E. Zoidis

Localized insulin-like growth factor I delivery to enhance new bone formation

Insulin-like growth factor I (IGF I) exerts an important role during skeletal growth and bone formation. Therefore, its localized delivery appears attractive for the treatment of bone defects. To prolong IGF I delivery, we entrapped the protein into biodegradable poly(lactide-co-glycolide) microspheres (PLGA MS) and evaluated the potential of this delivery system for new bone formation in two defect models of ovine long bones, i.e., a 8-mm methaphyseal drill hole and a 10-mm segmental tibia defect. Administration of 100 microg of IGF I in PLGA MS resulted in new bone formation within 3 weeks in the drill hole and bridging of the segmental defect within 8 weeks. The observed increase of 12% newly formed bone in the drill hole defect after 3 weeks was substantial, compared to the measured morphometric bone-to-total area ratio of 31% bone in normal cancellous bone. Bone regeneration was further explored by measuring gene expression of typical markers for local mediators and growth factors by real-time polymerase chain reaction. Inflammation was reduced in presence of IGF I and this in vivo observation was corroborated in vitro by quantifying gene expression of inflammatory proteins and by assessing the activation of the NF-kappaB pathway, playing an important role in the regulation of inflammation. Administration of the IGF I delivery system downregulated inflammatory marker gene expression at the site of bone injury, induced new bone formation and reduced bone resorption, and resulted in bridging of 10-mm segmental tibial defects within 8 weeks.

Selenoproteins and maternal nutrition.

Selenium (Se) is an essential trace element of fundamental importance to health due to its antioxidant, anti-inflammatory and chemopreventive properties attributed to its presence within at least 25 selenoproteins (Sel). Sel include but not limited to glutathione peroxidases (GPx1-GPx6), thioredoxin reductases (TrxR1-TrxR3), iodothyronine deiodinases (ID1-ID3), selenophosphate synthetase 2 (SPS2), 15-kDa Sel (Sel15), SelH, SelI, SelK, SelM, SelN, SelO, SelP, SelR, SelS, SelT, SelV, SelW, as well as the 15-kDa Sel (Fep15), SelJ and SelU found in fish. In this review, we describe some of the recent progress in our understanding of the mechanisms of Sel synthesis. The impact of maternal Se intake on offspring is also discussed. The key regulatory point of Sel synthesis is Se itself, which acts predominantly at post-transcriptional levels, although recent findings indicate transcriptional and redox regulation. Maternal nutrition affects the performance and health of the progeny. Both maternal and offspring Se supplementations are essential for the antioxidant protection of the offspring. Prenatal Se supplementation provides an effective antioxidant system that is already in place at the time of birth while, postnatal Se supplementation becomes the main determinant of progeny Se status after the first few days of progeny life.

Selenium affects the expression of GPx4 and catalase in the liver of chicken

A total of 128 chickens (Gallus gallus, broilers) were used to investigate the effect of organic selenium (Se) in expression of catalase (CAT) and phospholipid hydroperoxidase 4 (GPx4) genes. There were 4 replicates of 4 dietary treatments: T1 (basal diet with no added Se), T2 (T1 with 0.15 ppm Se added), T3 (T1 with 0.3 ppm Se) and T4 (T1 with 3.0 ppm Se). At 4th and 6th week, 2 chickens per replicate pen were sacrificed for whole blood and liver sample collections. Samples were analyzed for total Se by ICP-MS and gene expression by RT-PCR. Dietary supplementation with organic Se (Se-yeast) readily elevated its concentration in the tissues. GPx4 mRNA levels, pooled for both ages, of chickens fed T3 and T4 diets were significantly reduced compared to those fed diet T1 by 47% and 77% respectively, while that of T2 did not differ. Liver CAT mRNA levels at 4th week were significantly decreased as Se supplementation increased, while at 6th week, were not significantly affected by Se. The study showed that liver GPx4 mRNA levels could be down-regulated by excess of Se. It is possible that reserves built by excess of Se meet antioxidant requirements and no additional GPx4 transcription is necessary.

Influence of organic selenium supplementation on the accumulation of toxic and essential trace elements involved in the antioxidant system of chicken.

The aim of the study was to investigate the interactions between selenium (Se) and various trace elements, both toxic and essential, involved in the antioxidant system. A total of 128 day-old chicks (Gallus gallus, broilers) were used to investigate the effect of Se yeast supplementation on the accumulation of cadmium (Cd), copper (Cu) iron (Fe) and zinc (Zn). There were four replicates of four dietary treatments: T1 (basal diet with no added Se, analyzed to contain 0.21 mg kg−1), T2 (T1 with 0.15 mg kg−1 Se added), T3 (T1 with 0.3 mg kg−1 Se) and T4 (T1 with 3.0 mg kg−1 Se). At week 4 and 6, two chickens per replicate pen were sacrificed for whole blood, breast muscle and liver sampling. Samples were analyzed by ICP–MS. Supplementation with Se-yeast, not only increased Se concentration but also reduced Cd concentration in the tissues. Selenium was negatively correlated with Cd and positively correlated with Zn, Cu and Fe. Cadmium was negatively correlated with Zn and Cu. Zinc was positively correlated with Cu. Iron was negatively correlated with Cu and uncorrelated with Zn and Cd. The balance between Se, Cu, Fe and Zn is important for proper antioxidant defense since they are an integral part of various antioxidant enzymes.

Phex cDNA cloning from rat bone and studies on phex mRNA expression: tissue-specificity, age-dependency, and regulation by insulin-like growth factor (IGF) I in vivo.

Phosphate regulating gene with homology to endopeptidases on the X chromosome (Phex) inactivating mutations cause X-linked hypophosphatemia (XLH). The disorder is characterized by decreased renal phosphate (Pi) reabsorption in both humans and mice, in the latter shown to be due to a reduction in mRNA and protein of type II sodium-dependent phosphate cotransporter (NadPi-II). To gain insight into the physiological role of Phex, we cloned the rat cDNA and examined tissue-specific and age-dependent mRNA expression. The rat full-length cDNA (2247 nucleotides) shares 96 and 90% identity with the mouse and human cDNA, respectively. We found 6.6 kb Phex transcripts in calvarial bone and lungs, and a weaker signal in liver of newborn rats. In adult animals, Phex mRNA signals were weaker in bone and lungs and absent in liver. Phex mRNA expression in bones and NadPi-I and -II cotransporter mRNA expression in kidney were also determined in hypophysectomized rats. These rats, which lack GH and IGF I, stop growing and exhibit decreased serum Pi levels. Treatment during 6 days with IGF I stimulated growth and increased serum Pi. Phex and NadPi-II cotransporter mRNA levels were higher in IGF I than in vehicle-treated animals, while mRNA expression of NadPi-I, 1alpha-hydroxylase and 24-hydroxylase and serum levels of calcitriol remained unaffected. Age-dependency of Phex expression suggests a role for Phex in Pi retention during growth. Moreover, our findings indicate that an increase in Phex expression in bones under the influence of IGF I may contribute to increased serum Pi by enhancing renal phosphate reabsorption. Because IGF I treatment increased NadPi-II mRNA expression and serum Pi, IGF I appears to act at least partially at pretranslational levels to increase NadPi-II mediated renal Pi retention in growing rats.

The role of selenium in cadmium toxicity: interactions with essential and toxic elements

1. The study was part of a project designed to investigate if organic selenium (Se) can ameliorate the toxic effects of cadmium (Cd). The main objective of the present study was to investigate, in the chicken, the interactions between Se, Cd and the following elements: Sb, Ca, Cr, Co, Cu, Fe, Pb, Mg, Mn, Mo, Ni, V and Zn. 2. A total of 300 1-d-old chickens (broilers) were randomly distributed among 4 dietary treatments with 5 replicate pens per treatment. In T1, chickens were fed on a diet with 0·3 mg/kg added Se, without added Cd. In T2, chickens were fed on a diet with 0·3 mg/kg Se and 10 mg/kg Cd. In T3, chickens were fed on a diet with 0·3 mg/kg Se and 100 mg/kg of Cd added and in T4 treatment, chickens were fed on a diet with 3 mg/kg Se and 100 mg/kg Cd added. Se was added as Se-yeast. Cd was added as cadmium chloride (CdCl2). On d 28 and 42, two chickens per replicate pen were killed for collection of whole blood, liver, kidney and breast muscle samples. Samples were analysed by ICP-MS. The data were analysed using a multivariate linear model. 3. While low Cd concentrations in the diet led only to an increase of Cd concentration in the examined tissues, addition of high concentrations of Cd increased the concentration of Cd, Cu, Sb and V and decreased that of Se, Mn and Fe. Addition of high Se concentrations did not significantly reduce Cd concentration. 4. Prior to model application, correlations of 78 elements were noted, while after model application 39 correlations were noted. Most notably, Cd was correlated with Ca, Co, Cu and Mg, while Se was correlated with Mn. 5. The present study revealed several correlations between essential, probably essential and toxic elements illustrating the importance of the balance between pro-oxidants and antioxidants.

Supranutritional selenium level affects fatty acid composition and oxidative stability of chicken breast muscle tissue.

A total of 128 broilers were used to investigate the effect of selenium (Se) on fatty acid (FA) composition and oxidative stability of lipids in the breast muscle tissue. There were 4 replicates of 4 dietary treatments: T1 (basal diet with no added Se), T2 (T1 with 0.15 mg Se added per kg diet), T3 (T1 with 0.3 mg Se added per kg diet) and T4 (T1 with 3.0 mg Se added per kg diet). A yeast source was used for added Se. Breast muscle tissue was collected from two chickens per replicate pen for the determination of Se concentration by ICP-MS, FA profile by GC and lipid oxidation using thiobarbituric acid reactive substances method. Addition of supranutritional Se levels to chicken diets leads to the production of Se-enriched meat. Consumption of 100 g of breast meat from chickens fed diets supplemented with 0.15, 0.3 and 3 mg Se per kg of diet can provide 26, 41 and 220 μg of Se, respectively. Long-chain polyunsaturated fatty acids namely C20:3n-6, C20:4n-6, C20:5n-3, C22:5n-3 and C22:6n-3 increased linearly (p = 0.047, p < 0.001, p = 0.023, p = 0.003 and p = 0.002, respectively) as the Se inclusion levels in the diets increased. At slaughter, a linear decrease in lipid oxidation (p = 0.019) was observed with Se addition, possibly attributed to the antioxidant properties of Se. Addition of supranutritional Se to chicken diets, at levels well below those causing toxicity, leads to production of Se-enriched meat, protection of health-promoting long-chain FA like C20:5n-3 and C22:6n-3 and protection of meat quality from oxidation at day 1 after slaughter.

Impact of IGF-I release kinetics on bone healing: A preliminary study in sheep

Spatiotemporal release of growth factors from a delivery device can profoundly affect the efficacy of bone growth induction. Here, we report on a delivery platform based on the encapsulation of insulin-like growth factor I (IGF-I) in different poly(D,L-lactide) (PLA) and poly(D,L-lactide-co-glycolide) (PLGA) microsphere (MS) formulations to control IGF-I release kinetics. In vitro IGF-I release profiles generally exhibited an initial burst (14-36% of total IGF-I content), which was followed by a more or less pronounced dormant phase with little release (2 to 34 days), and finally, a third phase of re-increased IGF-I release. The osteoinductive potential of these different IGF-I PL(G)A MS formulations was tested in studies using 8-mm metaphyseal drill hole bone defects in sheep. Histomorphometric analysis at 3 and 6 weeks after surgery showed that new bone formation was improved in the defects locally treated with IGF-I PL(G)A MS (n=5) as compared to defects filled with IGF-I-free PL(G)A MS (n=4). The extent of new bone formation was affected by the particular release kinetics, although a definitive relationship was not evident. Local administration of IGF-I resulted in down-regulation of inflammatory marker genes in all IGF-I treated defects. The over-expression of growth factor genes in response to IGF-I delivery was restricted to formulations that produced osteogenic responses. These experiments demonstrate the osteoinductive potential of sustained IGF-I delivery and show the importance of delivery kinetics for successful IGF-I-based therapies.

Combined GWAS and ‘guilt by association’-based prioritization analysis identifies functional candidate genes for body size in sheep

BackgroundBody size in sheep is an important indicator of productivity, growth and health as well as of environmental adaptation. It is a composite quantitative trait that has been studied with high-throughput genomic methods, i.e. genome-wide association studies (GWAS) in various mammalian species. Several genomic markers have been associated with body size traits and genes have been identified as causative candidates in humans, dog and cattle. A limited number of related GWAS have been performed in various sheep breeds and have identified genomic regions and candidate genes that partly account for body size variability. Here, we conducted a GWAS in Frizarta dairy sheep with phenotypic data from 10 body size measurements and genotypic data (from Illumina ovineSNP50 BeadChip) for 459 ewes.ResultsThe 10 body size measurements were subjected to principal component analysis and three independent principal components (PC) were constructed, interpretable as width, height and length dimensions, respectively. The GWAS performed for each PC identified 11 significant SNPs, at the chromosome level, one on each of the chromosomes 3, 8, 9, 10, 11, 12, 19, 20, 23 and two on chromosome 25. Nine out of the 11 SNPs were located on previously identified quantitative trait loci for sheep meat, production or reproduction. One hundred and ninety-seven positional candidate genes within a 1-Mb distance from each significant SNP were found. A guilt-by-association-based (GBA) prioritization analysis (PA) was performed to identify the most plausible functional candidate genes. GBA-based PA identified 39 genes that were significantly associated with gene networks relevant to body size traits. Prioritized genes were identified in the vicinity of all significant SNPs except for those on chromosomes 10 and 12. The top five ranking genes were TP53, BMPR1A, PIK3R5, RPL26 and PRKDC.ConclusionsThe results of this GWAS provide evidence for 39 causative candidate genes across nine chromosomal regions for body size traits, some of which are novel and some are previously identified candidates from other studies (e.g. TP53, NTN1 and ZNF521). GBA-based PA has proved to be a useful tool to identify genes with increased biological relevance but it is subjected to certain limitations.

Meta-analysis of selenium accumulation and expression of antioxidant enzymes in chicken tissues

A meta-analysis integrating results of 40 selenium (Se) supplementation experiments that originated from 35 different controlled randomized trials was carried out in an attempt to identify significant factors that affect tissue Se accumulation in chicken. Examined factors included: Se source (12 different sources examined), type of chicken (laying hens or broilers), age of birds at the beginning of supplementation, duration of supplementation, year during which the study was conducted, sex of birds, number of chickens per treatment, method of analysis, tissue type, concentration of Se determined and Se added to feed. A correlation analysis was also carried out between tissue Se concentration and glutathione peroxidase activity. Data analysis showed that the factors significantly affecting tissue Se concentration include type of chicken (P=0.006), type of tissue (P<0.001) and the analytical method used (P=0.014). Although Se source was not found to affect tissue Se concentration (overall P>0.05), certain inorganic (sodium selenite), calcium selenite, sodium selenate and organic sources (B-Traxim Se), Se-yeast, Se-malt, Se-enriched cabbage and Se-enriched garlic as well as background Se level from feed ingredients were found to significantly affect tissue Se concentration. The Se accumulation rate (estimated as linear regression coefficient of Se concentrations to Se added to feed) discriminated between the various tissues with highest values estimated in the leg muscle and lowest in blood plasma. Correlation analysis has also shown that tissue Se concentration (pooled data) was correlated to Se added to feed (r=0.529, P<0.01, log values) and to glutathione peroxidase activity (r=0.332, P=0.0478), with the latter not being correlated with Se added to feed. Although significant factors affecting Se concentration were reported in the present study, they do not necessarily indicate the in vivo function of the antioxidant system or the level of accumulated Se as other factors, not examined in the present study, may interact at the level of trace element absorption, distribution and retention.