Reduction of the major porcine xenoantigen Galoc(1,3)Gal by expression of α(1,2)fucosyltransferase

Abstract: Although removal of the Galα(1,3)Gal antigen from pigs would prevent hyperacute graft rejection, the technique of homologous recombination to knock out the α 1,3 galactosyltransferase gene is not available for pigs, and an alternative strategy is presented. As both α 1,3 galactosyltransferase and α 1,2 fucosyltransferase use the same substrate (N-acetyl lacto-samine), competition between the transferases in vitro and in vivo was examined. The data show that there is indeed a hierarchy of these gly-cosyltransferases competing for the same substrate, and that α 1,2 fuco-syltransferase takes precedence over α 1,3 galactosyltransferase: a) COS cells simultaneously transfected with cDNA clones encoding α, 2 fuco-syltransferase and α 1,3 galactosyltransferase show preferential expression of the H substance (synthesised by α 1,2fucosyltransferase) rather than Galα(1,3)Gal (synthesised by α 1,3galactosyltransferase), even though α 1,3galactosyltransferase mRNA and functional enzyme was present, b) In a pig kidney cell line that expressed both the Galα(1,3)Gal and H, the increased expression of H induced by the transfection and stable expression of α 1,2fucosyltransferase resulted in decreased expression of Galα(1,3)Gal. c) Coexpression of α 1,2fucosyltransferase and α 1,3galactosyltransferase in either COS cells or the pig cell line resulted in decreased human antibody binding and complement-mediated cell lysis, d) Transgenic mice, ubiquitously expressing α 1,2fucosyltransferase show a major decrease in Galα-(1,3)Gal expression and a decrease in natural human antibody binding. These findings have important implications for xenotransplantation in that α,2fucosyltransferase transgenic pigs could be a source of donors for xenotransplantation to humans.     

Inhibition of liver metastasis by all-trans retinoic acid incorporated into O/W emulsions in mice

All-trans retinoic acid (ATRA) was incorporated into lipid emulsions in an attempt to alter its distribution characteristics and improve its inhibition of liver cancer metastasis. Lipid emulsions composed of egg phosphatidylcholine, cholesterol, and soybean oil were the optimized carriers for ATRA delivery, as shown by the submicron particle size and high incorporation efficiency. The particle size and zeta potential of ATRA incorporated into emulsions were about 133 nm and -11 mV, respectively. In vitro drug release study demonstrated that the release of ATRA from emulsions was sustained in the absence and present of bovine serum albumin, suggesting that ATRA was stable when incorporated in emulsions. After intravenous administration in mice, [3H]cholesteryl hexadecyl ether incorporated into emulsion, which is the inherent distribution of emulsions, accumulated gradually mainly in the liver. The blood concentration and hepatic accumulation of [3H]ATRA incorporated into emulsion was significantly higher than that of serum dissolving [3H]ATRA, which represent the original distribution characteristic of free ATRA. In a murine liver metastasis model by colon adenocarcinoma, the liver metastasis number and liver weight were significantly reduced and the survival time of mice was prolonged following intravenous injection of ATRA incorporated into emulsions.     

Regional citrate anticoagulation reduces polymorphonuclear cell degranulation in critically ill patients treated with continuous venovenous hemofiltration

Citrate which chelates ionized calcium can be used as regional anticoagulation in continuous venovenous hemofiltration (CVVH). This is the first study conducted to examine the potentially additive benefit effect of regional citrate anticoagulation (RCA) on polymorphonuclear (PMN) cell degranulation of myeloperoxidase (MPO) and cytokines production in patients with critically acute kidney injury (AKI) undergoing CVVH treatment. This prospective randomized controlled trial was conducted in 20 critically ill patients with AKI who underwent CVVH. The patients were randomized into regional citrate group (n=10) and heparin group (n=10). The pre-dilution CVVH with polyethersulfone dialyzers were utilized in both groups. The levels of pre-filter and post-filter MPO as well as inflammatory and anti-inflammatory cytokines were measured at baseline, 6h, and 24 h after initiating CVVH. In the heparin group, the post-filter serum MPO levels were significantly higher than the pre-filter (median 49.0 vs. 60.5 ng/mL, P<0.05) at 6 h. There were no significant differences between pre- and post-dialyzer MPO levels in the citrate group. Citrate could significantly decrease systemic pre-filter serum MPO levels from baseline at 6 h (median 43.5 vs. 17.3 ng/mL, P<0.01) as well as IL-8 levels (P<0.05) whereas heparin provided only significant TNF-α reduction (P<0.05). The CVVH circuit survival in the citrate group was longer than the heparin group. In conclusion, citrate, utilized as a regional anticoagulant in CVVH, can reduce both membrane bioincompatibility-induced and systemic oxidative stress and inflammation, and can prolong CVVH circuit survival time.     

Intermediate-Term Effects of Transcatheter Secundum Atrial Septal Defect Closure on Cardiac Remodeling in Children and Adults

The study aimed to investigate the intermediate-term effects of transcatheter atrial septal defect (ASD) closure on cardiac remodeling in children and adult patients. Between December 2003 and February 2009, 117 patients (48 males, 50 adults) underwent transcatheter ASD closure with the Amplatzer septal occluder (ASO). The mean age of the patients was 15 years, and the mean follow-up period was 25.9 ± 12.4 months. New York Heart Association (NYHA) class, electrocardiographic parameters, and transthoracic echocardiographic (TTE) examination were evaluated before the ASD closure, then 1 day, 1 month, 6 months, 12 months, and yearly afterward. Transcatheter ASD closure was successfully performed for 112 (96%) of the 117 patients. The mean ASD diameter measured by transesophageal echocardiography (TEE) was 14.0 ± 4.2 mm, and the mean diameter stretched with a sizing balloon was 16.6 ± 4.8 mm. The mean size of the implanted device was 18.6 ± 4.9 mm. The Qp/Qs ratio was 2.2 ± 0.8. The mean systolic pulmonary artery pressure was 40 ± 10 mmHg. At the end of the mean follow-up period of 2 years, the indexed right ventricular (RV) end-diastolic diameter had decreased from 36 ± 5 to 30 ± 5 mm/m² (p = 0.005), and the indexed left ventricular (LV) end-diastolic diameter had increased from 33 ± 5 to 37 ± 6 mm/m² (p = 0.001), resulting in an RV/LV ratio decreased from 1.1 ± 0.2 to 0.8 ± 0.2 (p = 0.001). The New York Heart Association (NYHA) functional capacity of the patients was improved significantly 24 months after ASD closure (1.9 ± 0.5 to 1.3 ± 0.5; p = 0.001). At the 2-year follow up electrocardiographic examination, the P maximum had decreased from 128 ± 15 to 102 ± 12 ms (p = 0.001), the P dispersion had decreased from 48 ± 11 to 36 ± 9 ms (p = 0.001), and the QT dispersion had decreased from 66 ± 11 to 54 ± 8 ms (p = 0.001). Five of six patients experienced resolution of their preclosure arrhythmias, whereas the remaining patient continued to have paroxysmal atrial fibrillation. A new arrhythmia (supraventricular tachycardia) developed in one patient and was well controlled medically. Transcatheter ASD closure leads to a significant improvement in clinical status and heart cavity dimensions in adults and children, as shown by intermediate-term follow-up evaluation. Transcatheter ASD closure can reverse electrical and mechanical changes in atrial myocardium, resulting in a subsequent reduction in P maximum and P dispersion times.     

Carotid artery thickness in children and young adults with end stage renal disease

Atherosclerosis is a major cause of morbidity and mortality for ESRD patients and we have little knowledge about the presence and risk factors of atherosclerosis in children with CRF. The measurement of carotid artery intima-media thickness (cIMT) using high-resolution ultrasonography is suggested as an excellent marker of subclinical atherosclerosis. In this study, we aimed to investigate the presence of atherosclerosis and to determine the relationship between atherosclerosis and some risk factors in children and young adults with ESRD. Thirty-four patients with ESRD and 20 controls were included in this study. The measurement of cIMT was performed by using a linear B-mode 7.5-MHz ultrasound transducer. We determined anemia, abnormal calcium/phosphate metabolism, hyperhomocysteinemia, hypertriglyceridemia and increased lipoprotein (a) levels in the ESRD group. The cIMT in the ESRD group was higher than in the control group (P<0.05). SBP, DBP, MAP, LVMI and LVH prevalence were statistically higher in the ESRD group (P<0.05). There were significant positive correlations between cIMT and LVMI, MBP, whereas a significant negative correlation was determined between cIMT and PTH in the ESRD group (P<0.05). When a multiple linear regression analysis was performed with cIMT as a dependent variable and LVMI, MBP, PTH, as independent variables, a significant positive correlation was determined between cIMT and LVMI (P<0.05). In conclusion, we think that arteriopathy occurs in children with ESRD. Left ventricular hypertrophy and hypertension may associate with vascular changes in children and young adults with ESRD. Further investigations are necessary to explain association of LVMI index with cIMT.     

Insulin resistance and atherosclerosis

The epidemic of obesity in the developed world over the last two decades is driving a large increase in type 2 diabetes and consequentially setting the scene for an impending wave of cardiovascular morbidity and mortality. It is only now being recognized that the major antecedent of type 2 diabetes, insulin resistance with its attendant syndrome, is the major underlying cause of the susceptibility to type 2 diabetes and cardiovascular disease. In metabolic tissues, insulin signaling via the phosphatidylinositol-3-kinase pathway leads to glucose uptake so that in insulin resistance a state of hyperglycemia occurs; other factors such as dyslipidemia and hypertension also arise. In cardiovascular tissues there are two pathways of insulin receptor signaling, one that is predominant in metabolic tissues (mediated by phosphatidylinositol-3-kinase) and another being a growth factor-like pathway (mediated by MAPK); the down-regulation of the former and continued activity of the latter pathway leads to atherosclerosis. This review addresses the metabolic consequences of the insulin resistance syndrome, its relationship with atherosclerosis, and the impact of insulin resistance on processes of atherosclerosis including insulin signaling in cells of the vasculature.     

The effect of high dose melatonin on cardiac ischemia- reperfusion Injury

Purpose

Melatonin, the most potent scavenger of toxic free radicals, has been found to be effective in protecting against pathological states due to the release of reactive oxygen species. This study was performed to establish the effect of high dose melatonin on protection against ischemia-reperfusion (I/R) injury in rat hearts.

Materials and Methods

Forty male Sprague-Dawley rats were used in this study. They were separated into four groups of ten rats each. A left coronary artery occlusion was induced in the rats by ligating the artery for 20 minutes and then releasing the ligation (reperfusion) afterwards. The control group was Group A. Group B was subjected to myocardial ischemia-reperfusion without any treatment, while Group C underwent myocardial ischemia-reperfusion with a melatonin treatment before the ischemia. Group D was subjected to myocardial ischemia-reperfusion with a melatonin treatment before the reperfusion. After 20 minutes of reperfusion, blood samples were obtained from each group for biochemical studies, and the animals were sacrificed for histological and, immunohistochemical examinations of the myocardial tissue.

Results

We found that the cardiac troponin T(cTn-T) levels were significantly increased in Group B when all groups were compared. In the Group C rats treated with melatonin, the cTn-T values were significantly lower than those in Groups B and D. In addition, malondialdehyde (MDA) and antioxidant enzymes including, superoxide dismutase (SOD) and myeloperoxidase (MPO) were lower than those in Group B in the melatonin treated groups. The differences were statistically significant (p < 0.05). Histopathologic and immunohistopathologic studies also supported the effectiveness of melatonin.

Conclusion

Our study suggests that high dose melatonin, appears to offer protection against cardiac ischemia-reperfusion injuries in rats by scavenging the free radicals and could have a potential clinical use in the management of myocardial ischemia.     

Surface ultrastrucure of larva and puparia of blow fly Hypopygiopsis tumrasvini Kurahashi (Diptera: Calliphoridae)

Flies of the genus Hypopygiopsis are forensically important, as their larvae are found to associate with human corpses. In this study, the ultrastructure of larvae and puparia of Hypopygiopsis tumrasvini Kurahashi is presented using scanning electron microscopy (SEM). The larvae are vermiform-shaped, creamy white, and have a smooth integument. The pseudocephalon of larvae bears sensory structures, i.e., antenna, maxillary palpus, and ventral organ. Two tufts of fresh outgrowths (or cerri) were observed along the dorsal margin of the mouth opening of the first instar; whereas a strong mouth hook was apparent in the second and third instars. Keilin??s organ, the sensory structure, was noticeable on the ventral surface of the thoracic segments in all instars. In the second and third instars, six conspicuous tubercles were present along the peripheral rims of the last abdominal segment. The puparia were relatively large, measuring 7.77?C9.51?mm in length and 3.10?C3.97?mm in width. At the latero-dorsal edge of the first abdominal segment, a cluster of ??125 bubble membranes was seen in young puparia; whereas a minute pupal respiratory horn was observed in old one. An SEM image revealed antler-like projections lined within a chamber of a broken pupal respiratory horn. Comparison on number of the bubble membranes of the other blow fly species was shown and the role of pupal respiratory horn compared with other dipterans was discussed.