Different role of advanced glycation end products in pathology of transplanted heart in patients with or without diabetes mellitus type 2.

BACKGROUND: Aim of the study was to localize advanced glycation end products (AGEs) in late endomyocardial biopsies (EMBs) of orthotopic heart transplant (OHT) recipients with and without diabetes mellitus (DM) to correlate their presence with acute rejection episodes (ARE) and cardiac allograft vasculopathy (CAV). MATERIALS AND METHODS: Elective EMBs were performed at 3 years post-OHT in 64 subjects, with DM (59 M/5 F), of overall mean age of 49 +/- 8 years and 24 patients, without DM (21 M/3 F), of overall mean age of 42 +/- 10y. Localization of myocardial AGEs in paraffin sections was assessed immunochemically using mouse monoclonal anti-AGE antibodies (clone 6d12) on cardiomyocytes, stromal cells, connective tissue elements and capillaries. RESULTS: The occurrence of AGEs was similar in DM versus non-DM subjects: namely, cardiocytes 73% versus 63%, stroma 33% versus 33%, connective tissue 13% versus 9%, and capillaries 31% versus 33%, respectively. Only in the DM group. The acute rejection episodes and mean EMB score significantly correlated with AGE presence in cardiomyocytes (r = 0.29/0.3; P = .02/.02; Spearman). There was no relation between AGE occurrence and CAV diagnosis among DM subjects, while the time free from angiographically confirmed CAV or a CAV-related event was significantly shorter among non-DM recipients without AGEs in capillaries and/or cardiocytes (P = .014/.017/.014/.03, respectively; log-rank). CONCLUSION: AGE occurrence in OHT recipients with DM was related to ARE, but not to CAV; in contrast, among non-DM patients it was not correlated with ARE, but their absence predicted CAV.

Safety evaluation of 1,4-alpha-glucan branching enzymes from Bacillus stearothermophilus and Aquifex aeolicus expressed in Bacillus subtilis.

1,4-alpha-Glucan branching enzyme (BE; EC 2.4.1.18) is a key biocatalyst in the synthesis of polysaccharides, and is therefore useful in the production of food ingredients. The BEs evaluated in this study (BE-01 and BE-02) are obtained by fermentation of Bacillus subtilis expressing the BE gene from either Bacillus stearothermophilus strain TRBE14 or Aquifex aeolicus strain VF5. The safety of BE-01 and BE-02 have not been previously evaluated, and therefore, both were subjected to standard toxicological testing. In a battery of standard Salmonella typhimurium strains (TA98, TA100, TA1535, and TA1537) and in Escherichia coli WP2uvrA, both with and without metabolic activation, neither BE-01 nor BE-02 exhibited mutagenic activity. Similarly, neither was associated with clastogenic properties in Chinese hamster ovary cells in an in vitro chromosomal aberration assay. In rats, oral administration of BE-01 or BE-02 at doses of up to 15 mL/kg body weight/day (approximately 870 and 900 mg/kg body weight/day, respectively) for 13 weeks did not produce compound-related clinical signs or toxicity, changes in body weight gain, food consumption, hematology, clinical chemistry, urinalysis, organ weights, or in any gross and microscopic findings. The results of this study support the safety of BE-01 and BE-02 in food production.

Advanced glycation end products in the development of ischemic and dilated cardiomyopathy in patients with diabetes mellitus type 2.

INTRODUCTION: Hyperglycemia intensifies nonenzymatic glucose coupling to tissues, resulting in myocardial stiffness and formation of advanced glycation end products (AGE). The aim of this study was to assess seeking AGE in the myocardium from patients with type 2 diabetes (DM2) subjected to orthotopic heart transplantation (OHT), seeking to establish whether AGE play a role in the development of cardiomyopathies leading to OHT. MATERIAL: The 2 studied groups consisted of 11 hearts explanted from patients with ischemic cardiomyopathy+DM2 (ICM+DM2, 55 +/- 6.5 years) and 8 from dilated cardiomyopathy+DM2 (DCM+DM2, 49.6 +/- 4.5 years). Comparative subgroups were composed of nondiabetic explanted hearts, 41 with ICM (52.8 +/- 5.8 years) and 41 with DCM (52.7 +/- 4.2 years). All patients were males. METHODS: We examined immunohistochemical localization of AGE using a semiquantitative scale of reaction intensity in cardiomyocytes, fibroblasts, capillaries, arterioles, and arteries. Additionally, we calculated the scores for cardiocytes (AGE(Cardiocyte)) and all left ventricular components (AGE(LV)). RESULTS: The cytoplasmic AGE deposits in cardiomyocytes were predominantly diffuse-granular in DM2 groups, whereas nondiabetic groups showed a lack of a reaction or a diffuse pattern. There were no differences in the reaction intensity between the 2 studied groups, or 2 comparative groups. All myocardial constituents showed higher AGE intensity in DM2 than nondiabetic groups. Only in the ICM+DM2 group did the DM2 duration correlate with AGE staining in selected myocardial layers and with AGE(Cardiocyte) and AGE(LV). CONCLUSIONS: The presence of AGE in the hearts of patients requiring transplantation was related to the duration of DM2. The deposition of AGE in left ventricular myocardium was enhanced by DM2 particularly in patients with ICM.

Cardiocyte nuclear chromatin density correlates with transplanted heart left ventricular mass.

INTRODUCTION: Cardiocyte hypertrophy is accompanied by polyploidy, seen as a decrease in chromatin density in the enlarged nucleus. Repeated biopsies of a transplanted heart offer the possibility of a dynamic evaluation of these phenomena. The aim of this work was an evaluation of cardiocyte nuclear chromatin density in transplanted hearts during long-term follow-up. MATERIALS AND METHODS: The material encompassed myocardial biopsy specimens taken during the first week, first month, and then on an annual basis up to 10 years after surgery. Only biopsy specimens with no rejection were considered (grade "0" International Society for Heart and Lung Transplantation [ISHLT] 122 biopsy specimens). The control group consisted of 7 donor heart specimens. We evaluated the optical density-mean gray level-of cardiomyocyte nuclear chromatin. We determined correlations of this index with the nuclear area, and with left ventricle ultrasound measurements, using correlation analysis. RESULTS: The chromatin mean gray level decreased with time, correlating positively with interventricular septum thickness, left ventricle posterior wall diameter, and left ventricular mass. Analysis of individual periods showed a significant positive correlation of the mean grey level with the cardiocyte nuclear surface in year 3, 4, and 9 after transplantation, thereby suggesting the occurrence of polyploidy at those times. The significant negative correlation of these values (1 week and 1 year) indicated normalization of early cardiocyte hypertrophy. CONCLUSIONS: With the passage of time chromatin condenses, leading to pyknosis. The activity of cardiocyte chromatin correlated with left ventricular hypertrophy. Compensatory cardiomyocyte polyploidy is a periodical phenomenon.

Evaluation of the potential in vivo genotoxicity of quercetin.

Quercetin, a naturally occurring flavonol commonly detected in apples, cranberries, blueberries, and onions, has been reported to possess antioxidant, anti-carcinogenic, anti-inflammatory, and cardioprotective properties. While positive results have been consistently reported in numerous in vitro mutagenicity and genotoxicity assays of quercetin, tested in vivo, quercetin has generally produced negative results in such studies. Furthermore, no evidence of carcinogenicity related to the oral administration of quercetin was observed in chronic rodent assays. In order to further define the in vivo genotoxic potential of quercetin, a bone marrow micronucleus assay and an unscheduled DNA synthesis (UDS) assay were conducted in Wistar rats. Administered orally to male rats at dose levels of up to 2000 mg/kg body weight, quercetin did not increase the number of micronucleated polychromatic erythrocytes (MN-PCE) 24 or 48 h following dosing in the micronucleus assay. Likewise, orally administered quercetin (up to 2000 mg/kg body weight) did not induce UDS in hepatocytes of male or female rats. While measurable levels of metabolized quercetin were observed in rat plasma samples for up to 48 h after dosing, peaking at 1h following treatment administration, the unmetabolized aglycone was not identified in either plasma or bone marrow. With the exception of only a few rats, the aglycone was also not detected in liver tissue. These results demonstrate that quercetin is not genotoxic under the conditions of these assays and further support the negative results of previously conducted in vivo assays.

The Threshold of Toxicological Concern (TTC) in risk assessment.

The Threshold of Toxicological Concern (TTC) is a level of human intake or exposure that is considered to be of negligible risk, despite the absence of chemical-specific toxicity data. The TTC approach is a form of risk characterisation in which uncertainties arising from the use of data on other compounds are balanced against the low level of exposure. The approach was initially developed by the FDA for packaging migrants, and used a single threshold value of 1.5 microg/day (called the threshold of regulation). Subsequent analyses of chronic toxicity data resulted in the development of TTC values for three structural classes with different potentials for toxicity (1,800, 540 and 90 microg/day). These TTC values have been incorporated into the procedure that is used internationally for the evaluation of flavouring substances. Further developments included additional TTC values for certain structural alerts for genotoxicity (0.15 microg/day), and for the presence of an organophosphate group (18 microg/day). All of these TTC values were incorporated into an extended decision tree for chemicals, such as contaminants, which might be present in human foods. The TTC approach has been shown to have potential applications to risk assessments of cosmetic ingredients, household products and impurities in therapeutic drugs.

Thymoglobulin administered early after heart transplantation reduces early myocardial hypertrophy assessed by morphometric studies.

UNLABELLED: The aim of this study was to assess the effects of early thymoglobulin administration on cardiocyte nuclear status in orthotopic heart transplant (OHT) recipients. MATERIAL AND METHODS: We investigated endomyocardial biopsies (EMBs) from 31 OHT recipients and 10 control cases. OHT patients were divided into the standard group who were treated without thymoglobuline; an ATG group who received thymoglobulin electively, and a standard+ATG group who were administered thymoglobulin upon a drop in renal function. We evaluated only EMBs obtained at 1 and 4 weeks after OHT showing no significant rejection (ISHLT grades 0 to 1B). The morphometric studies were performed using a computerized, automated Quantimet image analysis system. Overall, 1750 cardiocyte nuclei were quantitated for area, length, breadth, perimeter, chromatin median grey level, and fullness factor. Statistical analysis was performed using the Mann- Whitney U test, the Wilcoxon test, and discriminant analysis. RESULTS: All OHT groups showed significantly higher values (indicating nucleus enlargement) than the control group. All factors suggesting myocardial hypertrophy were significantly higher in the standard group; however, they decreased significantly with time. In contrast, the nuclear geometric parameters were significantly lower and stable throughout the study in the ATG group. The results of the standard+ATG group were intermediate, and their normalization as incomplete at the week 4 examination. Discriminant analysis revealed the closest Mahalanobis distance between control and ATG groups both at and weeks 1 and 4 after OHT. CONCLUSION: Thymoglobulin administered early after surgery protected cardiocyte hypertrophy in heart transplant recipients, mitigating graft ischemic damage.

Remodeling of human transplanted myocardium in ten-year follow-up: a clinical pathology study.

UNLABELLED: Remodeling taking place in transplanted myocardium leads to a change in the number of cardiocytes. Ultrasound measurements and biopsy evaluation should reflect their loss and compensation. We sought to evaluate the morphology of the transplanted heart upon long-term follow-up. MATERIAL AND METHODS: Myocardial biopsies were obtained in the first week, first month, and then annually for 10 years from transplantation that did not show rejection (grade "0" ISHLT, 122 biopsies). The control group encompassed 7 donor heart fragments. Proliferation in biopsies was evaluated with Ki67 (M7240, DAKO), cardiocyte hypertrophy by measuring their diameter, the surface area of the nuclei, nuclear-sarcoplasmic index, and stromal fibrosis evaluated as the surface area fraction. Ultrasound measurements included diastolic thickness of the interventricular septum, posterior wall of the left ventricle, and left ventricular mass. The correlation of measurements with time from transplantation was evaluated using Spearman's test. RESULTS: A positive Ki67 reaction was observed in fibroblasts and endothelial cells. The increased cardiocyte nuclear area correlated with the time elapsed since transplantation (r = 0.2; P < .05) with a simultaneous decrease in cardiocyte thickness (r = -0.3; P < .05), without changes in the nuclear-cytoplasmic index (r = 0.02; P > .05). Stromal fibrosis also increased (r = 0.1; P < .05). Ultrasound measurements of the left ventricle showed a decreased tendency with the passage of time (r = -0.2 to -0.3; P < .05). CONCLUSION: A transplanted heart does not undergo hypertrophy but rather fibrous atrophy with apparent compensatory hypertrophy of the cardiomyocytes.

Transplanted heart cardiomyocytes reveal continuous expression of antiapoptotic Bcl-2 protein.

BACKGROUND: Apoptotic mechanisms take place in cardiocyte death during acute heart graft rejection and remodeling by the mitochondrial pathway. This process is suppressed by Bcl-2 protein. Besides that, knowledge about cardiocyte antiapoptotic responses after heart transplantation is scanty. We sought to estimate Bcl-2 expression in the absence of rejection. MATERIAL: The study group included endomyocardial biopsies taken at 1 week, 1 month, 1 through to 10 years after heart transplant, which showed rejection grade "0"; the control group were donor heart fragments. METHOD: Bcl-2 expression was determined immunohistochemically by NP030 antibody (DAKO) and Envision-DAB. The intensity of staining was assessed semiquantitatively. RESULTS: No Bcl-2 expression was seen in the controls; in the posttransplant groups, the significantly strongest sarcoplasmic reaction was observed at 1 week after heart transplant. Thereafter, the reaction decreased, and was weakest in the 3- and 5-year groups. From this time, Bcl-2 expression increased albeit without statistical significance. The intensity of the reaction showed no correlation with the time elapsed from heart transplant (Spearman r = 0.05; P > .05). CONCLUSION: The expression of antiapoptotic Bcl-2 protein occurs during the entire posttransplant period, being probably a preservative and adaptative response.

Cardiocyte nucleus shape as an indicator of heart graft aging.

UNLABELLED: Morphometric publications based on the measurement of cardiocyte nuclei indicated their progressive hypertrophy ignoring, however, their shape, which is a deciding factor for the microscopic-based diagnosis of hypertrophy. We sought was to demonstrate how the shapes of cardiocyte nuclei change over time and correlate them with the thickness of the interventricular septum, (IVS) the biopsy site. MATERIAL: We evaluated myocardial biopsies taken in the first week, first month, and then annually until posttransplant year 10. Only biopsies with no rejection were considered: grade "0" ISHLT (122 biopsies). The control group encompassed fragments from seven donor hearts. METHODS: Cardiomyocyte nuclei were evaluated morphometrically. We calculated the length, breadth, perimeter, roundness, elongation, and fullness factors for correlation with the IVS thickness, and selected indices. The relationships between karyometry and IVS thickness (measured by ultrasound) as well as time were calculated by Spearman's correlation test. RESULTS: Among the examined indices, only nuclear length did not correlate significantly with follow-up time. Among the remaining indices, the strongest correlations with time were observed with regard to breadth (r = 0.214), perimeter (r = 0.150), roundness (r = -0.06) and fullness (r = 0.06), and finally elongation (r = 0.02). The decreasing thickness of the interventricular septum (r = -0.31) showed a weak correlation only with the cardiocyte nuclear length (r = -0.05). CONCLUSION: Graft aging imitates hypertrophy inasmuch as cardiocyte nuclei become wider despite the decreased thickness of the interventricular septum. Therefore, karyometric measurements do not reflect myocardial morphology.

A critical review of the data related to the safety of quercetin and lack of evidence of in vivo toxicity, including lack of genotoxic/carcinogenic properties.

Quercetin is a naturally-occurring flavonol (a member of the flavonoid family of compounds) that has a long history of consumption as part of the normal human diet. Because a number of biological properties of quercetin may be beneficial to human health, interest in the addition of this flavonol to various traditional food products has been increasing. Prior to the use of quercetin in food applications that would increase intake beyond that from naturally-occurring levels of the flavonol in the typical Western diet, its safety needs to be established or confirmed. This review provides a critical examination of the scientific literature associated with the safety of quercetin. Results of numerous genotoxicity and mutagenicity, short- and long-term animal, and human studies are reviewed in the context of quercetin exposure in vivo. To reconcile results of in vitro studies, which consistently demonstrated quercetin-related mutagenicity to the absence of carcinogenicity in vivo, the mechanisms that lead to the apparent in vitro mutagenicity, and those that ensure absence of quercetin toxicity in vivo are discussed. The weight of the available evidence supports the safety of quercetin for addition to food.

Comparison of estimated daily intakes of flavouring substances with no-observed-effect levels.

This study was conducted to determine the margins of safety between no-observed-effect levels (NOELs) and estimates of daily intake for 809 flavouring substances evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) between 2000 and 2004. Estimates of daily intake were calculated using two methods, the maximized survey-derived daily intake (MSDI) and the possible average daily intake (PADI). The MSDI estimates were based on the production volume of flavouring agents as reported by industry, whereas the higher more conservative PADI estimates were derived by multiplying the anticipated average use level of a flavouring substance in each of 33 food categories by the average amount of food consumed daily from that food category and summing the intake over all 33 food categories. These intake estimates were used to calculate the margins of safety for the flavouring agents to determine whether adequate margins of safety would still exist in the event that the MSDIs used by JECFA to evaluate the safety of flavouring substances underestimated daily intakes. Based on the calculation of the margins of safety using the MSDI values, 99.9% of the 809 flavouring substances evaluated by JECFA have margins of safety of greater than 100. In comparison, 98% of flavouring substances have margins of safety of greater than 100 when the margins of safety were calculated from PADI values. The results indicate that if the MSDI estimates used by JECFA for the evaluation of the safety of flavouring substances were underestimated, a wide margin of safety exists for all but a few of the flavouring substances even when intakes were estimated from PADI values.