High‐Resolution Genome Screen for Bone Mineral Density in Heterogeneous Stock Rat

We previously demonstrated that skeletal mass, structure, and biomechanical properties vary considerably in heterogeneous stock (HS) rat strains. In addition, we observed strong heritability for several of these skeletal phenotypes in the HS rat model, suggesting that it represents a unique genetic resource for dissecting the complex genetics underlying bone fragility. The purpose of this study was to identify and localize genes associated with bone mineral density in HS rats. We measured bone phenotypes from 1524 adult male and female HS rats between 17 and 20 weeks of age. Phenotypes included dual‐energy X‐ray absorptiometry (DXA) measurements for bone mineral content and areal bone mineral density (aBMD) for femur and lumbar spine (L₃–L₅), and volumetric BMD measurements by CT for the midshaft and distal femur, femur neck, and fifth lumbar vertebra (L₅). A total of 70,000 polymorphic single‐nucleotide polymorphisms (SNPs) distributed throughout the genome were selected from genotypes obtained from the Affymetrix rat custom SNPs array for the HS rat population. These SNPs spanned the HS rat genome with a mean linkage disequilibrium coefficient between neighboring SNPs of 0.95. Haplotypes were estimated across the entire genome for each rat using a multipoint haplotype reconstruction method, which calculates the probability of descent for each genotyped locus from each of the eight founder HS strains. The haplotypes were tested for association with each bone density phenotype via a mixed model with covariate adjustment. We identified quantitative trait loci (QTLs) for BMD phenotypes on chromosomes 2, 9, 10, and 13 meeting a conservative genomewide empiric significance threshold (false discovery rate [FDR] = 5%; p < 3 × 10^?6). Importantly, most QTLs were localized to very small genomic regions (1–3 megabases [Mb]), allowing us to identify a narrow set of potential candidate genes including both novel genes and genes previously shown to have roles in skeletal development and homeostasis. © 2014 American Society for Bone and Mineral Research.     

An Alternative Approach to Selecting Patients for High‐risk Screening with Breast MRI

Current guidelines for adding breast MRI to annual screening mammography are based entirely upon stratification of risk, with a heavy focus on lifetime calculations. This approach is fraught with difficulty due to the reliance on mathematical models that vary widely in their calculations, the inherent age discrimination of using lifetime risks rather than short‐term incidence, and the failure to incorporate mammographic density, the latter being an independent risk as well as the greatest predictor of mammographic failure. By utilizing a system of patient selection similar to what was used in the American College of Radiology Imaging Network (ACRIN) 6666 trial for multi‐modality imaging, 33 women without a prior diagnosis of breast cancer were found to harbor mammographically occult carcinoma through MRI screening. These 33 patients represent a 2% yield, closely approximating the yields seen in prospective MRI screening trials of women at very high risk of breast cancer. Using the “~20–25%” minimum established by the American Cancer Society and later the National Comprehensive Cancer Network, the Gail model would have prompted the use of MRI in only 9 of 33 (27.3%) patients, the Claus model 1 of 33 (3%), and the Tyrer–Cuzick model 12 of 33 (36.4%). Using all three models and opting for the highest calculated risk, then including BRCA‐positivity, still would have identified only 16 of 33 (48.5%) patients with occult breast cancer discovered by MRI. Only one patient was BRCA‐positive, and none had lobular carcinoma in situ, while 6 of 33 patients (18.2%) had atypical ductal hyperplasia (ADH). Measures are proposed to refine patient selection for MRI screening through the use of short‐term or categorical risks, mammographic density, while maintaining cost‐effectiveness through longer MRI screening intervals.     

Application of High‐Resolution Skeletal Imaging to Measurements of Volumetric BMD and Skeletal Microarchitecture in Chinese‐American and White Women: Explanation of a Paradox

Asian women have lower rates of hip and forearm fractures despite lower areal BMD (aBMD) by DXA compared with white women and other racial groups. We hypothesized that the lower fracture rates may be explained by more favorable measurements of volumetric BMD (vBMD) and microarchitectural properties, despite lower areal BMD. To address this hypothesis, we used high‐resolution pQCT (HRpQCT), a new method that can provide this information noninvasively. We studied 63 premenopausal Chinese‐American (n = 31) and white (n = 32) women with DXA and HRpQCT. aBMD by DXA did not differ between groups for the lumbar spine (1.017 ± 0.108 versus 1.028 ± 0.152 g/cm²; p = 0.7), total hip (0.910 ± 0.093 versus 0.932 ± 0.134 g/cm²; p = 0.5), femoral neck (0.788 ± 0.083 versus 0.809 ± 0.129 g/cm²; p = 0.4), or one‐third radius (0.691 ± 0.052 versus 0.708 ± 0.047 g/cm²; p = 0.2). HRpQCT at the radius indicated greater trabecular (168 ± 41 versus 137 ± 33 mg HA/cm³; p = <0.01) and cortical (963 ± 46 versus 915 ± 42 mg HA/cm³; p < 0.0001) density; trabecular bone to tissue volume (0.140 ± 0.034 versus 0.114 ± 0.028; p = <0.01); trabecular (0.075 ± 0.013 versus 0.062 ± 0.009 mm; p < 0.0001) and cortical thickness (0.98 ± 0.16 versus 0.80 ± 0.14 mm; p < 0.0001); and lower total bone area (197 ± 34 versus 232 ± 33 mm²; p = <0.001) in the Chinese versus white women and no difference in trabecular number, spacing, or inhomogeneity before adjustment for covariates. Similar results were observed at the weight‐bearing tibia. At the radius, adjustment for covariates did not change the direction or significance of differences except for bone, which became similar between the groups. However, at the tibia, adjustment for covariates attenuated differences in cortical BMD and bone area and accentuated differences in trabecular microarchitecture such that Chinese women additionally had higher trabecular number and lower trabecular spacing, as well as inhomogeneity after adjustment. Using the high‐resolution technology, the results provide a mechanistic explanation for why Chinese women have fewer hip and forearm fractures than white women.     

3D Assessment of Cortical Bone Porosity and Tissue Mineral Density Using High‐Resolution µCT: Effects of Resolution and Threshold Method

Current micro–computed tomography (µCT) systems allow scanning bone at resolutions capable of three‐dimensional (3D) characterization of intracortical vascular porosity and osteocyte lacunae. However, the scanning and reconstruction parameters along with the image segmentation method affect the accuracy of the measurements. In this study, the effects of scanning resolution and image threshold method in quantifying small features of cortical bone (vascular porosity, vascular canal diameter and separation, lacunar porosity and density, and tissue mineral density) were analyzed. Cortical bone from the tibia of Sprague‐Dawley rats was scanned at 1‐µm and 4‐µm resolution, reconstructions were density‐calibrated, and volumes of interest were segmented using approaches based on edge‐detection or histogram analysis. In 1‐µm resolution scans, the osteocyte lacunar spaces could be visualized, and it was possible to separate the lacunar porosity from the vascular porosity. At 4‐µm resolution, the vascular porosity and vascular canal diameter were underestimated, and osteocyte lacunae were not effectively detected, whereas the vascular canal separation and tissue mineral density were overestimated compared to 1‐µm resolution. Resolution had a much greater effect on the measurements than did threshold method, showing partial volume effects at resolutions coarser than 2 µm in two separate analyses, one of which assessed the effect of resolution on an object of known size with similar architecture to a vascular pore. Although there was little difference when using the edge‐detection versus histogram‐based threshold approaches, edge‐detection was somewhat more effective in delineating canal architecture at finer resolutions (1–2 µm). In addition, use of a high‐resolution (1 µm) density‐based threshold on lower resolution (4 µm) density‐calibrated images was not effective in improving the lower‐resolution measurements. In conclusion, if measuring cortical vascular microarchitecture, especially in small animals, a µCT resolution of 1 to 2 µm is appropriate, whereas a resolution of at least 1 µm is necessary when assessing osteocyte lacunar porosity. © 2014 American Society for Bone and Mineral Research.     

Adult Living Donor Liver Transplantation for Acute‐on‐Chronic Liver Failure in High–Model for End‐Stage Liver Disease Score Patients

The large volume of adult living donor liver transplantations (ALDLTs) at our center affords a unique opportunity to examine the impact of acute‐on‐chronic liver failure (ACLF) among high–Model for End‐Stage Liver Disease MELD score patients. From February 1998 to March 2010, 1958 cirrhotic recipients were analyzed to study the relationship between MELD scores and ALDLT outcomes. A total of 327 high‐MELD score recipients were categorized into ACLF and non‐ACLF groups, and their outcomes were compared. The 5‐year graft and patient survival in the high‐MELD group were 75.2% and 76.4%, respectively, which were significantly worse than the low and intermediate MELD groups. The presence of ACLF associated with higher MELD scores appeared to be the dominant factor responsible for the inferior results of patients with MELD score of 30–34 points. The 5‐year graft survivals in the ACLF group was 70.5% and in the non‐ACLF group it was 81.0% (p = 0.035). Therefore, ALDLT should be performed as soon as possible in high‐MELD score patients prior to ACLF development. Moreover, ACLF patients should be separately categorized when analyzing the outcomes of ALDLT. ALDLT for ACLF patients should not be discouraged because favorable outcomes can be expected through timely ALDLT and comprehensive management.     

Transplant Accommodation in Highly Sensitized Patients: A Potential Role for Bcl-xL and Alloantibody

Transplantation of renal allografts into recipients with circulating anti-HLA antibodies results in hyperacute rejection. In some cases, however, antibodies return without causing harm; this phenomenon has been termed 'accommodation'. We have investigated this process in human allotransplantation. We removed anti-HLA antibodies by immunoadsorption in seven highly sensitized dialysis patients who subsequently underwent renal transplantation. Immunohistochemistry of renal biopsies for IgG and antiapoptotic proteins was performed. We also developed a model of 'accommodation' using anti-HLA antibodies eluted from sensitized patients and incubated with human umbilical vein endothelial cells (HUVECs) at different concentrations. Their effect on HUVEC phenotype was then analysed. Anti-donor antibody returned in 4/7 patients, without evidence of hyperacute rejection. Three out of four of these 'accommodated' grafts showed specific endothelial up-regulation of Bcl-xL and 2/2 tested positive for endothelial IgG deposition. HUVECs incubated with subsaturating concentrations of anti-HLA antibody showed increased expression of Bcl-xL, were rendered refractory to endothelial cell activation and became resistant to complement-mediated lysis. In contrast, HUVECs incubated with saturating concentrations underwent activation and expressed low levels of Bcl-xL. In conclusion, endothelial Bcl-xL expression defines the accommodation process in human allografts and this phenotype may be initiated by exposure of endothelium to low concentrations of anti-donor HLA antibodies.     

The Opioid Haemorphin‐7 in Horses During Low‐speed and High‐speed Treadmill Exercise to Fatigue

The opioid neuropeptide haemorphin‐7 was measured, by immunoreactivity, in Standardbred horses during low‐speed (7 m/s) and high‐speed (10 m/s) endurance exercises, lasting 49–58 and 12–16 min respectively. In parallel, heart rate, muscle temperature and plasma lactate concentrations were measured. The profile of the low‐speed exercise showed significantly increased heart rate after 10 min [154 beats per minute (bpm)]. After the exercise, muscle temperature (42.1°C) and plasma lactate (4.8 mmol/l) were significantly increased. The profile of the high‐speed exercise was comparatively characterized by a higher increase of heart rate after 5 min (194 bpm) and higher increases of muscle temperature (43.2°C) and lactate levels (15.8 mmol/l) after the exercise. The horses were probably exhausted by glycogen depletion in the low‐speed exercise and by muscle pH decrease in the high‐speed exercise. Haemorphin‐7 increased significantly during the high‐speed exercise (274.8 fmol/ml) but not during low speed (108.3 fmol/ml), coincident with the results of lactate. These results suggest that plasma haemorphin‐7 is measurable in the horse by immunoreactivity, and that intense exercise stimulates release of this opioid. Such endogenous opioids are most likely involved in regulatory functions associated with pain, physical effort, inflammation, and blood pressure variation in horses, as have been established in other species.     

Staging of Bilateral Lung Transplantation for High‐Risk Patients With Interstitial Lung Disease: One Lung at a Time

The choice of a single or bilateral lung transplant for interstitial lung disease (ILD) is controversial, as surgical risk, long‐term survival and organ allocation are competing factors. In an effort to balance risk and benefit, our center adopted a staged bilateral lung transplant approach for higher surgical risk ILD patients where the patient has a single lung transplant followed by a second single transplant at a later date. We sought to understand the surgical risk, organ allocation and early outcomes of these staged bilateral recipients as a group and in comparison to matched single and bilateral recipients. Our analysis demonstrates that staged bilateral lung transplant recipients (n = 12) have a higher lung allocation score (LAS), lower pulmonary function tests and a lower glomerular filtration rate prior to the first transplant compared to the second (p < 0.01). There was a shorter length of hospital stay for the second transplant (p = 0.02). The staged bilateral compared to the single and bilateral case‐matched controls had comparable short‐term survival (p = 0.20) and pulmonary function tests at 1 year. There was a higher incidence of renal injury in the conventional bilateral group compared to the single and staged bilateral groups. The staged bilateral procedure is a viable option in select ILD patients.     

Discovery of non‐HLA antibodies associated with cardiac allograft rejection and development and validation of a non‐HLA antigen multiplex panel: From bench to bedside

We analyzed humoral immune responses to nonhuman leukocyte antigen (HLA) after cardiac transplantation to identify antibodies associated with allograft rejection. Protein microarray identified 366 non‐HLA antibodies (>1.5 fold, P < .5) from a discovery cohort of HLA antibody–negative, endothelial cell crossmatch–positive sera obtained from 12 cardiac allograft recipients at the time of biopsy‐proven rejection. From these, 19 plasma membrane proteins and 10 autoantigens identified from gene ontology analysis were combined with 48 proteins identified through literature search to generate a multiplex bead array. Longitudinal sera from a multicenter cohort of adult cardiac allograft recipients (samples: n = 477 no rejection; n = 69 rejection) identified 18 non‐HLA antibodies associated with rejection (P < .1) including 4 newly identified non‐HLA antigenic targets (DEXI, EMCN, LPHN1, and SSB). CART analysis showed 5/18 non‐HLA antibodies distinguished rejection vs nonrejection. Antibodies to 4/18 non‐HLA antigens synergize with HLA donor‐specific antibodies and significantly increase the odds of rejection (P < .1). The non‐HLA panel was validated using an independent adult cardiac transplant cohort (n = 21 no rejection; n = 42 rejection, >1R) with an area under the curve of 0.87 (P < .05) with 92.86% sensitivity and 66.67% specificity. We conclude that multiplex bead array assessment of non‐HLA antibodies identifies cardiac transplant recipients at risk of rejection.     

High‐order observers‐based LQ control scheme for wind speed and uncertainties estimation in WECSs

Tip speed ratio control is a popular method in wind energy conversion systems in order to capture the maximum power. This method, however, requires wind speed information, which is difficult in practice to accurately measure it. Therefore, estimation methods are usually applied, where a high‐precision estimate leads to a high‐efficient system. Based on the fact that the wind speed varies in a random way, this paper proposes a generalized high‐order observer to estimate the aerodynamic torque and the wind speed accordingly. This observer algorithm releases the assumption that the wind speed should be slowly varying, which is required in previous observer designs. Moreover, two other generalized high‐order observers are also applied to estimate the uncertainties, which depend on state variables and cannot be considered as slow‐varying disturbances. Using the outputs of these observers, a robust high‐performance optimal control system is developed for the rotor speed to keep the optimal tip speed ratio. The stability analysis of the designed control system is fully presented. The effectiveness of the proposed technique is validated via simulation studies.     

High‐dose rocuronium for rapid‐sequence induction and reversal with sugammadex in two myasthenic patients

The anesthetic management of patients affected by myasthenia gravis is usually challenging in elective surgery and even more so in emergency procedures. The difficulties involved are several‐fold, ranging from the choice of an appropriate muscle relaxant (i.e. one that enables safe and rapid airway management) to neuromuscular monitoring and normal muscular recovery. Additionally, optimizing patient conditions – either pharmacologically or with plasmapheresis – before intervention is well beyond the realm of possibility. We discuss the anesthetic management of two myasthenic patients undergoing emergency surgery (for sigmoid perforation and upper gastrointestinal bleeding respectively). In both cases, we opted for rapid‐sequence induction with high‐dose rocuronium to prevent inhalation of gastric contents. We also report on the implication of neuromuscular monitoring. We found that the rocuronium–sugammadex combination was a useful and effective option in the emergency setting.     

Histological picture of antibody‐mediated rejection without donor‐specific anti‐HLA antibodies: Clinical presentation and implications for outcome

In this cohort study (n = 935 transplantations), we investigated the phenotype and risk of graft failure in patients with histological criteria for antibody‐mediated rejection (ABMR) in the absence of circulating donor‐specific anti‐human leukocyte antigen (HLA) antibodies (DSA), and compared this to patients with definite ABMR and HLA‐DSA‐positivity. The histological picture did not differ between HLA‐DSA‐positive (n = 85) and HLA‐DSA‐negative (n = 123) cases of ABMR histology, apart from increased complement split product 4d (C4d) deposition in the peritubular capillaries in HLA‐DSA‐positive cases. Histology of ABMR without HLA‐DSA was more transient than DSA‐positive ABMR, and patients with ABMR histology without HLA‐DSA had graft survival superior to that of HLA‐DSA‐positive patients, independent of concomitant T cell–mediated rejection (38.2%) or borderline changes (17.9%). Multivariate analysis showed that the risk of graft failure was not higher in patients with histological picture of ABMR (ABMR_h) in the absence of HLA‐DSA, compared to patients without ABMR_h. Despite an association between C4d deposition and HLA‐DSA‐positivity, using C4d deposition as alternative for the DSA criterion in the diagnosis of ABMR, as proposed in Banff 2017, did not contribute to the prognosis of graft function and graft failure. We concluded that biopsies with ABMR_h but without detectable HLA‐DSA represent a distinct, often transient phenotype with superior allograft survival.     

Calculated PRA: Initial Results Show Benefits for Sensitized Patients and a Reduction in Positive Crossmatches

The calculated panel reactive antibody (CPRA), which is based upon unacceptable HLA antigens listed on the waitlist form for renal transplant candidates, replaced PRA as the measure of sensitization among US renal transplant candidates on October 1, 2009. An analysis of the impact of this change 6 months after its implementation shows an 83% reduction in the number of kidney offers declined nationwide because of a positive crossmatch. The increasing acceptance and utilization of unacceptable HLA antigens to avoid offers of predictably crossmatch‐positive donor kidneys has increased the efficiency of kidney allocation, resulting in a significant increase in the percentage of transplants to broadly sensitized (80+% PRA/CPRA) patients from 7.3% during the period 07/01/2001–6/30/2002 to 15.8% of transplants between 10/1/09–3/31/10. The transplant rates per 1000 active patient‐years on the waitlist also increased significantly for broadly sensitized patients after October 1, 2009. These preliminary results suggest that ‘virtual’ positive crossmatch prediction based on contemporary tools for identifying antibodies directed against HLA antigens is effective, increases allocation efficiency and improves access to transplants for sensitized patients awaiting kidney transplantation.