Method for Evaluating Cortical Bone Young's Modulus: Numerical Twin Reconstruction, Finite Element Calculation, and Microstructure Analysis.

The determination of bone mechanical properties remains crucial, especially to feed up numerical models. An original methodology of inverse analysis has been developed to determine the longitudinal elastic modulus of femoral cortical bone. The method is based on a numerical twin of a specific three-point bending test. It has been designed to be reproducible on each test result. In addition, the biofidelity of the geometric acquisition method has been quantified. As the assessment is performed at the scale of a bone shaft segment, the Young's modulus values obtained (between 9518.29 MPa and 14181.15 MPa) are considered average values for the whole tissue, highlighting some intersubject variability. The material microstructure has also been studied through histological analysis, and bone-to-bone comparisons highlighted discrepancies in quadrants microstructures. Furthermore, significant intrasubject variability exists since differences between the bone's medial-lateral and anterior-posterior quadrants have been observed. Thus, the study of microstructures can largely explain the differences between the elastic modulus values obtained. However, a more in-depth study of bone mineral density would also be necessary and would provide some additional information. This study is currently being setup, alongside an investigation of the local variations of the elastic modulus.

Macrocrack propagation in a notched shaft segment of human long bone: Experimental results and mechanical aspects.

Experimenting with crack propagation in human cortical tissue is a necessary prerequisite for developing a cracking model. A three-point bending test on a shaft section of a notched human long bone is presented. A procedure for carrying out the experimental test, including unloading/reloading cycles, is implemented. The results obtained are analyzed regarding the physical mechanisms which occur in the different phases of the test, and during the cycles. The prominent role of cracking is highlighted. In addition a hypothesis is proposed concerning the potential effect of initial internal residual stresses, due to bone remodelling, on the significant residual notch openings after unloading and on the cycles' shape.

Effect of neonatal bacille Calmette-Guérin on the tuberculin skin test reaction in the first 2 years of life.

SETTING: Latent tuberculous infection (LTBI) is an important reservoir of disease reactivation that is sufficient to generate new cases for decades. The tuberculin skin test (TST) is an important tool to diagnose LTBI; however, neonatal bacille Calmette-Guérin (BCG) vaccination may impact interpretation of TST data. OBJECTIVES: To analyse the effect of the neonatal BCG vaccine on TST reaction in the first 2 years of life in children with no identified contact with tuberculosis (TB). DESIGN: This was a cross-sectional study in children up to 2 years of age who received neonatal BCG vaccination. In the absence of baseline comorbidities or contact with the bacillus, the children were given the TST. RESULTS: Seventy-nine children participated in the study. A decline in TST reactivity was observed in the first 12-24 months of age in patients who had been vaccinated with neonatal BCG but with no contact with TB. After the age of 10 months, no patient showed a TST reaction of >5 mm. CONCLUSION: BCG had low impact on the TST in children with no TB contact. This finding suggests the need to reassess the cut-off point to 5 mm of induration to improve TST specificity in LTBI identification.

Effects of transgenic expression of dopamine beta hydroxylase (Dbh) gene on blood pressure in spontaneously hypertensive rats.

The spontaneously hypertensive rat (SHR) is the most widely used animal model of essential hypertension and left ventricular hypertrophy. Catecholamines play an important role in the pathogenesis of both essential hypertension in humans and in the SHR. Recently, we obtained evidence that the SHR harbors a variant in the gene for dopamine beta hydroxylase (Dbh) that is associated with reduced adrenal expression of Dbh mRNA and reduced DBH enzymatic activity which correlated negatively with blood pressure. In the current study, we used a transgenic experiment to test the hypothesis that reduced Dbh expression predisposes the SHR to hypertension and that augmentation of Dbh expression would reduce blood pressure. We derived 2 new transgenic SHR-Dbh lines expressing Dbh cDNA under control of the Brown Norway (BN) wild type promoter. We found modestly increased adrenal expression of Dbh in transgenic rats versus SHR non-transgenic controls that was associated with reduced adrenal levels of dopamine and increased plasma levels of norepinephrine and epinephrine. The observed changes in catecholamine metabolism were associated with increased blood pressure and left ventricular mass in both transgenic lines. We did not observe any consistent changes in brainstem levels of catecholamines or of mRNA levels of Dbh in the transgenic strains. Contrary to our initial expections, these findings are consistent with the possibility that genetically determined decreases in adrenal expression and activity of DBH do not represent primary determinants of increased blood pressure in the SHR model.

Long-term pioglitazone treatment enhances lipolysis in rat adipose tissue.

OBJECTIVES: The insulin-sensitizing effects of thiazolidinediones are believed to depend at least in part on reductions in circulating levels of nonesterified fatty acids (NEFA). The mechanisms that mediate the reductions in NEFA are not fully understood and could involve reductions in adipose tissue lipolysis, increases in glyceroneogenesis and NEFA reesterification in triglycerides in adipose tissue and increases in NEFA metabolism by oxidative tissues. METHODS: In a congenic strain of spontaneously hypertensive rats that fed a high-sucrose diet to promote features of the metabolic syndrome, we studied the effects of chronic pioglitazone treatment over 4 months on adipose tissue lipolysis and NEFA metabolism. RESULTS: We observed significant increases in basal and adrenaline-stimulated NEFA and glycerol release, and near-total suppression of NEFA reesterification in epididymal adipose tissue isolated from rats chronically treated with pioglitazone. However, pioglitazone-treated rats also exhibited significant increases in mitochondrial DNA levels in adipose tissue (3.2-fold increase, P=0.001) and potentially greater sensitivity to the antilipolytic effects of insulin than untreated controls. In addition, chronic pioglitazone treatment was associated with increased palmitate oxidation in soleus muscle, reduced fasting levels of serum NEFA and triglycerides, as well as reduced serum levels of insulin and increased serum levels of adiponectin. CONCLUSIONS: Despite suppressing NEFA reesterification and increasing basal and adrenaline-stimulated lipolysis, chronic pioglitazone treatment may decrease circulating NEFA levels in part by increasing adipose tissue sensitivity to the antilipolytic effects of insulin and by enhancing NEFA oxidation in skeletal muscle.

Fat-specific transgenic expression of resistin in the spontaneously hypertensive rat impairs fatty acid re-esterification.

OBJECTIVE: To investigate the mechanism by which fat-specific transgenic expression of resistin affects fatty acid metabolism in the spontaneously hypertensive rat (SHR). DESIGN: Basal- and adrenaline-stimulated lipolysis, basal- and insulin-stimulated lipogenesis as well as the site (glycerol versus acyl moiety) of glucose incorporated into triglycerides were determined in adipose tissue isolated from SHR-Resistin transgenic and SHR control rats. RESULTS: A moderate expression of transgenic resistin in adipose tissue was associated with significant increase in the FFA/glycerol ratio during adrenaline-stimulated lipolysis in the SHR-Resistin transgenic rats (3.27+/-0.26) compared to SHR controls (2.11+/-0.10, P=0.0005). Transgenic SHR also exhibited a significant decrease in FFA re-esterification in adipose tissue (approximately by 23%). CONCLUSION: These findings raise the possibility that the prodiabetic effects of transgenic resistin may be partly mediated by increased FFA release from adipose tissue due to impaired FFA re-esterification in adipocytes.

Treating the metabolic syndrome: telmisartan as a peroxisome proliferator-activated receptor-gamma activator.

Hypertension commonly occurs as part of a genetically complex disorder of carbohydrate and lipid metabolism known as the metabolic syndrome. Most current antihypertensive drugs appear ineffective against the metabolic syndrome, which is a strong predictor of cardiovascular disease and death in affected patients. Angiotensin II can influence the activity of certain genes and cellular and biochemical pathways that may contribute to the pathogenesis of the metabolic syndrome. However, as a class, angiotensin II receptor blockers (ARBs) have proven only minimally to modestly effective in ameliorating the disturbances in carbohydrate and lipid metabolism that characterise the metabolic syndrome. Recent preclinical studies indicate that the ARB telmisartan acts as a selective peroxisome proliferators-activated receptor-gamma (PPARgamma) modulator when tested at concentrations that might be achievable with oral doses recommended for treatment of hypertension; this property does not appear to be shared by other ARBs. PPARgamma is a nuclear receptor that influences the expression of multiple genes involved in carbohydrate and lipid metabolism and is an attractive therapeutic target for the prevention and control of insulin resistance, type 2 diabetes and atherosclerosis. In cellular transactivation assays, telmisartan functioned as a partial agonist of PPARgamma and achieved 25-30% of maximal receptor activation attained with conventional PPARgamma ligands. Preclinical and clinical studies indicate that administration of telmisartan can improve carbohydrate and lipid metabolism without causing the side effects that accompany full PPARgamma activators. If the preliminary data are supported by the results of ongoing large-scale clinical studies, telmisartan could have a central role in the prevention and treatment of metabolic syndrome, diabetes and atherosclerosis.

Transgenic expression of CD36 in the spontaneously hypertensive rat is associated with amelioration of metabolic disturbances but has no effect on hypertension.

Spontaneously hypertensive rats (SHR/NIH strain) harbor a deletion variant in the Cd36 fatty acid transporter and display defective fatty acid metabolism, insulin resistance and hypertension. Transgenic rescue of Cd36 in SHR ameliorates insulin resistance and improves dyslipidemia. However, the role of Cd36 in blood pressure regulation remains controversial due to inconsistent blood pressure effects that were observed with transgenic expression of Cd36 on the SHR background. In the current studies, we developed two new SHR transgenic lines, which express wild type Cd36 under the control of the universal Ef-1 alpha promoter, and examined the effects of transgenic expression of wild type Cd36 on selected metabolic and cardiovascular phenotypes. Transgenic expression of Cd36 in the new lines was associated with significantly decreased serum fatty acids, amelioration of insulin resistance and glucose intolerance but failed to induce any consistent changes in blood pressure as measured by radiotelemetry. The current findings confirm the genetic association of defective Cd36 with disordered insulin action and fatty acid metabolism in the SHR/NIH strain and suggest that Cd36 is linked to other gene(s) on rat chromosome 4 that regulate blood pressure.

The CD36 protein functions as an immunogenic domain of the RT8 alloantigen.

Completely concordant distributions of Cd36 and Rt8 deletion/null and wild-type alleles among inbred and congenic strains, together with Western blot analysis of RT8/CD36 proteins, indicated that the CD36 protein functions as an immunogenic domain of the RT8 alloantigen.

Genetic isolation of quantitative trait loci for blood pressure development and renal mass on chromosome 5 in the spontaneously hypertensive rat.

Total genome scans of genetically segregating populations derived from spontaneously hypertensive rats (SHR) and other rat models of essential hypertension suggested a presence of quantitative trait loci (QTL) regulating blood pressure on multiple chromosomes, including chromosome 5. The objective of the current study was to test directly a hypothesis that chromosome 5 of the SHR carries a blood pressure regulatory QTL. A new congenic strain was derived by replacing a segment of chromosome 5 in the SHR/Ola between the D5Wox20 and D5Rat63 markers with the corresponding chromosome segment from the normotensive Brown Norway (BN/Crl) rat. Arterial pressures were directly monitored in conscious, unrestrained rats by radiotelemetry. The transfer of a segment of chromosome 5 from the BN strain onto the SHR genetic background was associated with a significant decrease of systolic blood pressure, that was accompanied by amelioration of renal hypertrophy. The heart rates were not significantly different in the SHR compared to SHR chromosome 5 congenic strain. The findings of the current study demonstrate that gene(s) with major effects on blood pressure and renal mass exist in the differential segment of chromosome 5 trapped within the new SHR.BN congenic strain.

Genetic susceptibility to renal injury in hypertension.

Substantial evidence indicates that hypertension plays a predominant role in the progression of most chronic renal diseases including diabetic nephropathy. Nevertheless, significant differences are observed in the susceptibility to develop hypertension-associated renal damage between individuals, racial groups and animal strains despite comparable hypertension. Recent studies employing a variety of genetic methods both in humans and in experimental models, have provided strong support for the potential importance of genetic factors and have suggested that genes influencing susceptibility to renal damage may be inherited separately from genes that influence blood pressure. However, due to the genetic complexity involved in a multifactorial trait such as the susceptibility to hypertensive renal damage, very limited progress has been achieved thus far in attempts to link such susceptibility to specific genetic mechanisms, chromosome regions and/or candidate genes. It is anticipated that the rapid recent advances in molecular genetic techniques and the simultaneous use of multiple complementary strategies, as is currently under way, will greatly facilitate this search and provide fundamental new insights into the pathogenesis of hypertensive renal damage.

Kidney-specific chromosome transfer in genetic hypertension: the Dahl hypothesis revisited.

BACKGROUND: A central dogma in the field of essential hypertension research is that the genetic transmission of increased blood pressure is determined solely by the genotype of the kidney. This concept is based in large part on studies in experimental rat models of spontaneous hypertension in which transplantation of a kidney from a hypertensive strain into a normotensive strain was reported to increase blood pressure, and transplantation of a kidney from a normotensive strain into a hypertensive strain was reported to decrease blood pressure. The enduring interpretation of these now classic experiments remains virtually unchanged from the view originally espoused a quarter century ago by Lewis Dahl, one of the founding fathers of the field of genetic hypertension research: "Blood pressure is determined by the genotype of the donor kidney and not the genotype of the recipient." METHODS: To test the Dahl hypothesis, we determined the blood pressure effects of selective intrarenal versus extrarenal exchange of single chromosome regions between the spontaneously hypertensive rat (SHR) and the normotensive Brown Norway (BN) rat. RESULTS: The replacement of a defined segment of chromosome 1 in the SHR with the corresponding chromosome region of the BN rat was sufficient to attenuate hypertension when selectively achieved either inside the kidney or outside the kidney. CONCLUSIONS: The current finding (1) demonstrates that naturally occurring genetic variants exist that can regulate blood pressure when selectively expressed outside the kidney as well as inside the kidney, and (2) compels reconsideration of the long-held view that in essential hypertension, the genetic transmission of increased blood pressure is determined solely by the genotype of the kidney.

Genetic isolation of a blood pressure quantitative trait locus on chromosome 2 in the spontaneously hypertensive rat.

OBJECTIVES: Total genome scans of genetically segregating populations derived from the spontaneously hypertensive rat (SHR) and other rat models of hypertension have suggested the presence of quantitative trait loci (QTL) regulating blood pressure and cardiac mass on multiple chromosomes, including chromosome 2. The objective of the current study was to directly test for the presence of a blood pressure QTL on rat chromosome 2. DESIGN: A new congenic strain was derived by replacing a segment of chromosome 2 in the SHR between D2Rat171 and D2Arb24 with the corresponding chromosome segment from the normotensive Brown Norway rat. Arterial pressures were directly monitored in conscious rats by radiotelemetry. RESULTS: We found that the SHR congenic strain (SHR-2) carrying a segment of chromosome 2 from the Brown Norway rat had significantly lower systolic and diastolic blood pressures than the SHR progenitor strain. The attenuation of hypertension in the SHR-2 congenic strain versus the SHR progenitor strain was accompanied by significant amelioration of cardiac hypertrophy. CONCLUSIONS: These findings demonstrate that gene(s) with major effects on blood pressure exist in the differential segment of chromosome 2 trapped within the new SHR.BN congenic strain.

Differential salt-sensitivity in the pathogenesis of renal damage in SHR and stroke prone SHR.

The spontaneously hypertensive rat (SHR) and the stroke prone SHR (SHRsp) display contrasting susceptibilities to the development of the severe hypertensive lesions of malignant nephrosclerosis, both with aging and after the provision of a high salt intake on the background of a Japanese style "stroke prone" rodent diet. The SHR is relatively resistant, whereas the SHRsp is markedly susceptible. The responsible mechanisms remain controversial. Blood pressure (BP) radiotelemetry was used to investigate the interrelationship between salt intake, systolic BP, and renal damage in 8- to 12-week-old male SHR and SHRsp given a standard North American style diet for 6 weeks, a standard diet plus 1% NaCl as drinking water for 6 weeks, or an 8% NaCl diet plus tap water for 4 weeks. After 4 weeks, BP was significantly greater in the SHRsp compared to the SHR and was significantly more sensitive to supplemental salt in the SHRsp than in SHR. Average systolic pressures during week 5 (after 4 weeks on standard diet plus tap water, standard diet plus 1% NaCl, and 8% NaCl diet plus tap water) were 188.0 +/- 3.0 mm Hg, 207.3 +/- 5.6 mm Hg, and 226 +/- 9.4 mm Hg in SHRsp compared with 171.4 +/- 3.8 mm Hg, 180.6 +/- 3.8 mm Hg, and 190.3 +/- 5.0 mm Hg in SHR. In the absence of supplemental NaCl, both strains exhibited minimal evidence of hypertensive renal damage until about 16 weeks of age. A high salt intake resulted in the development of lesions of malignant nephrosclerosis (fibrinoid necrosis and thrombosis of small vessels and glomeruli) in the SHRsp but not in the SHR; semiquantitative histologic renal damage scores in SHRsp versus SHR being 10.4 +/- 2.0 versus 0.7 +/- 0.2 after 6 weeks of standard diet plus 1% NaCl, and 32.1 +/- 2.5 versus 0.7 +/- 0.4 after 4 weeks of 8% NaCl diet plus tap water; P < .001 for both comparisons. The development of more severe hypertension in salt-supplemented SHRsp could only partly account for the severity of renal damage in SHRsp, the increase in which was disproportionate to the increase in absolute BP. However, the rate of increase of BP was greater in the SHRsp and this might have contributed to the greater renal damage observed in the SHRsp. These data indicate that the contrasting genetic susceptibility to renal damage between SHR and SHRsp is mediated, at least in part, by a differential BP salt sensitivity.

Identification of a mutation in ADD1/SREBP-1 in the spontaneously hypertensive rat.

It has recently been proposed that primary mutations in genes involved in fatty acid and lipid metabolism may contribute to the pathogenesis of insulin resistance and dyslipidemia often observed in spontaneous forms of hypertension. In the current study in the spontaneously hypertensive rat (SHR), we mapped and sequenced the gene encoding a key transcription factor known as ADD1 (adipocyte determination and differentiation factor 1) or SREBP-1c (sterol regulatory element binding protein- c) that has recently been identified as a master regulator of genes involved in the hepatic control of lipid and carbohydrate metabolism. We found that (1) the gene for ADD1/SREBP-1c maps to a region of rat Chromosome 10 previously reported to contain a quantitative trait locus involved in the regulation of hepatic cholesterol levels and (2) the SHR harbors a valine-to-methionine substitution in the COOH terminal portion of the ADD1/SREBP-1 protein that is not present in 44 other strains of laboratory rats. These findings, together with previous studies showing that transgenic expression of SREBP-1 isoforms has major effects on hepatic fatty acid and cholesterol biosynthesis, suggest that naturally occurring variation in the gene encoding the SREBP-1 isoforms might contribute to inherited variation in lipid metabolism in the SHR versus other strains of rats. These results should serve to motivate future transfection studies of the effect of the SHR mutant on SREBP-1 expression and activation in vitro, as well as the development of congenic and transgenic strains of SHR to investigate the effects of different variants of SREBP-1 on carbohydrate and lipid metabolism in vivo.

Defective fatty acid uptake in the spontaneously hypertensive rat is a primary determinant of altered glucose metabolism, hyperinsulinemia, and myocardial hypertrophy.

Genetic linkage studies implicated deficiency of CD36, a membrane fatty acid (FA) transporter, in the hypertriglyceridemia and hyperinsulinemia of the spontaneously hypertensive rat (SHR). In this study we determined whether loss of CD36 function in FA uptake is a primary determinant of the SHR phenotype. In vivo, tissue distribution of iodinated, poorly oxidized beta-methyliodophenyl pentadecanoic acid (BMIPP) was examined 2 h after its intravenous injection. Fatty acid transport was also measured in vitro over 20 to 120 s in isolated adipocytes and cardiomyocytes obtained from SHR and from a congenic line (SHRchr4) that incorporates a piece of chromosome 4 containing wild-type CD36. SHR heart and adipose tissue exhibited defects in FA uptake and in conversion of diglycerides to triglycerides that are similar to those observed in the CD36 null mouse. However, a key difference in SHR tissues is that fatty acid oxidation is much more severely impaired than fatty acid esterification, which may underlie the 4-5-fold accumulation of free BMIPP measured in SHR muscle. Studies with isolated adipocytes and cardiomyocytes directly confirmed both the defect in FA transport and the fact that it is underestimated by BMIPP. Heart, oxidative muscle, and adipose tissue in the SHR exhibited a large increase in glucose uptake measured in vivo using [(18)F]fluorodeoxyglucose. Supplementation of the diet with short-chain fatty acids, which do not require CD36-facilitated transport, eliminated the increase in glucose uptake, the hyperinsulinemia, and the heart hypertrophy in the SHR. This indicated that lack of metabolic energy consequent to deficient FA uptake is the primary defect responsible for these abnormalities. Hypertension was not alleviated by the supplemented diet suggesting it is unrelated to fuel supply and any contribution of CD36 deficiency to this trait may be more complex to determine. It may be worth exploring whether short-chain FA supplementation can reverse some of the deleterious effects of CD36 deficiency in humans, which may include hypertrophic cardiomyopathy.

Transgenic rescue of defective Cd36 ameliorates insulin resistance in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) display several features of the human insulin-resistance syndromes. Cd36 deficiency is genetically linked to insulin resistance in SHR. We show that transgenic expression of Cd36 in SHR ameliorates insulin resistance and lowers serum fatty acids. Our results provide direct evidence that Cd36 deficiency can promote defective insulin action and disordered fatty-acid metabolism in spontaneous hypertension.

Preparation, characterization, and performance of magnetic iron-carbon composite microparticles for chemotherapy.

Magnetic microcarrier particles useful for delivering chemotherapeutic drug molecules are described. The particles are formed by joint deformation of iron and carbon in a ball mill. Physical, chemical, and functional characterization has been carried out on the particles. Physical characteristics include microscopy, particle size analysis (0.5-5 microm), surface area (250 m2/g), water vapor adsorption isotherm (hydrophobic surface), and analysis of the iron-carbon interface by Mössbauer spectroscopy, X-ray diffraction, and differential thermal analysis. Chemical analysis was used to identify elements in the particles other than carbon and iron. Functional characteristics measured included the particles' ability to adsorb and desorb doxorubicin, cytotoxicity, and their magnetic susceptibility.

Ligands for the peroxisome proliferator-activated receptor-gamma and the retinoid X receptor-alpha exert synergistic antiproliferative effects on human coronary artery smooth muscle cells.

In human coronary artery vascular smooth muscle (hcaVSM) cells, the mechanisms that mediate the antiproliferative effects of ligands for the peroxisome proliferator-activated receptor-gamma (PPAR gamma) and the retinoid X receptor-alpha (RXR alpha) are unclear. Dimerization of PPAR gamma with RXR alpha and occupancy by both ligands is required for maximal activation. Accordingly, we determined whether the antiproliferative activity of the PPAR gamma ligands, troglitazone or 15-deoxy-Delta-12,14-prostaglandin J2 (15d-PGJ2), was enhanced with the RXR alpha ligand, 9-cis-retinoic acid (9-cis-RA). Incubation of actively proliferating hcaVSM cells with either troglitazone or 15d-PGJ2 resulted in a dose-dependent inhibition of proliferation with half-maximal inhibitory concentrations (IC(50)s) of 13 and 2 microM, respectively. Quiescent cells incubated with troglitazone or 15d-PGJ2 and subsequently stimulated with PDGF-BB showed a concentration-dependent decrease in the active form of MAP kinase, suggesting that inhibition of cell growth by troglitazone may involve the MAP kinase pathway, an important growth activation pathway in VSM cells. Incubation of cells with either 0.1 or 1.0 microM 9-cis-RA inhibited cell growth to a similar degree. Addition of troglitazone or 15d-PGJ2 to cells in combination with either concentration of 9-cis-RA resulted in a striking increase in growth inhibition, and was accompanied by an approximately 4-fold reduction in the IC(50)s for both PPAR gamma ligands. These findings imply that RXR alpha activation by 9-cis-RA synergistically enhanced inhibition of hcaVSM cell growth. The precise nature of this cooperative interaction between PPAR gamma and RXR alpha remains to be determined.