Nucleotide metabolic mismatches in mammalian hearts: implications for transplantation

Introduction

Human donor organ shortages have led surgeons and scientists to explore the use of animals as alternative organ sources. Acute thrombovascular rejection (AVR) is the main hurdle in xenotransplantation. Disparities in nucleotide metabolism in the vessels of different species may contribute significantly to the microvascular component of AVR.

Methods

We evaluated the extent of nucleotide metabolism mismatch in selected organs and endothelial cells of different mammals with particular focus on the changes in activity of ecto-5’-nucleotidase (E5’N) elicited by exposure of porcine hearts or endothelial cells to human blood (ex vivo) or human plasma (in vitro).

Results

E5’N activity in the rat heart was significantly higher than in other species. We noted a significant difference (p<0.001) in E5’N activity between human and pig endothelial cell lines. Initial pig aortic endothelial E5’N activity decreased in vitro after a three-hour exposure to human and porcine plasma while remaining constant in controls. Ex vivo perfusion with fresh human blood for four hours resulted in a significant decrease of E5’N activity in both wild type and transgenic pig hearts overexpressing human decay accelerating factor (p<0.001).

Conclusions

This study provides evidence that mismatches in basal mammalian metabolic pathways and humoral immunity interact in a xenogeneic environment. Understanding the role of nucleotide metabolism and signalling in xenotransplantation may identify new targets for genetic modifications and may lead to the development of new therapies extending graft survival.     

Laminar flow reduces cases of surgical site infections in vascular patients

Introduction

Numerous strategies are employed routinely in an effort to lower rates of surgical site infections (SSIs). A laminar flow theatre environment is generally used during orthopaedic surgery to reduce rates of SSIs. Its role in vascular surgery, especially when arterial bypass grafts are used, is unknown.

Methods

A retrospective review of a prospectively maintained database was undertaken for all vascular procedures performed by a single consultant over a one-year period. Cases were performed, via random allocation, in either a laminar or non-laminar flow theatre environment. Demographic data, operative data and evidence of postoperative SSIs were noted. A separate subgroup analysis was undertaken for patients requiring an arterial bypass graft. Univariate and multivariate logistical regression was undertaken to identify significant factors associated with SSIs.

Results

Overall, 170 procedures were analysed. Presence of a groin incision, insertion of an arterial graft and a non-laminar flow theatre were shown to be predictive of SSIs in this cohort. In the subgroup receiving arterial grafts, only a non-laminar flow theatre environment was shown to be predictive of an SSI.

Conclusions

This study suggests that laminar flow may reduce incidences of SSI, especially in the subgroup of patients receiving arterial grafts.     

The effects of primer precuring on the shear bond strength between gold alloy surfaces and metal brackets

The objective of this study was to investigate the effects of precuring of primer coated on bracket bases on the strength of bonds between metal brackets and gold alloy. Square type III gold alloy plates were sandblasted with 30 μm silicon dioxide. After silica coating, excessive particles were removed gently with air. Silane was then applied, and maxillary central incisor metal brackets were bonded to each conditioned alloy surface with Transbond XT. Half of the specimens were precured at the bracket base after primer coating and the other half was not precured before bonding to the alloy surface. After bracket positioning, samples were cured using a light emitting diode (LED) for 40 seconds. Shear bond strengths were tested and adhesive remnant index (ARI) was evaluated after 1 hour and 24 hours. The primer precuring and 24 hours group exhibited highest bond strength (12.53 MPa) and the no precuring and 1 hour group showed lowest bond strength (5.58 MPa). Precured groups showed lower ARI scores. Due to the shallow curing depth of LED light and inhibition of transillumination at the metal surface, primer precuring at the bracket base is required for secure bracket bonding on gold alloy surfaces using LED curing units.     

Ultrastructural localization of human platelet thrombospondin, fibrinogen, fibronectin, and von Willebrand factor in frozen thin section

We have investigated the localization of thrombospondin (TSP), fibrinogen, fibronectin, and von Willebrand factor in human platelets by transmission electron microscopy of antibody-stained ultrathin frozen sections. In negatively stained thin sections, alpha granules were identified on the basis of their smooth, roughly spherical shape, size, single limiting electron-lucent 100 A membrane, and frequent presence of electron-dense nucleoid. In contrast, mitochondria exhibited characteristic double membranes and cristae. Sections were separately stained with affinity-purified polyclonal antibodies to these proteins as well as with three monoclonal anti-TSP antibodies. Antibody specificity was documented in radioimmunoassays, by immunofluorescent cross-blocking, and by staining of bands of appropriate mobility in Western blots of whole platelets. Bound antibody was visualized using a 5-nm colloidal gold-avidin conjugate. In resting cells, staining of virtually all alpha granules was observed for all four proteins. In contrast, consistent staining was absent from other organelles, including plasma membranes, mitochondria, and vacuolar structures that may represent the open canalicular system.     

Fibronectin binding to thrombin-stimulated platelets: evidence for fibrin(ogen) independent and dependent pathways

Plasma fibronectin binds in a specific and saturable manner to thrombin- stimulated platelets. gamma-Thrombin stimulated 80% as much fibronectin binding to platelets as alpha-thrombin with conversion of less than or equal to 1% of platelet fibrinogen to fibrin. Afibrinogenemic and normal platelets bound similar quantities of fibronectin in the presence of calcium or magnesium-ethylene glycol tetra-acetic acid (EGTA). These observations indicate that fibronectin can interact with platelets without involvement of fibrin or fibrinogen. Nevertheless, two different effects of fibrin(ogen) on fibronectin binding were observed. First, exogenous fibrinogen inhibited fibronectin binding to thrombin-stimulated platelets. This inhibition was unidirectional, as fibronectin did not inhibit fibrinogen binding to ADP or thrombin- stimulated cells. Second, formaldehyde-fixed cells with surface- associated fibrin bound significant quantities of fibronectin. This interaction required calcium and did not occur on fixed cells with or without surface-bound fibrinogen. A portion of the ligand bound to fixed cells with surface-associated fibrin was modified to form a derivative with a molecular weight identical to that of the fibronectin subunit cross-linked to the alpha-chain of fibrin. This high mol wt derivative was also observed to a variable extent with living cells in the presence of magnesium or calcium but not in the presence of magnesium-EGTA. Thus, fibronectin binds to platelets by at least two mechanisms: (1) a fibrin(ogen)-independent pathway that requires divalent ions and is inhibited by exogenous fibrinogen; and (2) a fibrin-dependent pathway with an absolute calcium requirement. With nonaggregated, thrombin-stimulated platelets, the former pathway appears to predominate.     

Evaluation of Disposal Stability for Cement Solidification of Lime Waste

The Korea Atomic Energy Research Institute (KAERI) obtains UO₂ powder using the ammonium uranyl carbonate (AUC) wet process. Hydrated lime (Ca(OH)₂) is used to neutralize liquid wastes produced from the AUC process, and the resulting byproduct is known as lime waste. The purpose of this study is to determine optimum operating conditions for cementation of radioactive lime waste produced from the AUC process, and to evaluate the structural stability and leaching stability of cement waste form. The waste acceptance criteria (WAC) of a waste disposal facility in Korea were used to evaluate the cement waste form samples. The maximum lime waste content guaranteeing the shape stability of cement waste form was found to be 80 wt.% or less. Considering the economic feasibility and error of the cementation process, the optimum operating conditions were achieved at a lime waste content of 75 wt.% and a water-to-cement (w/c) ratio of 2.0. The compressive strength of cement waste form samples prepared under optimal operating conditions was 61.4, 76.3, and 61.0 kgf/cm² after the thermal cycling test, water immersion test, and irradiation, respectively, satisfying the compressive strength of 35.2 kgf/cm² specified in WAC. A leaching test was performed on the samples, and the leachability indexes (LX) of Cs, Sr, and Co nuclides were 7.63, 8.02, and 10.89, respectively, which are all higher than the acceptance criterion of 6. The results showed that the cement waste forms prepared under optimal operating conditions satisfied the WAC in terms of structural stability and leaching stability. As such, the proposed cement solidification method for lime waste disposal can be effective in solidifying lime waste powder produced during the neutralization of liquid wastes in the AUC process.     

Effects of Dietary Fat to Carbohydrate Ratio on Obesity Risk Depending on Genotypes of Circadian Genes

Although the impacts of macronutrients and the circadian clock on obesity have been reported, the interactions between macronutrient distribution and circadian genes are unclear. The aim of this study was to explore macronutrient intake patterns in the Korean population and associations between the patterns and circadian gene variants and obesity. After applying the criteria, 5343 subjects (51.6% male, mean age 49.4 ± 7.3 years) from the Korean Genome and Epidemiology Study data and nine variants in seven circadian genes were analyzed. We defined macronutrient intake patterns by tertiles of the fat to carbohydrate ratio (FC). The very low FC (VLFC) was associated with a higher risk of obesity than the optimal FC (OFC). After stratification by the genotypes of nine variants, the obesity risk according to the patterns differed by the variants. In the female VLFC, the major homozygous allele of CLOCK rs11932595 and CRY1 rs3741892 had a higher abdominal obesity risk than those in the OFC. The GG genotype of PER2 rs2304672 in the VLFC showed greater risks for obesity and abdominal obesity. In conclusion, these findings suggest that macronutrient intake patterns were associated with obesity susceptibility, and the associations were different depending on the circadian clock genotypes of the CLOCK, PER2, and CRY1 loci.