Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth.

The development of life-threatening cancer metastases at distant organs requires disseminated tumour cells' adaptation to, and co-evolution with, the drastically different microenvironments of metastatic sites. Cancer cells of common origin manifest distinct gene expression patterns after metastasizing to different organs. Clearly, the dynamic interaction between metastatic tumour cells and extrinsic signals at individual metastatic organ sites critically effects the subsequent metastatic outgrowth. Yet, it is unclear when and how disseminated tumour cells acquire the essential traits from the microenvironment of metastatic organs that prime their subsequent outgrowth. Here we show that both human and mouse tumour cells with normal expression of PTEN, an important tumour suppressor, lose PTEN expression after dissemination to the brain, but not to other organs. The PTEN level in PTEN-loss brain metastatic tumour cells is restored after leaving the brain microenvironment. This brain microenvironment-dependent, reversible PTEN messenger RNA and protein downregulation is epigenetically regulated by microRNAs from brain astrocytes. Mechanistically, astrocyte-derived exosomes mediate an intercellular transfer of PTEN-targeting microRNAs to metastatic tumour cells, while astrocyte-specific depletion of PTEN-targeting microRNAs or blockade of astrocyte exosome secretion rescues the PTEN loss and suppresses brain metastasis in vivo. Furthermore, this adaptive PTEN loss in brain metastatic tumour cells leads to an increased secretion of the chemokine CCL2, which recruits IBA1-expressing myeloid cells that reciprocally enhance the outgrowth of brain metastatic tumour cells via enhanced proliferation and reduced apoptosis. Our findings demonstrate a remarkable plasticity of PTEN expression in metastatic tumour cells in response to different organ microenvironments, underpinning an essential role of co-evolution between the metastatic cells and their microenvironment during the adaptive metastatic outgrowth. Our findings signify the dynamic and reciprocal cross-talk between tumour cells and the metastatic niche; importantly, they provide new opportunities for effective anti-metastasis therapies, especially of consequence for brain metastasis patients.

Solid anthropomorphic infant whole-body DXA phantom: design, evaluation, and multisite testing.

BACKGROUND: Dual-energy X-ray absorptiometry (DXA) requires phantoms for quality control and cross-calibration. No commercially available phantoms are designed specifically for whole-body scanning of infants. METHODS: We fabricated a phantom closely matching a 7-kg human infant in body habitus using polyvinyl chloride (PVC), nylon mix, and polyethylene for bone, lean tissue, and fat, respectively, for evaluating the comparability of instruments used in studies on infant body composition. We scanned the phantom multiple times for short- and long-term repeatability and then shipped it to six other sites for comparison scans. All instruments were Hologic Delphi or Discovery models. Scan analyses were in-house procedures (Hologic V12.1). RESULTS: Short- and long-term results were not significantly different. Nylon mix underrepresented expected lean mass values by 5%, PVC underrepresented bone by 12%, and polyethylene overrepresented fat by 30%. Precision values were as follows: lean mass ≈ 3%; bone ≈ 3.5%; and fat = 5.5-7.5%. Instruments differed significantly for bone mineral content and density results in most instances. Three instruments differed in fat and lean mass. The two Hologic models differed significantly in all compartments except bone density. CONCLUSION: The phantom design came close to emulating bone, lean tissue, and fat and showed good reproducibility. Significant differences among various DXA instruments highlight the necessity of cross-calibration for any multicenter studies.

Decreased bone mineralization in children with Noonan syndrome: another consequence of dysregulated RAS MAPKinase pathway?

INTRODUCTION: Noonan syndrome (NS) is a disorder of RAS- mitogen activated protein kinase (MAPK) pathway with clinical features of skeletal dysplasia. This pathway is essential for regulation of cell differentiation and growth including bone homeostasis. Currently, limited information exists regarding bone mineralization in NS. MATERIALS AND METHODS: Using dual-energy X-ray absorptiometry (DXA), bone mineralization was evaluated in 12 subjects (mean age 8.7 years) with clinical features of NS. All subjects underwent genetic testing which showed mutations in PTPN11 gene (N=8) and SOS1 gene (N=1). In a subgroup of subjects with low bone mass, indices of calcium-phosphate metabolism and bone turnover were obtained. RESULTS: 50% of subjects had low bone mass as measured by DXA. Z-scores for bone mineral content (BMC) were calculated based on age, gender, height, and ethnicity. Mean BMC z-score was marginally decreased at -0.89 {95% CI -2.01 to 0.23; p=0.1}. Mean total body bone mineral density (BMD) z-score was significantly reduced at -1.87 {95% CI -2.73 to -1.0; p=0.001}. Mean height percentile was close to - 2 SD for this cohort, thus total body BMD z-scores were recalculated, adjusting for height age. Adjusted mean total body BMD z-score was less reduced but still significant at -0.82 {95% CI -1.39 to -0.25; p=0.009}. Biochemical evaluation for bone turnover was unremarkable except serum IGF-I and IGF-BP3 levels which were low-normal for age. DISCUSSION: Children with NS have a significantly lower total body BMD compared to age, gender, ethnicity and height matched controls. In addition, total BMC appears to trend lower in children with NS compared to controls. We conclude that the metabolic bone disease present resulted from a subtle variation in the interplay of osteoclast and osteoblast activity, without clear abnormalities being defined in the metabolism of either. Clinical significance of this finding needs to be validated by larger longitudinal studies. Also, histomorphometric analysis of bone tissue from NS patients and mouse model of NS may further elucidate the relationship between the RAS-MAPK pathway and skeletal homeostasis.

Assessment of bone mineral status in children with Marfan syndrome.

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder with skeletal involvement. It is caused by mutations in fibrillin1 (FBN1) gene resulting in activation of TGF-ß, which developmentally regulates bone mass and matrix properties. There is no consensus regarding bone mineralization in children with MFS. Using dual-energy X-ray absorptiometry (DXA), we evaluated bone mineralization in 20 children with MFS unselected for bone problems. z-Scores were calculated based on age, gender, height, and ethnicity matched controls. Mean whole body bone mineral content (BMC) z-score was 0.26±1.42 (P=0.41). Mean bone mineral density (BMD) z-score for whole body was -0.34±1.4 (P=0.29) and lumbar spine was reduced at -0.55±1.34 (P=0.017). On further adjusting for stature, which is usually higher in MFS, mean BMC z-score was reduced at -0.677±1.37 (P=0.04), mean BMD z-score for whole body was -0.82±1.55 (P=0.002) and for lumbar spine was -0.83±1.32 (P=0.001). An increased risk of osteoporosis in MFS is controversial. DXA has limitations in large skeletons because it tends to overestimate BMD and BMC. By adjusting results for height, age, gender, and ethnicity, we found that MFS patients have significantly lower BMC and BMD in whole body and lumbar spine. Evaluation of diet, exercise, vitamin D status, and bone turnover markers will help gain insight into pathogenesis of the reduced bone mass. Further, larger longitudinal studies are required to evaluate the natural history, incidence of fractures, and effects of pharmacological therapy.

A comparison of the growth responses following intramuscular GHRH plasmid administration versus daily growth hormone injections in young pigs.

The efficacy of daily porcine growth hormone (GH) injections versus plasmid-driven porcine GH-releasing hormone (pGHRH) production to promote growth was assessed. Ten-day-old piglets were injected intramuscularly with 0.1, 1, or 3 mg pGHRH, or a control plasmid followed by electroporation. Plasmid constructs were driven by a synthetic muscle-specific promoter. A fifth group received daily injections of GH [0.15 mg/(kg.day)]. Control and pGHRH-treated pigs were pair-fed to GH-treated pigs. Body composition was assessed by dual-energy X-ray absorptiometry (DXA). Weight gains of GH- and pGHRH-treated pigs were greater than of controls (P < 0.001) due to greater lean mass accretion; fat accretion was similar across all treatments. Weight gain of pGHRH- and GH-treated pigs was similar for 6 weeks, but over the final 10 days, only pigs administered the highest plasmid dose maintained higher growth rates. Serum insulin-like growth factor-I (IGF-I) levels were two- to threefold higher in GH- and pGHRH-treated pigs than in controls after 4 weeks (P = 0.05), but subsequently decreased to control levels in the pGHRH-treated group. Organ weights were greater in GH- than pGHRH-treated and control piglets (P < 0.02). These results demonstrate that pGHRH transfer is effective for promoting growth and avoids the need for the frequent injections necessitated with peptide hormone use.

Chronic parenteral nutrition induces hepatic inflammation, steatosis, and insulin resistance in neonatal pigs.

Prematurity and overfeeding in infants are associated with insulin resistance in childhood and may increase the risk of adult disease. Total parenteral nutrition (TPN) is a major source of infant nutritional support and may influence neonatal metabolic function. Our aim was to test the hypothesis that TPN induces increased adiposity and insulin resistance compared with enteral nutrition (EN) in neonatal pigs. Neonatal pigs were either fed enteral formula orally or i.v. administered a TPN mixture for 17 d; macronutrient intake was similar in both groups. During the 17-d period, we measured body composition by dual-energy X-ray absorptiometry scanning; fasting i.v. glucose tolerance tests (IVGTT) and hyperinsulinemic-euglycemic clamps (CLAMP) were performed to quantify insulin resistance. On d 17, tissue was collected after 1-h, low-dose CLAMP for tissue insulin signaling assays. TPN pigs gained less lean and more body fat and developed hepatic steatosis compared with EN pigs. After 7 and 13 d, IVGTT showed evidence of insulin resistance in the TPN compared with the EN group. Fasting plasma glucose and insulin also were higher in TPN pigs. CLAMP showed that insulin sensitivity was markedly lower in TPN pigs than in EN pigs. TPN also reduced the abundance of the insulin receptor, insulin receptor substrate 1, and phosphatidylinositol 3 kinase in skeletal muscle and liver and the proliferation of total pancreatic cells and ß-cells. Hepatic proinflammatory genes as well as c-Jun-N-terminal kinase 1 phosphorylation, plasma interleukin 6, and tumor necrosis factor-α were all higher in TPN pigs than in EN pigs. The results demonstrate that chronic TPN induces a hepatic inflammatory response that is associated with significant insulin resistance, hepatic steatosis, and fat deposition compared with EN in neonatal pigs. Further studies are warranted to establish the mechanism of TPN-induced insulin resistance and hepatic metabolic dysfunction and whether there are persistent metabolic consequences of this lifesaving form of infant nutritional support.

Cross-validation of generalised body composition equations with diverse young men and women: the Training Intervention and Genetics of Exercise Response (TIGER) Study.

Generalised skinfold equations developed in the 1970s are commonly used to estimate laboratory-measured percentage fat (BF%). The equations were developed on predominately white individuals using Siri's two-component percentage fat equation (BF%-GEN). We cross-validated the Jackson-Pollock (JP) generalised equations with samples of young white, Hispanic and African-American men and women using dual-energy X-ray absorptiometry (DXA) as the BF% referent criterion (BF%-DXA). The cross-sectional sample included 1129 women and men (aged 17-35 years). The correlations between BF%-GEN and BF%-DXA were 0.85 for women and 0.93 for men. Analysis of measurement error showed that BF%-GEN underestimated BF%-DXA of men and women by 1.3 and 3.0 %. General linear models (GLM) confirmed that BF%-GEN systematically underestimated BF%-DXA of Hispanic men and women, and overestimated BF%-DXA of African-American men. GLM were used to estimate BF%-DXA from the JP sum of skinfolds and to account for race/ethnic group bias. The fit statistics (R and standard error of the estimate; see) of the men's calibration model were: white, R 0.92, see 3.0 %; Hispanic, R 0.91, see 3.0 %; African-American, R 0.95, see 2.6 %. The women's statistics were: white and African-American, R 0.86, see 3.8 %; Hispanic, R 0.83, see 3.4 %. These results showed that BF%-GEN and BF%-DXA were highly correlated, but the error analyses documented that the generalised equations lacked accuracy when applied to these racially and ethnically diverse men and women. The inaccuracy was linked to the body composition and race/ethnic differences between these Training Intervention and Genetics of Exercise Response (TIGER) study subjects and the men and women used to develop the generalised equations in the 1970s and using BF%-DXA as the referent criterion.

Body mass index bias in defining obesity of diverse young adults: the Training Intervention and Genetics of Exercise Response (TIGER) study.

The BMI cut-score used to define overweight and obesity was derived primarily using data from Caucasian men and women. The present study evaluated the racial/ethnic bias of BMI to estimate the adiposity of young men and women (aged 17-35 years) using dual-energy X-ray absorptiometry (DXA) determination of percentage body fat (DXA-BF%) as the referent standard. The samples were 806 women and 509 men who were tested from one to three times over 9 months providing 1300 observations for women and 820 observations for men. Linear mixed models (LMM) regression showed that with age and BMI controlled, DXA-BF% of African-American (AA) men and women, Asian-Indian men and women, Hispanic women and Asian women significantly differed from non-Hispanic white (NHW) men and women. For the same BMI of NHW women, the DXA-BF% of AA women was 1.76 % lower, but higher for Hispanic (1.65 %), Asian (2.65 %) and Asian-Indian (5.98 %) women. For the same BMI of NHW men, DXA-BF% of AA men was 4.59 % lower and 4.29 % higher for Asian-Indian men. Using the recommended BMI cut-scores to define overweight and obesity systematically overestimated overweight and obesity prevalence for AA men and women, and underestimated prevalence for Asian-Indian men and women, Asian women and Hispanic women. The present study extends the generalisability of research documenting the racial/ethnic bias of the universal overweight and obesity BMI cut-scores.

Generalized metabolic bone disease in Neurofibromatosis type I.

Skeletal abnormalities are a recognized component of Neurofibromatosis type I (NF1) but a generalized metabolic bone defect in NF1 has not been fully characterized thus far. The purpose of this study was to characterize at the densitometric, biochemical and pathological level the bone involvement in NF1 patients. Using dual energy X-ray absorptiometry (DXA) we analyzed bone status in 73 unselected NF1 subjects, 26 males and 47 females, mainly children and adolescents (mean age: 16.6 years). In a subgroup of subjects with low bone mass, we measured indices of calcium-phosphate metabolism, bone turnover, and bone density before and after vitamin D and calcium treatment. We found statistically significant and generalized reduction in bone mass with the mean lumbar bone mineral density (BMD) z-score being -1.38+/-1.05 (CI 95% -1.62 to -1.13), and whole body bone mineral content (BMC) z-score -0.61+/-1.19 (CI 95% -0.94 to -0.29), both significantly reduced compared to normal controls (p<.001). PTH was moderately elevated and after 4 months of supplemental therapy with calcium and vitamin D, it decreased to the normal range. However, BMD z-scores did not significantly improve after 2 years of follow-up. Histological analysis of bone samples from NF1 patients revealed substantial alteration of bone microarchitecture due mainly to reduced trabecular bone. Our observations are consistent with a generalized bone metabolic defect due to loss of the function of neurofibromin. Early identification of patients with osteoporosis may permit more timely and aggressive treatments to prevent the likely substantial morbidity associated with increased fracture risk later in life.

Bone mineral content and bone mineral density are lower in older than in younger females with Rett syndrome.

Although bone mineral deficits have been identified in Rett syndrome (RTT), the prevalence of low bone mineral density (BMD) and its association with skeletal fractures and scoliosis has not been characterized fully in girls and women with RTT. Accordingly, we measured total body bone mineral content (BMC) and BMD using dual energy x-ray absorptiometry in a cross-sectional group of 50 females, aged 2-38 y, with RTT. Methyl-CpG-binding 2 (MECP2) mutations, skeletal fractures, and scoliosis were documented. The prevalence of BMC and BMD z scores < or-2 SD was 59 and 45%, respectively. Although absolute BMC and BMD increased significantly with increasing age, BMC, and BMD z scores were significantly lower in older than in younger females. The prevalence of fractures and scoliosis was 28 and 64%, respectively. Low BMD z scores were positively associated with fractures and scoliosis. Deficits in BMD were identified across a broad range of MECP2 mutations. This study identified associations among low BMD, fractures, and scoliosis, and underscored the need for better understanding of the molecular mechanisms of MECP2 in the regulation of bone mineral metabolism.

Body-composition assessment in infancy: air-displacement plethysmography compared with a reference 4-compartment model.

BACKGROUND: A better understanding of the associations of early infant nutrition and growth with adult health requires accurate assessment of body composition in infancy. OBJECTIVE: This study evaluated the performance of an infant-sized air-displacement plethysmograph (PEA POD Infant Body Composition System) for the measurement of body composition in infants. DESIGN: Healthy infants (n = 49; age: 1.7-23.0 wk; weight: 2.7-7.1 kg) were examined with the PEA POD system. Reference values for percentage body fat (%BF) were obtained from a 4-compartment (4-C) body-composition model, which was based on measurements of total body water, bone mineral content, and total body potassium. RESULTS: Mean (+/- SD) reproducibility of %BF values obtained with the PEA POD system was 0.4 +/- 1.3%. Mean %BF obtained with the PEA POD system (16.9 +/- 6.5%) did not differ significantly from that obtained with the 4-C model (16.3 +/- 7.2%), and the regression between %BF for the 4-C model and that for the PEA POD system (R2 = 0.73, SEE = 3.7%BF) did not deviate significantly from the line of identity (y = x). CONCLUSIONS: The PEA POD system provided a reliable, accurate, and immediate assessment of %BF in infants. Because of its ease of use, good precision, minimum safety concerns, and bedside accessibility, the PEA POD system is highly suitable for monitoring changes in body composition during infant growth in both the research and clinical settings.

Metabolic and behavioral predictors of weight gain in Hispanic children: the Viva la Familia Study.

BACKGROUND: Despite the high prevalence of overweight among Hispanic children in the United States, definitive predictors of weight gain have not been identified in this population. OBJECTIVE: The study objective was to test sociodemographic, metabolic, and behavioral predictors of 1-y weight gains in a large cohort of Hispanic children studied longitudinally. DESIGN: Subjects (n = 879) were siblings from 319 Hispanic families enrolled in the Viva la Familia Study. Families were required to have at least one overweight child aged 4-19 y. One-year changes in weight and body composition by dual-energy X-ray absorptiometry were measured. Data were from parental interviews, birth certificates, multiple-pass 24-h dietary recalls, 3-d accelerometry, 24-h respiration calorimetry, measurements of eating in the absence of hunger, and measurement of fasting blood biochemistry indexes by radioimmunoassay. Generalized estimating equations and principal component analysis were applied. RESULTS: Weight gain increased with age (P = 0.001), peaking at approximately 10 y of age in girls and approximately 11 y of age in boys. Mean (+/-SD) weight gain was significantly higher in overweight (7.5 +/- 3.7 kg/y) than in nonoverweight (4.4 +/- 2.4 kg/y) children and in boys than in girls. When adjusted for age, age squared, sex, and Tanner stage, the final model indicated a child's body mass index (BMI; kg/m2) status, maternal BMI, energy expenditure (total energy expenditure, basal metabolic rate, and sleeping metabolic rate), and fasting blood biochemistry indexes (total triiodothyronine, insulin, leptin, and ghrelin) as independent, positive predictors of weight gain (P = 0.01-0.001). CONCLUSION: Knowledge of the metabolic and behavioral predictors of weight gain in Hispanic children will inform prevention and treatment efforts to address this serious public health problem in the United States.

Effect of prebiotic supplementation and calcium intake on body mass index.

OBJECTIVE: To assess the effects of a prebiotic supplement and usual calcium intake on body composition changes during pubertal growth. STUDY DESIGN: We measured anthropometry and body fat with dual-energy X-ray absorptiometry in 97 young adolescents who were randomized to receive either a daily prebiotic supplement or maltodextrin (control) for 1 year. RESULTS: Subjects who received the prebiotic supplement had a smaller increase in body mass index (BMI) compared with the control group (BMI difference 0.52 +/- 0.16 kg/m2, P = .016), BMI Z-score (difference 0.13 +/- 0.06, P = .048) and total fat mass (difference 0.84 +/- 0.36 kg, P = .022). The prebiotic group maintained their baseline BMI Z-score (0.03 +/- 0.01, paired t test, P = .30), although BMI Z-score increased significantly in the control group (0.13 +/- 0.03, P < .001). In considering subjects whose usual calcium intake was > or = 700 mg/d, those who received the prebiotic supplement had a relative change in BMI that was 0.82 kg/m2 less than control subjects (P < .01), and BMI Z-score that was 0.20 less than control subjects (P = .003). Differences tended to be maintained 1 year after supplementation was stopped. CONCLUSION: Prebiotic supplementation and avoidance of a low calcium intake can have significant effects in modulating BMI and other body composition changes during puberty.

Evaluation of body composition in neonates and infants.

A better understanding of the nutritional needs of both healthy and sick infants is important. Not only does too much or too little nutrition during early life have long-term effects on health, but periods of rapid growth during the first year of life also have long-term consequences. Knowledge of the changes in body composition in early life can help to better define nutritional needs at these ages. Several methods are available for measuring body composition of neonates and infants. Most focus on an assessment of either body fatness or bone mineralization; only a few can monitor the quality of the non-fat lean tissues. This paper provides an evaluation of the different approaches currently available to monitor infant body composition, identifying both their strengths and limitations.

Physiologic growth hormone replacement improves fasting lipid kinetics in patients with HIV lipodystrophy syndrome.

BACKGROUND: HIV lipodystrophy syndrome (HLS) is characterized by accelerated lipolysis, inadequate fat oxidation, increased hepatic reesterification, and a high frequency of growth hormone deficiency (GHD). The effect of growth hormone (GH) replacement on these lipid kinetic abnormalities is unknown. OBJECTIVE: We aimed to measure the effects of physiologic GH replacement on lipid kinetics in men with HLS and GHD. DESIGN: Seven men with HLS and GHD were studied with the use of infusions of [13C1]palmitate, [2H5]glycerol, and [2H3]leucine to quantify total and net lipolysis, palmitate and free fatty acid (FFA) oxidation, and VLDL apolipoprotein B-100 synthesis before and after 6 mo of GH replacement (maximum: 5 microg x kg(-1) x d(-1)). RESULTS: GH replacement decreased the rates of total lipolysis [FFA(total) rate of appearance (x +/- SE): from 4.80 +/- 1.24 to 3.32 +/- 0.76 mmol FFA x kg fat(-1) x h(-1); P < 0.05] and net lipolysis (FFA(net) rate of appearance: from 1.87 +/- 0.34 to 1.20 +/- 0.25 mmol FFA x kg fat(-1) x h(-1); P < 0.05). Fat oxidation decreased (from 0.28 +/- 0.02 to 0.20 +/- 0.02 mmol FFA x kg lean body mass(-1) x h(-1); P < 0.002), as did the rate of appearance of FFAs available for intrahepatic reesterification (from 0.50 +/- 0.13 to 0.29 +/- 0.09 mmol FFA x kg fat(-1) x h(-1); P < 0.03). Fractional and absolute synthetic rates of VLDL apolipoprotein B-100 were unaltered. These kinetic changes were associated with a decrease in the waist-to-hip ratio but no significant change in fasting plasma lipid concentrations. Fasting plasma glucose concentrations increased after treatment (from 5.2 +/- 0.2 to 5.8 +/- 0.3 mmol/L; P < 0.01). CONCLUSIONS: Physiologic GH replacement has salutary effects on abnormal lipid kinetics in HLS. The effects are mediated by diminished lipolysis and hepatic reesterification rather than by increased fat oxidation.

Vitamin D receptor Fok1 polymorphisms affect calcium absorption, kinetics, and bone mineralization rates during puberty.

UNLABELLED: Few studies of the VDR polymorphisms have looked at calcium metabolism or long-term effects. We measured bone mineralization and calcium metabolic parameters longitudinally in a group of 99 adolescents. We found a significant relationship between calcium absorption and skeletal calcium accretion and the Fok1, but not other VDR or related, genetic polymorphisms. It seems that the Fok1 polymorphism directly affects bone mineralization during pubertal growth through an effect on calcium absorption. INTRODUCTION: There are few data regarding the relationship between genetic markers for low bone mass and changes in calcium metabolism in childhood or adolescence. We sought to identify the effects of polymorphisms of the vitamin D receptor (VDR) on calcium and bone mineral metabolism in a longitudinal study of pubertal adolescents. MATERIALS AND METHODS: Adolescents (n = 99) received comprehensive stable isotope studies of calcium absorption, bone calcium kinetics, and bone mineralization. Studies were repeated 12 months later. Polymorphisms of putative genetic markers were determined and related to bone mineralization and calcium metabolic finding. Results were analyzed by ANOVA in which changes over time were determined using the initial value as a covariate. RESULTS: Polymorphisms of the Fok1 gene of the VDR were significantly related to calcium absorption (p = 0.008) and whole body BMC (p = 0.03) and BMD (p = 0.006). The Fok1 effect on whole body BMD was significant for those with Ca intake >800 mg/day (p < 0.001), whereas for those with Ca intake < or = 800 mg/day, the Fok1 genotype did not have a significant effect on whole body BMD (p = 0.40). The Fok1 genotype was significantly related to the changes during the year in whole body calcium accretion, with the ff genotype having a 63 +/- 20 mg/day deficit compared with the FF genotype (p = 0.008). CONCLUSIONS: The Fok1 polymorphism of the VDR receptor seems to directly affect bone mineral accretion during pubertal growth through an effect on calcium absorption. The relationship between different genetic polymorphisms and bone mineral metabolism may vary by life stage as well as diet.

A combination of prebiotic short- and long-chain inulin-type fructans enhances calcium absorption and bone mineralization in young adolescents.

BACKGROUND: Short-term studies in adolescents have generally shown an enhancement of calcium absorption by inulin-type fructans (prebiotics). Results have been inconsistent; however, and no studies have been conducted to determine whether this effect persists with long-term use. OBJECTIVE: The objective was to assess the effects on calcium absorption and bone mineral accretion after 8 wk and 1 y of supplementation with an inulin-type fructan. DESIGN: Pubertal adolescents were randomly assigned to receive 8 g/d of a mixed short and long degree of polymerization inulin-type fructan product (fructan group) or maltodextrin placebo (control group). Bone mineral content and bone mineral density were measured before randomization and after 1 y. Calcium absorption was measured with the use of stable isotopes at baseline and 8 wk and 1 y after supplementation. Polymorphisms of the Fok1 vitamin D receptor gene were determined. RESULTS: Calcium absorption was significantly greater in the fructan group than in the control group at 8 wk (difference: 8.5 +/- 1.6%; P < 0.001) and at 1 y (difference: 5.9 +/- 2.8%; P = 0.04). An interaction with Fok1 genotype was present such that subjects with an ff genotype had the least initial response to fructan. After 1 y, the fructan group had a greater increment in both whole-body bone mineral content (difference: 35 +/- 16 g; P = 0.03) and whole-body bone mineral density (difference: 0.015 +/- 0.004 g/cm(2); P = 0.01) than did the control group. CONCLUSION: Daily consumption of a combination of prebiotic short- and long-chain inulin-type fructans significantly increases calcium absorption and enhances bone mineralization during pubertal growth. Effects of dietary factors on calcium absorption may be modulated by genetic factors, including specific vitamin D receptor gene polymorphisms.

High-efficiency growth hormone-releasing hormone plasmid vector administration into skeletal muscle mediated by electroporation in pigs.

We report here a very efficient method for the in vivo transfer of therapeutic plasmid DNA into porcine muscle fibers by using electric pulses of low field intensity. We evaluated delivery of 0.1-3 mg of plasmid vectors that encode reporter secreted-embryonic alkaline phosphatase (SEAP) or therapeutic growth hormone releasing hormone (GHRH). Reporter gene studies showed that internal needle electrodes give a 25-fold increase in expression levels compared with caliper electrodes in skeletal muscle in swine. Dose and time courses were performed. Pigs injected with 0.1 mg plasmid had significantly greater weight gain than controls over 53 days (22.4 +/- 0.8 kg vs. 19.7 +/- 0.03 kg, respectively; P<0.01). The group treated with GHRH-expressing plasmid at 14 days of age demonstrated greater weight gain than controls at every time point (25.8 +/- 1.5 kg vs. 19.7 +/- 0.03 kg; P<0.01). Body composition studies by dual X-ray absorbitometry showed a 22% decrease in fat deposition (P<0.05) and a 10% increase in bone mineral density (P<0.004). Our studies demonstrate that by optimizing the electroporation method, favorable physiological changes, such as enhanced weight gain and improved body composition, can be obtained at extremely low plasmid doses in a large mammal.

Enhanced animal growth via ligand-regulated GHRH myogenic-injectable vectors.

Regulated animal growth occurred following a single electroporated injection of a mixture of two plasmids (10 microg of DNA), one expressing the GeneSwitch regulator protein, the other an inducible growth hormone releasing hormone (GHRH) gene, into the tibialis anterior muscles of adult SCID mice. Administration of the ligand mifepristone (MFP) up-regulated GHRH expression, as shown by elevations of IGF-I levels, and when MFP dosing was withdrawn, IGF-I returned to baseline levels. Five cycles of IGF-I induction were observed during a five-month period. Chronic MFP dosing for 25 days increased lean body mass, weight gain, and bone mineral density significantly compared with non-MFP treated controls. In summary, long-term drug-regulated GHRH expression was achieved following plasmid-based gene therapy, and chronic induction of GHRH expression in adult animals led to improvements in weight gain and body composition.

Growth hormone improves bone mineral content in children with cystic fibrosis.

AIM: Osteoporosis and osteopenia have been reported as common complications of cystic fibrosis (CF); however, little is known about accrual of bone mineral in CF. The goal of our study was to measure bone mineral content (BMC) in non-acutely-ill, but poorly growing children with CF, and to determine the relationship between height, lean body mass and BMC. Our second aim was to evaluate the effect of one year of treatment with human recombinant growth hormone (GH) on total body BMC. METHODS: We measured total-body BMC using dual energy X-ray absorptiometry in 32 poorly growing (height < or =10th percentile for age) prepubertal Caucasian children (ages 7 years 6 months-12 years 9 months, 17 M and 15, F) with CF. BMC and lean tissue mass (LTM) were measured at baseline, at 6 months and one year. One half of the children were randomly assigned to receive treatment with GH (GHTX). Results were compared to reference data maintained for healthy children matched for age and ethnicity. Sex steroid and IGF-I levels were also measured. RESULTS: Children with CF exhibited lower total body BMC and LTM than age-, ethnicity- and gender-matched controls. This was still apparent when the data were matched for height and bone age. BMC correlated with height, LTM, and IGF-I levels. Although at baseline the groups were similar, the GHTX group demonstrated significantly greater increase in height, weight, LTM and BMC than the NonTX group. These differences remained despite correction for increase in height CONCLUSION: Our study is the first to evaluate BMC in children with CF and suggests that poor accumulation of bone mineral is a problem. We have further demonstrated that GH treatment improves accumulation of bone mineral.