used to evaluate the role of the CX3CL1-CX3CR1 axis in osteoclast recruitment and osteoclastogenesis. Ionizing radiation (IR) promoted the migration of circulating CD11b+ cells to irradiated bones and dose-dependently increased the number of differentiated osteoclasts in irradiated bones. Notably, CX3CL1 was dramatically upregulated in the vascular endothelium after IR. IR-induced production of CX3CL1 by skeletal vascular endothelium promoted chemoattraction of circulating CX3CR1+/CD11b+ cells and triggered homing of these osteoclast precursor cells toward the bone remodeling surface, a specific site for osteoclast differentiation. CX3CL1 also increased the endothelium-derived expression of other chemokines including stromal cell-derived factor-1 (CXCL12) and macrophage inflammatory protein-2 (CXCL2) by activating the hypoxia-inducible factor-1 α pathway. These effects may further enhance osteoclastogenesis. A series of in vivo experiments confirmed that knockout of CX3CR1 in bone marrow-derived cells and functional inhibition of CX3CL1 using a specific neutralizing antibody significantly ameliorated osteoclastogenesis and prevented bone loss after IR. These results demonstrate that the de novo CX3CL1-CX3CR1 axis plays a pivotal role in osteoclast recruitment and subsequent bone resorption, and verify its therapeutic potential as a new target for anti-resorptive treatment.

C-reactive protein induces G2/M phase cell cycle arrest and apoptosis in monocytes through the upregulation of B-cell translocation gene 2 expression.

We hypothesized that C-reactive protein (CRP) may affect the cell cycle and induce apoptotic changes of monocytes. CRP (∼25 µg/ml) significantly increased expressions of B-cell translocation gene 2 (BTG2) mRNA and protein in human monocytes through pathways involving CD32/NADPH oxidase 2/p53, which eventually induced G2/M phase arrest and apoptotic cell death. Such pro-apoptotic effect of CRP was not found in thioglycollate-elicited intraperitoneal monocytes/macrophages harvested from BTG2-knockout male C57BL/6 mice (n=5). Within atheromatous plaques obtained from CRP-transgenic male LDLR(-/-) C57BL/6 mice (n=5) and human coronary arteries, BTG2 co-localized with CRP, p53 and monocytes/macrophages. Therefore the pro-apoptotic pathway of CRP-CD32-Nox2-p53-BTG2 may contribute to the retardation of the atherogenic process.

The changes in plasma serotonin levels after hormone therapy and their relationship with estrogen responsiveness on bone in postmenopausal women.

CONTEXT: Selective serotonin reuptake inhibitors have shown to be associated with an increased risk of fractures. It has been suggested that circulating serotonin is an important regulatory factor and that estrogen may regulate bone metabolism through the serotonin pathway. OBJECTIVE: Our objective was to determine the association between plasma serotonin level and bone turnover before and after hormone therapy (HT) in postmenopausal women. PARTICIPANTS AND DESIGN: In this parallel comparative study using age-matched controls, 80 postmenopausal women (21 control, 59 receiving HT) aged 46-64 yr were assessed. The plasma levels of serotonin, serum concentrations of osteocalcin and carboxyterminal telopeptides, and bone mineral density (BMD) were measured at baseline and after 3 months and 1 yr of HT. RESULTS: The plasma serotonin level was significantly correlated with serum total alkaline phosphatase level at baseline (r = -0.223, P = 0.048) but not with serum osteocalcin (r = -0.217, P = 0.056) or carboxyterminal telopeptides (r = -0.217, P = 0.054). There was no significant association between baseline serotonin and BMD measured at the spine or femur. The median decrements of circulating serotonin from baseline were -9.3% (interquartile range -34.0 to 53.6%) and -7.2% (-25.5 to 64.5%) at 3 months and 1 yr of HT, respectively. These changes were not significantly different from those in the control group. The short-term changes of circulating serotonin at 3 months after HT did not show significant association with the changes in BMD measured at the lumbar spine or proximal femur 1 year after HT. CONCLUSIONS: Our results suggest that circulating serotonin may reflect bone turnover status, but it is not a strong enough predictor of bone loss to use as a bone marker. Moreover, serial measurements of plasma serotonin after short-term treatment with estrogen cannot predict the long-term responsiveness of bone to estrogen, suggesting that the bone-preserving effect of estrogen is independent of the peripheral action of serotonin on bone.

Nutritional risk factors of early development of postpartum prediabetes and diabetes in women with gestational diabetes mellitus.

OBJECTIVE: Early detection of prediabetes and diabetes after delivery helps prevent and delay the development of overt type 2 diabetes in women with gestational diabetes mellitus (GDM). We sought to identify modifiable risk factors for the early development of postpartum type 2 diabetes in women with GDM that may help establish interventions for preventing or delaying the subsequent onset of type 2 diabetes. METHODS: Three hundred eighty-one women who developed GDM during pregnancy were tested for 1) antepartum anthropometric and biochemical measurements, 2) pregnancy outcome, 3) oral glucose tolerance test at 6 to 12 wk after delivery, and 4) postpartum anthropometric, biochemical, and nutritional measurements. The subjects were divided into three groups on the basis of the postpartum oral glucose tolerance test results: normal glucose tolerance group (n=193), prediabetes (n=161), and diabetes (n=27). RESULTS: The incidences of postpartum prediabetes and diabetes at 6 to 12 wk follow-up in Korean women with GDM were 44.8% and 5.2%, respectively. Antepartum modifiable risk factors for developing type 2 diabetes at early postpartum included higher body mass index, lower ß-cell function, insulin dosage during late pregnancy, and the non-modifiable risk factor of family history of diabetes (R2=0.14). Postpartum risk factors included higher body mass index, serum triacylglycerols, hemoglobin A1c, and energy intake and lower insulin secretion capacity (R2=0.43). Animal fat intake was higher in the prediabetes and diabetes groups than in the normal glucose tolerance group, whereas breast-feeding did not alter the risk for the development of postpartum diabetes. CONCLUSION: This study strongly suggests that the development of postpartum type 2 diabetes in women with GDM can be prevented and/or delayed by lifestyle and nutritional intervention during antepartum and postpartum.

Quantitation of fatty acid ethyl esters in human meconium by an improved liquid chromatography/tandem mass spectrometry.

This paper reports the development and validation of an improved assay for quantitation of fatty acid ethyl esters (FAEEs) in human meconium using liquid chromatography/tandem mass spectrometry (LC-MS/MS). FAAEs (ethyl laurate, ethyl myristate, ethyl palmitate, ethyl palmitoleate, ethyl stearate, ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate) and the internal standard (I.S.), ethyl heptadecanoate, were separated by reverse phase HPLC and quantified by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ionization mode. The absolute recovery of FAEEs varied from 55+/-10% for 0.33 nmol/g (100 ng/g) of ethyl linoleate up to 86+/-8% for 1.55 nmol/g (500 ng/g) of ethyl miristate. The LODs and LOQs varied from 0.01 to 0.08 nmol/g and from 0.02 to 0.27 nmol/g, respectively. The assay has been successfully applied to examine the FAEE levels in 81 meconium samples from babies born to mothers reporting alcohol consumption, to varying degrees, during pregnancy.

Negative association between metabolic syndrome and bone mineral density in Koreans, especially in men.

Cardiovascular disease and osteoporosis are thought to share common risk factors, and metabolic syndrome (MS) is composed of major risk factors for cardiovascular disease. This study was performed to investigate the relationships between specific MS components and bone mineral density (BMD). BMD was measured at the femoral neck of Korean men aged 40 years or more (n = 1,780) and postmenopausal women (n = 1,108) using dual-energy X-ray absorptiometry. We identified subjects with MS as defined by two criteria, International Diabetes Federation (IDF) and American Heart Association/National Heart, Lung, and Blood Institute (AHA/NHLBI). Body fat and lean mass were measured via bioimpedance analysis. The prevalence of MS was 19.8% and 7.7% in men and 20.8% and 11.6% in postmenopausal women according to the AHA/NHLBI definition and the IDF definition, respectively. After multivariate adjustment, femoral neck BMD was significantly lower in subjects with MS regardless of diagnostic criteria. BMD decreased as the number of MS components increased (P < 0.001 for trends in both sexes). Among MS components, waist circumference was the most important factor in this negative association. When multiple linear regression models were applied to each 5-kg weight stratum to test for a linear trend, waist circumference and fat mass were negatively associated with BMD and lean mass was positively associated with BMD in men but not in women. MS was associated with a lower BMD in Korean men and postmenopausal women, suggesting that visceral fat may lead to bone loss, especially in men.

Genetic polymorphism of geranylgeranyl diphosphate synthase (GGSP1) predicts bone density response to bisphosphonate therapy in Korean women.

PURPOSE: Genetic factor is an important predisposing element influencing the susceptibility to osteoporosis and related complications. The purpose of the present study is to investigate whether genetic polymorphisms of farnesyl diphosphate synthase (FDPS) or geranylgeranyl diphosphate synthase (GGPS) genes were associated with the response to bisphosphonate therapy. MATERIALS AND METHODS: In the present study, 144 Korean women with osteoporosis were included. Among 13 genetic polymorphisms found within the FDPS and GGPS1 gene, 4 genetic polymorphisms with frequencies > 5% were selected for further study. Bone mineral density (BMD) response after 1 year treatment of bisphosphonate therapy was analyzed according to the genotypes. RESULTS: Women with 2 deletion allele of GGPS1 -8188A ins/del (rs3840452) had significantly higher femoral neck BMD at baseline compared with those with one or no deletion allele (0.768 +/- 0.127 vs. 0.695 +/- 0.090 respectively; p = 0.041). The response rate of women with 2 deletion allele of GGPS1 -8188A ins/del (28.6%) was significantly lower than the rate of women with one (81.4%) or no deletion allele (75.0%) (p = 0.011). Women with 2 deletion allele of GGPS1 -8188A ins/del had 7-fold higher risk of non-response to bisphosphonate therapy compared with women with other genotypes in GGPS1 -8188 after adjusting for baseline BMD (OR = 7.48; 95% CI = 1.32-42.30; p = 0.023). Other polymorphisms in FDPS or GGPS1 were not associated with lumbar spine BMD or femoral neck BMD. CONCLUSION: Our study suggested that GGPS1 -8188A ins/del polymorphism may confer susceptibility to femoral neck BMD response to bisphosphonate therapy in Korean women. However, further study should be done to confirm the results in a larger population.

Marked individual variation in isoflavone metabolism after a soy challenge can modulate the skeletal effect of isoflavones in premenopausal women.

Soy-isoflavones may act as estrogenic agonists or antagonists depending on the endogenous hormone status. These clinical effects can be exerted variably in individuals by the metabolic ability to produce a more potent metabolite than precursors. The objective of this randomized, double-blind, placebo-controlled study was to investigate the skeletal effect of isoflavones according to their metabolic variability in premenopausal women. Volunteers were randomly assigned to receive either soy-extract isoflavones (n=32) or lactose (n=21) once a day for three menstrual cycles. After intervention, the urinary excretions of isoflavones and their metabolites were significantly higher in the soy group than in the placebo group and showed a large inter-individual variation. Women in the soy group were divided into subgroups according to their ability to excrete more potent metabolites. Serum osteocalcin and urine deoxypyridinoline showed a tendency to increase after a challenge in equol high-excretors. Serum osteocalcin concentration in the genistein high-excretors increased significantly after a challenge (P=0.04) but did not increase in either the placebo or genistein low-excretors. An estrogenic antagonistic effect of isoflavones on bone turnover was observed in premenopausal women who are able to produce more potent metabolites.

The prevalence of GAD antibodies in Korean women with gestational diabetes mellitus and their clinical characteristics during and after pregnancy.

BACKGROUND: We conducted this research in order to evaluate the prevalence of glutamic acid decarboxylase antibodies (GADA) in Korean women with gestational diabetes mellitus (GDM), to identify the clinical characteristics of these women, and to gauge the prevalence of diabetes among them after childbirth. METHODS: We studied 887 Korean women with GDM who were screened for GADA, and assessed their antepartum clinical characteristics and the outcomes of their pregnancies. At 6 weeks' postpartum, 75 g oral glucose tolerance tests were performed to determine the diabetic status of GDM women with GADA. RESULTS: The prevalence of GADA in Korean women with GDM was 1.7%. Plasma glucose levels at 0- and 3-h during oral glucose tolerance tests were significantly different between GADA-positive and GADA-negative women with GDM. There were no significant differences between them in terms of age, body mass index, family history of diabetes, fasting insulin, and lipid profiles. However, GADA-positive women with GDM required insulin treatment during pregnancy more frequently than GADA-negative patients. The development of diabetes at early postpartum was significantly higher in GADA-positive women with GDM than those who were GADA negative. CONCLUSIONS: The prevalence of GADA in Korean women with GDM was low. However, GADA-positive women with GDM are more susceptible to subsequently developing type 1 diabetes, even in the early postpartum period. Long-term follow up studies and intervention to prevent type 1 diabetes among GADA-positive GDM women are needed.

Effect of RGD-immobilized dual-pore poly(L-lactic acid) scaffolds on chondrocyte proliferation and extracellular matrix production.

Interactions between cell and polymer surface have great implications in tissue engineering. In this study, chondrocyte proliferation and matrix production were examined using porous poly(L-lactide) (PLLA) scaffolds that have different surface characteristics. PLLA scaffolds were prepared using a gas-foaming method, and subjected to surface modifications through plasma treatment and subsequent in situ grafting of hydrophilic acrylic acid (AA). To immobilize peptide ligands, the AA-grafted PLLA scaffolds (PLLA-PAA) were further reacted with either Gly-Arg-Asp-Gly (GRDG) or Gly-Arg-Gly-Asp (GRGD) to produce PLLA-PAA-GRDG or PLLA-PAA-GRGD scaffold, respectively. The average porosities of the scaffolds were more than 90%, and their pore sizes ranged from 200 approximately 300 to 10 approximately 50 microm for large and small pores, respectively. The concentrations of each bound component were 2.14 x 10(-4) mmol/cm(2) for AA, 1.87 nmol/g for GRDG, and 1.68 nmol/g for GRGD. When chondrocytes were seeded onto the different PLLA scaffolds, cell adhesion and proliferation were highly affected as the substrate types vary. The RGD-immobilized scaffolds resulted in higher cellularity and better accumulation of total glycosaminoglycan than the others. Histological staining of Safranin O showed that the deposited extracellular matrix was more intense and widely distributed in the PLLA-PAA-GRGD scaffold. The present data suggest that immobilization of RGD peptide on the AA-grafted PLLA scaffold can be an effective tool for chondrocyte attachment and proliferation, and that it may also be helpful to facilitate cartilaginous tissue formation.

Highly efficient gene transfer with degradable poly(ester amine) based on poly(ethylene glycol) diacrylate and polyethylenimine in vitro and in vivo.

BACKGROUND: Polyethylenimine (PEI) is toxic although it is one of the most successful and widely used gene delivery polymers with the aid of the proton sponge effect. Therefore, development of new novel gene delivery carriers having high efficiency with less toxicity is necessary. METHODS: In this study, a degradable poly(ester amine) carrier based on poly(ethylene glycol) diacrylate (PEGDA) and low molecular weight linear PEI was prepared. Furthermore, we compared the gene expression of the polymer/DNA complexes using two delivery methods: intravenous administration as an invasive method and aerosol as a non-invasive method. RESULTS: The synthesized polymer had a relatively small molecular weight (MW = 7980) with 25 h half-life in vitro. The polymer/DNA complexes were formed at an N/P ratio of 9. The particle sizes and zeta-potentials of the complexes were dependent on N/P ratio. Compared to PEI 25K, the newly synthesized polymer exhibited high transfection efficiency with low toxicity. Poly(ester amine)-mediated gene expression in the lung and liver was higher than that of the conventional PEI carrier. Interestingly, non-invasive aerosol delivery induced higher gene expression in all organs compared to intravenous method in an in vivo mice study. Such an expressed gene via a single aerosol administration in the lung and liver remained unchanged for 7 days. CONCLUSIONS: Our study demonstrates that poly(ester amine) may be applied as an useful gene carrier.

Hypercalcemia induced by ovarian clear cell carcinoma producing all transcriptional variants of parathyroid hormone-related peptide gene during pregnancy.

BACKGROUND: Parathyroid hormone-related peptide (PTHrP) appears as the major causative agent responsible for the humoral hypercalcemia of malignancy (HHM). However, the use of promoters and splicing patterns of PTHrP gene in HHM have not been reported yet. CASE: A 35-year-old woman was diagnosed as an ovarian clear cell carcinoma with HHM caused by elevated serum PTHrP after delivery. An immunohistochemical study showed PTHrP expression in the tumor tissue. The Southern blot analysis following RT-PCR confirmed the presence of all types of PTHrP mRNA transcripts produced by a combination of three promoters, one 5' alternative splicing and three alternative 3' splicing events. CONCLUSION: An ovarian clear cell carcinoma induced PTHrP-related HHM, which resulted from the high expression of all isoforms for PTHrP gene.

Isoflavone metabolites and their in vitro dual functions: they can act as an estrogenic agonist or antagonist depending on the estrogen concentration.

The major soy isoflavones are daidzin and genistin, the glycoside conjugates of daidzein (DZ) and genistein (GTN). After ingestion, they are metabolized into diverse compounds in the gut. The marked inter-individual variation has been suggested in their metabolism. The clinical effects may be modulated by the metabolic ability to produce a more potent metabolite than the precursor. Our study was, therefore, designed to analyze and compare in vitro biologic activities of their metabolites: DZ, GTN, dihydrogenistein (DGTN), dihydrodaidzein (DDZ), tetrahydrodaidzein (TDZ), O-desmethylangolensin (ODMA), and equol (EQL). Furthermore, we investigated their modulatory effects in the presence of estrogen using several in vitro systems. The intermediate metabolites, such as DGTN, DDZ, and TDZ, bind much weakly to both ERs and induce less potently in transcriptional activity, gene expression, and mammary cell proliferation than their precursors. EQL has the strongest binding affinities and estrogenic activities especially for ERbeta among the daidzin metabolites and shows the ability to suppress osteoclast formation at high doses. The test isoflavonoids act like estrogen antagonists with the premenopausal dose of E2 and thus inhibit estrogenic actions by E2, whereas they exert estrogen agonist activity with the lower dose of estrogen close to the serum levels of postmenopausal women. Our results suggest that phytoestrogens such as isoflavones may exert their effects as estrogen antagonists in a high estrogen environment, or they may act as estrogen agonists in a low estrogen environment.

Association of estrogen receptor alpha gene microsatellite polymorphism with annual changes in bone mineral density in Korean women with hormone replacement therapy.

Variation in drug response to hormone replacement therapy (HRT) may reflect genetic heterogeneity in the estrogen-related genes, possibly including estrogen receptor alpha (ERalpha) gene. However, only a few association studies of the drug response to HRT have been reported, focusing mainly on the intronic polymorphisms of the ERalpha gene. We therefore examined 284 postmenopausal women (mean age, 52.2 +/- 5.0 years) for the microsatellite thymine-adenine (TA) repeat polymorphism in the promoter of the ERalpha gene and its relationship to drug response by measuring changes in bone mineral density (BMD) after 1 year of HRT. In our study population, the most common number of TA repeats was 14, with a range of values between 11 and 27. At baseline, the number of TA repeats was neither associated with measured lumbar spine or femoral neck BMD nor with bone markers. When we categorized the subjects by the TA repeat numbers into an L group (n = 142), with a low mean number of repeats (TA < 16), and an H group (n = 142), with a high mean number of repeats (TA > or = 16), no significant genotypic differences were noted in spinal or femoral neck BMD or in bone markers. However, the drug response on lumbar spine BMD after 1 year of HRT correlated with the mean number of TA repeats (r = -0.131, P = 0.035) after adjustment for confounding factors such as body mass index and years since menopause. This correlation was also seen with the number of TA repeats on the shorter allele (r = -0.159, P = 0.012), which was defined as the allele with the lower number of TA repeats. However, this genotypic association was not found in the femoral neck BMD (r = 0.053, P = 0.396). When we defined the nonresponder group as women who had lost BMD even with HRT, 15.9% of the subjects were included, and this group was significantly younger and had higher initial BMD than the responder group. After further adjustment for age and initial BMD, the number of TA repeats on the shorter allele remained significantly associated with drug responsiveness (P = 0.005). These data indicate significant effects of the ERalpha TA repeat polymorphism on the estrogen responsiveness of lumbar spine BMD after 1 year of HRT in Korean women.

Co-localization and interaction of organic anion transporter 1 with caveolin-2 in rat kidney.

The organic anion transporters (OAT) have recently been identified. Although the some transport properties of OATs in the kidney have been verified, the regulatory mechanisms for OAT's functions are still not fully understood. The rat OAT1 (rOAT1) transports a number of negatively charged organic compounds between the cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. Therefore, we investigated the protein-protein interactions between rOAT1 and caveolin-2. In the rat kidney, the expressions of rOAT1 mRNA and protein were observed in both the cortex and the outer medulla. With respect to Cav-2, the expressions of mRNA and protein were observed in all portions of the kidney (cortex < outer medulla = inner medulla). The results of Western blot analysis using the isolated caveolae-enriched membrane fractions or the immunoprecipitates by respective antibodies from the rat kidney showed that rOAT1 and Cav-2 co-localized in the same fractions and they formed complexes each other. These results were confirmed by performing confocal microscopy with immunocytochemistry using the primary cultured renal proximal tubular cells. When the synthesized cRNA of rOAT1 along with the antisense oligodeoxynucleotides of Xenopus Cav-2 were co-injected into Xenopus oocytes, the [(14)C]p-aminohippurate and [(3)H]methotrexate uptake was slightly, but significantly decreased. The similar results were also observed in rOAT1 over-expressed Chinese hamster ovary cells. These findings suggest that rOAT1 and caveolin-2 are co-expressed in the plasma membrane and rOAT1's function for organic compound transport is upregulated by Cav-2 in the normal physiological condition.

Degradable polyethylenimine-alt-poly(ethylene glycol) copolymers as novel gene carriers.

An ideal gene carrier requires both safety and transfection efficiency. Polyethylenimine (PEI) is a well-known cationic polymer, which has high transfection efficiency owing to its buffering capacity. But it has been reported that PEI is cytotoxic in many cell lines and non-degradable. In this study, we synthesized degradable PEI-alt-poly(ethylene glycol) (PEG) copolymers using Michael-type addition reactions as a new gene carrier and characterized them. These copolymers were complexed with plasmid DNA and the resulting complexes were characterized by dynamic light scattering, gel retardation and atomic force microscopy to determine particle sizes, complex formation and complex shape, respectively. Cytotoxicity and transfection efficiency of the copolymers were also checked in cultured HeLa human cervix epithelial carcinoma cells, HepG2 human hepatoblastoma cell line and MG63 human osteosarcoma cells. PEG to PEI ratio in the copolymers was near 1 and the molecular weight of the copolymer ranged from around 8000 to 12,900. These copolymers degraded rapidly at 37 degrees C in 0.1 M phosphate buffered saline (PBS, pH 7.4). The complete copolymer/DNA complex was formed at an N/P ratio of 12, producing a complex resistant to DNase I. Particle sizes decreased with increasing N/P ratio and PEG molecular weight, exhibiting a minimum value of 75 nm at an N/P ratio of 45 with PEI-alt-PEG (700). Cytotoxicity study showed that copolymers exhibited no cytotoxic effects on cells even at high copolymer concentration. Also, transfection efficiency was influenced by PEG molecular weight and, in case of PEI-alt-PEG (258), the transfection efficiency was higher than that for PEI 25 K in HepG2 and MG63, whereas it was lower than that for PEI 25K in HeLa cells.

The changes in circulating osteoprotegerin after hormone therapy in postmenopausal women and their relationship with oestrogen responsiveness on bone.

OBJECTIVE: Oestrogen replacement reduces the increased rate of bone remodelling after the menopause. Osteoprotegerin (OPG) is a negative regulator of osteoclast-mediated bone resorption. In vitro studies have shown that oestrogen stimulates OPG production. However, the role of OPG in physiological bone remodelling and its regulation by oestrogen in vivo remain controversial. In this study, we analysed the association between changes in serum OPG levels and bone turnover status before and after hormone therapy (HT) in healthy postmenopausal women. PATIENTS AND MEASUREMENTS: Ninety-nine healthy postmenopausal women of Korean ethnicity, aged 42-64 years (52.3 +/- 4.9 years, mean +/- SD) were enrolled in our study. Serum OPG levels were assessed by a highly sensitive sandwich-type enzyme immunoassay. Serum concentrations of osteocalcin (OC) and carboxyterminal telopeptides (CTx) were determined by electrochemiluminescence immunoassays. Bone mineral density (BMD) at the lumbar spine and femoral neck was measured by dual-energy X-ray absorptiometry (DEXA). RESULTS: Baseline levels of OPG correlated neither a the bone formation marker, serum OC, nor with a bone resorption marker, serum CTx. No significant association of baseline OPG was found with baseline BMD measured at the lumbar spine and femoral neck. Serum OPG levels measured after 3 months and 1 year of HT decreased significantly compared to baseline (P < 0.001 in both). The changes in circulating OPG at 3 months of HT correlated with the changes in both serum OC (r = 0.226, P = 0.029) and serum CTx (r = 0.214, P = 0.038) at 3 months after HT. However, there was no significant association between the changes in circulating OPG after 3 months of HT and BMD values of the lumbar spine or femoral neck after 1 year of HT. CONCLUSIONS: Our results suggest that baseline OPG levels do not reflect bone turnover status and that serial measurements of serum OPG after HT are not a useful predictor of the long-term effects of oestrogen on bone density. The decrease in serum concentrations of OPG after HT may occur to compensate for the action of oestrogen in suppressing bone resorption.

Poly (4-vinylimidazole) as nonviral gene carrier: in vitro and in vivo transfection.

We explored poly(4-vinylimidazole) (P4V) as a nonviral gene carrier. We show that P4V can form DNA condensates of small size (<110 nm) using a dye-exclusion assay with ethidium bromide and dynamic light scattering, and that the complexes form in a pH-sensitive manner, due to the amphotericity of the polymer. P4V was demonstrated to lead to transfection in vitro as effectively as polyethyleneimine (PEI), but at lower cytotoxicity, under conditions where higher amounts of either polymer are required, using luciferase and green fluorescent protein as examples. Transfection in vivo was also explored, using a gene encoding yellow fluorescent protein and human osteoprotegerin injected in the tail vein of the rat. Transfection was observed, both at the gene and protein levels in lung and spleen tissue. Transfection in vivo appeared to be at least as effective using P4V as with PEI. Based upon this good transfection and low cytotoxicity, P4V seems to show promise as a nonviral gene transfer vector.

High transfection efficiency of poly(4-vinylimidazole) as a new gene carrier.

Poly(4-vinylimidazole) (P4V) was obtained by free radical polymerization of 4-vinylimidazole (4V) prepared by decarboxylation of urocanic acid. P4V formed a complex with DNA that exhibited higher transfection effiency on Hela cells than polyethylenimine (PEI), through the proton sponge mechanism of the imidazole groups in the side chain of the P4V, and low cell toxicity.