Comparison of continuous vs. pulsed focused ultrasound in treated muscle tissue as evaluated by magnetic resonance imaging, histological analysis, and microarray analysis

The purpose of this study was to investigate the effect of different application modes of high intensity focused ultrasound (HIFU) to muscle tissue. HIFU was applied to muscle tissue of the flank in C3H/Km mice. Two dose regimes were investigated, a continuous HIFU and a short-pulsed HIFU mode. Three hours after HIFU treatment pre- and post-contrast T1-weighted, T2-weighted images and a diffusion-weighted STEAM sequence were obtained. After MR imaging, the animals were euthenized and the treated, and the non-treated tissue was taken out for histology and functional genomic analysis. T2 images showed increased signal intensity and post-contrast T1 showed a decreased contrast uptake in the central parts throughout the tissue of both HIFU modes. A significantly higher diffusion coefficient was found in the muscle tissue treated with continuous wave focused ultrasound. Gene expression analysis revealed profound changes of 54 genes. For most of the analyzed genes higher expression was found after treatment with the short-pulse mode. The highest up-regulated genes encoded for the MHC class III (FC ≈84), HSP 70 (FC ≈75) and FBJ osteosarcoma related oncogene (FC ≈21). Immunohistology and the immunoblot analysis confirmed the presence of HSP70 protein in both applied HIFU modes. The use of HIFU treatment on muscle tissue results in dramatic changes in gene expression; however, the same genes are up-regulated after the application of continuous or pulsed HIFU, indicating that the tissue reaction is independent of the type of tissue damage. Hundt and Yuh contributed equally to this paper. Supported in part by the Lucas Foundation and the Phil Allen Truse.     

Hepatic reticuloendothelial function during parenteral nutrition including an MCT/LCT or LCT emulsion after liver transplantation – a double-blind study

It has been demonstrated that total parenteral nutrition (TPN) modulates the function of the hepatic reticuloendothelial system (RES). The objective of this study was to evaluate the impact of two different TPN lipid emulsions on the recovery of allograft RES function after orthotopic liver transplantation (OLTx). In a prospective, double-blind study, OLTx patients were randomly assigned to two treatment groups. Group I ( n=13) received a TPN regimen that included long-chain triglycerides (LCT). Group II ( n=9) received a TPN regimen that included a fat emulsion consisting of both medium-chain triglycerides (MCT) and LCT. At baseline, i.e., on days 2 or 3 after OLTx ( t1), before lipids for TPN were started, hepatic RES function was determined using the human serum albumin millimicrosphere technique (K-value, 1/min). A second measurement ( t2) was obtained after 7 days of TPN, including one of the study's two fat emulsions. The mean (+/- SD) K-value (1/min) was 0.48+/-0.16 in the LCT group and 0.55+/-0.28 in the MCT/LCT group at t1, and it improved to 0.62+/-0.21 in the LCT group and to 0.86+/-0.32 in the MCT/LCT group at t2. RES function recovery was significantly better in the MCT/LCT group ( P< or = 0.05). MCT/LCT emulsion appears to be the TPN fat emulsion of choice after OLTx as it seems to have less impact on hepatic RES recovery.     

Comparison between bovine bone and titanium interference screws for implant fixation in ACL reconstruction: a biomechanical study

Introduction

The application of interference screws for the fixation of bone-patellar tendon-bone (BPTB) grafts is a well-established technique in anterior-cruciate ligament reconstruction. Interference screws derived from bovine compact bone are a biological alternative to metallic or biodegradable polymer interference screws.

Materials and methods

In 60 porcine specimens, the tibial part of an anterior-cruciate ligament reconstruction was performed using a BPTB graft. To secure the graft, either an 8-mm titanium interference screw or a self-made bovine interference screw (BC), or a commercial bovine compact bone screw (Tutofix^®) was used. The maximum failure load was determined by means of a universal testing machine with computer interface at a testing speed of 50 mm/min. In a second test series, cyclic sub-maximal load was applied to the test specimen from 40 to 400 N with a number of 1,000 load cycles and a frequency of 1 Hz. Subsequently, the maximum failure load was determined. The stiffness of the test specimen was investigated in both test series. Each type of interference screw was tested 10 times.

Results

A secure fixation of the grafts was achieved with all interference screws. In the experiments on the maximum load to failures, the titanium screws showed significantly higher failure loads than the Tutofix^® screws (P = 0.005). The stiffness of the grafts fixed with BC screws was significantly higher as compared to the fixation with Tutofix^® screws (P = 0.005). After cyclic sub-maximal loading, the maximum failure load of the titanium screws was significantly higher than that of the Tutofix^® screws (P = 0.033). The fixation of the BC screws showed a significantly higher failure load (P = 0.021) and stiffness (P = 0.032) than the Tutofix^® screw fixation. Except for two screw head fractures and two intra-tendinous graft ruptures, the failure mode was slippage in the interface between interference screw and bone plug.

Conclusion

Interference screws derived from bovine compact bone show similar good results as the titanium interference screws. Therefore, the safety and in vivo performance of products derived from xenogenic bone should be the focus of further investigations.     

Combined Risk Allele Score of Eight Type 2 Diabetes Genes Is Associated With Reduced First-Phase Glucose-Stimulated Insulin Secretion During Hyperglycemic Clamps

OBJECTIVE

At least 20 type 2 diabetes loci have now been identified, and several of these are associated with altered β-cell function. In this study, we have investigated the combined effects of eight known β-cell loci on insulin secretion stimulated by three different secretagogues during hyperglycemic clamps.

RESEARCH DESIGN AND METHODS

A total of 447 subjects originating from four independent studies in the Netherlands and Germany (256 with normal glucose tolerance [NGT]/191 with impaired glucose tolerance [IGT]) underwent a hyperglycemic clamp. A subset had an extended clamp with additional glucagon-like peptide (GLP)-1 and arginine (n = 224). We next genotyped single nucleotide polymorphisms in TCF7L2, KCNJ11, CDKAL1, IGF2BP2, HHEX/IDE, CDKN2A/B, SLC30A8, and MTNR1B and calculated a risk allele score by risk allele counting.

RESULTS

The risk allele score was associated with lower first-phase glucose-stimulated insulin secretion (GSIS) (P = 7.1 × 10⁻⁶). The effect size was equal in subjects with NGT and IGT. We also noted an inverse correlation with the disposition index (P = 1.6 × 10⁻³). When we stratified the study population according to the number of risk alleles into three groups, those with a medium- or high-risk allele score had 9 and 23% lower first-phase GSIS. Second-phase GSIS, insulin sensitivity index and GLP-1, or arginine-stimulated insulin release were not significantly different.

CONCLUSIONS

A combined risk allele score for eight known β-cell genes is associated with the rapid first-phase GSIS and the disposition index. The slower second-phase GSIS, GLP-1, and arginine-stimulated insulin secretion are not associated, suggesting that especially processes involved in rapid granule recruitment and exocytosis are affected in the majority of risk loci.Type 2 diabetes is a polygenic disease in which the contribution of a number of detrimental gene variants in combination with environmental factors is thought to be necessary for the development of disease. In the past 2 years, results of several genome-wide association studies (GWASs) have been published (1–5), leading to a rapidly increasing number of detrimental type 2 diabetes susceptibility loci. More recently, it has indeed been shown that combining information from these diabetes loci into a risk allele score for all loci enhances diabetes risk (6–9). However, the predictive power of this combined risk allele score is yet insufficient to substitute or largely improve predictive power of known clinical risk factors (8,9). At present, little is known about how these gene variants in combination affect insulin secretion or insulin resistance. Based on recent data, mainly obtained from oral glucose tolerance tests (OGTTs), it was shown that a combined risk allele score from gene variants associated with type 2 diabetes is associated with insulin secretion and not with insulin sensitivity (10–13). However, the OGTT is unable to distinguish between first- and second-phase insulin secretion. Furthermore, other secretagogues, like glucagon-like peptide (GLP)-1 and arginine, were not included in these studies.It is thought that the rapid recruitment and release of insulin granules from the readily releasable pool (RRP) is responsible for the first phase of insulin secretion, whereas the slower prolonged second phase involves recruitment to the membrane of more distant granules and de novo insulin synthesis. Although the exact pathways regulating both phases of glucose-stimulated insulin secretion (GSIS) are not completely resolved, it seems logical that they are at least in part different. This is further corroborated by our recent observation that the heritability for both phases of GSIS in twins is derived from partly nonoverlapping sets of genes (13a).Also, other nonglucose, stimuli-like incretins and amino acids can evoke an insulin response. Detailed phenotypic investigations of the response to these different stimuli may help to elucidate which processes are primarily affected by these loci. Previously, we have already shown that type 2 diabetes genes/loci can have different effects on first- and second-phase GSIS, as measured using hyperglycemic clamps. Also, based on the method of stimulation (i.e., oral versus intravenous), the outcome may differ substantially (14–17), which provides further clues about the mechanism by which they affect insulin secretion.In this study, we genotyped gene variants in TCF7L2, KCNJ11, HHEX/IDE, CDKAL1, IGF2BP2, SLC30A8, CDKN2A/CDKN2B, and MTNR1B in 447 hyperglycemic clamped subjects (256 with normal glucose tolerance [NGT] and 191 with impaired glucose tolerance [IGT]) from four independent studies in the Netherlands and Germany. These eight loci were chosen based on the fact that they were reproducibly associated with β-cell function in various studies (rev. in 18,19). A combined risk allele score of all eight gene variants was calculated for each individual and tested against the various detailed measurements of β-cell function using the hyperglycemic clamp, generally considered to be the gold standard for quantification of first- and second-phase GSIS (20). Furthermore, we also assessed the combined effect of these eight genes on two other stimuli, GLP-1 and arginine-stimulated insulin secretion during hyperglycemia, in a subset of the study sample (n = 224). The latter test provides an estimation of the maximal insulin secretion capacity of a subject and may, according to animal studies, serve as a proxy for β-cell mass (21).     

Glycemia Determines the Effect of Type 2 Diabetes Risk Genes on Insulin Secretion

OBJECTIVE

Several single nucleotide polymorphisms (SNPs) in diabetes risk genes reduce glucose- and/or incretin-induced insulin secretion. Here, we investigated interactions between glycemia and such diabetes risk polymorphisms.

RESEARCH DESIGN AND METHODS

Insulin secretion was assessed by insulinogenic index and areas under the curve of C-peptide/glucose in 1,576 subjects using an oral glucose tolerance test (OGTT). Participants were genotyped for 10 diabetes risk SNPs associated with β-cell dysfunction: rs5215 (KCNJ11), rs13266634 (SLC30A8), rs7754840 (CDKAL1), rs10811661 (CDKN2A/2B), rs10830963 (MTNR1B), rs7903146 (TCF7L2), rs10010131 (WFS1), rs7923837 (HHEX), rs151290 (KCNQ1), and rs4402960 (IGF2BP2).Furthermore, the impact of the interaction between genetic variation in TCF7L2 and glycemia on changes in insulin secretion was tested in 315 individuals taking part in a lifestyle intervention study.

RESULTS

For the SNPs in TCF7L2 and WFS1, we found a significant interaction between glucose control and insulin secretion (all P ≤ 0.0018 for glucose × genotype). When plotting insulin secretion against glucose at 120 min OGTT, the compromising SNP effects on insulin secretion are most apparent under high glucose. In the longitudinal study, rs7903146 in TCF7L2 showed a significant interaction with baseline glucose tolerance upon change in insulin secretion (P = 0.0027). Increased glucose levels at baseline predicted an increase in insulin secretion upon improvement of glycemia by lifestyle intervention only in carriers of the risk alleles.

CONCLUSIONS

For the diabetes risk genes TCF7L2 and WFS1, which are associated with impaired incretin signaling, the level of glycemia determines SNP effects on insulin secretion. This indicates the increasing relevance of these SNPs during the progression of prediabetes stages toward clinically overt type 2 diabetes.Type 2 diabetes is a disorder characterized by chronically elevated blood glucose levels due to insulin resistance and a relative lack of compensatory pancreatic insulin secretion. Environmental triggers such as a sedentary lifestyle, physical inactivity, and increased body weight play an important role in the development of the disease. In this regard, genetics and especially gene-environment interactions play an important role. Recent research revealed more than 25 gene variants leading to a higher risk for the development of type 2 diabetes (1). Interestingly, most of the diabetes risk genes alter β-cell function (1). This supports the hypothesis that the main genetic effect in the development of type 2 diabetes could be impaired insulin secretion. Neither environmental triggers nor genetics alone can explain the multifactorial disease type 2 diabetes, thus a close interaction between both is presumed (2–4). Hence, environmental influences may determine an individual''s susceptibility for single nucleotide polymorphism (SNP) effects, or vice versa genotype may designate a person''s susceptibility toward environmental factors.One “environmental” factor that plays a role early in the pathogenesis of type 2 diabetes is elevated glucose. It is well known that years before type 2 diabetes occurs, glucose control is altered, as reflected by higher fasting glucose and/or higher postprandial glucose (5). High glucose exerts unfavorable effects on insulin sensitivity and secretion, known as glucotoxicity (6,7). On the other hand, elevated glucose levels are needed for the incretin effect. Glucagon-like peptide 1–induced insulin secretion becomes fully active only in the hyperglycemic range (8,9). Incretin-dependent insulin secretion might therefore be of particular importance when compensatory insulin hypersecretion is required.The aim of this study was to investigate whether glycemia influences the effects of genetic variation associated with type 2 diabetes on insulin secretion. We therefore studied 10 genome-wide association study–derived variants that were furthermore found to influence β-cell function in subsequent studies (rev. in 1,10). Of these, 2 (in the TCF7L2 and WFS1 loci) are associated with incretin-stimulated insulin secretion (1). As the magnitude of incretin-stimulated insulin secretion is dependent on elevated glucose levels (8,9), we hypothesized that glucose levels specifically interact with the effect of those SNPs on insulin secretion both in cross-sectional and longitudinal intervention studies.     

Human fetal bone cells associated with ceramic reinforced PLA scaffolds for tissue engineering

Fetal bone cells were shown to have an interesting potential for therapeutic use in bone tissue engineering due to their rapid growth rate and their ability to differentiate into mature osteoblasts in vitro. We describe hereafter their capability to promote bone repair in vivo when combined with porous scaffolds based on poly(l-lactic acid) (PLA) obtained by supercritical gas foaming and reinforced with 5 wt.% beta-tricalcium phosphate (TCP). Bone regeneration was assessed by radiography and histology after implantation of PLA/TCP scaffolds alone, seeded with primary fetal bone cells, or coated with demineralized bone matrix. Craniotomy critical size defects and drill defects in the femoral condyle in rats were employed. In the cranial defects, polymer degradation and cortical bone regeneration were studied up to 12 months postoperatively. Complete bone ingrowth was observed after implantation of PLA/TCP constructs seeded with human fetal bone cells. Further tests were conducted in the trabecular neighborhood of femoral condyles, where scaffolds seeded with fetal bone cells also promoted bone repair. We present here a promising approach for bone tissue engineering using human primary fetal bone cells in combination with porous PLA/TCP structures. Fetal bone cells could be selected regarding osteogenic and immune-related properties, along with their rapid growth, ease of cell banking and associated safety.     

Factors affecting renal hydrogen ion excretion capacity in healthy children

In 560 healthy German children and adolescents aged 2.8–22.0 years from the DONALD (Dortmund Nutritional and Anthropometric Longitudinally Designed) study, the relationship between urine pH and renal net acid excretion (mmol/day/1.73 m²) was analysed. A quadratic model showed the best fit (r ²=0.608). Using logistic regression analysis three parameters (urinary phosphorus excretion, total protein intake and urinary ratio of potassium and sodium) had a significant effect on renal hydrogen ion excretion capacity characterised by the probability of high or low net acid excretion with respect to the urine pH value. Urinary osmolality, in contrast to what has been seen in a previous experimental study with low birth weight infants, along with sex and age had no significant independent effects on renal net acid excretion with respect to the urine pH value over the range of osmolalities observed. In healthy children and adolescents a low fluid intake with high urinary osmolality does not at least substantially decrease the renal capacity of hydrogen ion excretion. Received: 7 April 2000 / Revised: 15 December 2000 / Accepted: 15 December 2000