Subnormothermic machine perfusion for non-heart-beating donor liver grafts preservation in a Swine model: a new strategy to increase the donor pool?

We previously reported that subnormothermic machine perfusion (sMP; 20°C) is able to improve the preservation of livers obtained from non-heart-beating donors (NHBDs) in rats. We have compared sMP and standard cold storage (CS) to preserve pig livers after 60 minutes of cardiac arrest. In the sMP group livers were perfused for 6 hours with Celsior at 20°C. In the CS group they were stored in Celsior at 4°C for 6 hours as usual. To simulate liver transplantation, both sMP- and CS-preserved livers were reperfused using a mechanical continuous perfusion system with autologus blood for 2 hours at 37°C. At 120 min after reperfusion aspartate aminotransferase levels in sMP versus CS were 499 ± 198 versus 7648 ± 2806 U/L (P < .01); lactate dehydrogenase 1685 ± 418 versus 12998 ± 3039 U/L (P < .01); and lactic acid 4.78 ± 3.02 versus 10.46 ± 1.79 mmol/L (P < .01) respectively. The sMP group showed better histopathologic results with significantly less hepatic damage. This study confirmed that sMP was able to resuscitate liver grafts from large NHBD animals.

Decreased apoptosis in fatty livers submitted to subnormothermic machine-perfusion respect to cold storage.

Machine perfusion at subnormothermic temperature (20°C), MP20, was developed by Vairetti et al. and showed to afford a better preservation of fatty livers respect to traditional cold storage (CS) in terms of enzyme release into the perfusate and bile, glycogen stores, energy charge and oxidative stress. Here we investigated whether it also caused decreased cell death by apoptosis. Fatty and lean Zucker rats were submitted to MP20 or CS for 6 h and reperfused normothermically for 2 h. Apoptotic cells were revealed by immunohistochemistry of activated caspase-3 and M30 (new epitope on CK18 degraded by caspase-3) and by the TUNEL assay. Portal pressure was also determined. A statistically significant reduction of hepatocyte apoptosis, but especially of sinusoidal cells was determined for fatty livers submitted to MP20 respect to CS. Portal pressure was significantly lower after MP20 respect to CS. The reduction of sinusoidal cell death by apoptosis without need for anti-apoptotic therapies appears particularly positive since apoptotic sinusoidal cells hinder microcirculation in the sinusoids and are thrombogenic. These results further confirm the potential of MP20 for preserving fatty livers that would be otherwise discarded as grafts, and thus for increasing the donor pool for liver transplantation.

New insights into fatty liver preservation using Institute Georges Lopez preservation solution.

Institute Georges Lopez preservation solution (IGL-1) has been demonstrated to be useful for fatty liver preservation. The mechanisms responsible for this effective graft protection against ischemia-reperfusion injury are pivotal actions on generation of nitric oxide a diffusible molecule with vasodilator properties, that facilitates the up-regulation of other well-known cytoprotective genes, such as hypoxia-inducible factor-1 alpha (HIF-1alpha) and heme-oxygenase 1 (HO-1). During normoxic reperfusion, the presence of nitric oxide permits HIF-1alpha accumulation to inhibit prolyl-hydoxylases, thus promoting an additional overexpression of the HO-1 in steatotic and nonsteatotic graft livers preserved in IGL-1.

A histochemical approach to the evaluation of the in vivo cytotoxicity of the nitrobutadienes (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene and methyl (2Z,4E)-2-methylsulfanyl-5-(1-naphthyl)-4-nitro-2,4-pentadienoate in mice liver and kidney.

Two new molecules (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene (1-Naph-DNB) and (2Z,4E)-2-methylsulfanyl-5-(1-naphthyl)-4-nitro-2,4-pentadienoate (1-Naph-NMCB) in previous studies showed interesting antiproliferative activity in vitro. Furthermore, toxicological tests and histological analysis provided promising results, in particular for 1-Naph-NMCB that displayed lower toxic activity both in terms of lethal effect and tissue damage of the main organs. Finally, studies of the antitumour activity in vivo confirmed the efficacy of both molecules, though with some differences in tumour selectivity and levels of activity. In this investigation the activities of some specific enzymes, acid phosphatase (AcPase), alkaline phosphatase (AlkPase), catalase (Cat), succinic dehydrogenase (SDH), glucose-6-phosphatase (G6Pase) and K+ p-nitrophenyl phosphatase (K+ pNPPase) were studied in the liver and kidney as histopathological biomarkers, to assess the effects of the two compounds in organs generally involved in the metabolism and excretion of different drugs. As oxidative stress may also develop as a consequence of the toxic effect of chemicals, reactive oxygen species (ROS) production was evaluated by a histochemical method. The results indicated that some enzyme activities and ROS expression changed in a dose-related manner. Nevertheless, neither in the liver nor in the kidney were dramatic toxic effects evident. By contrast, the variations of some enzyme activities (AlkPase, AcPase, Cat, K+ pNPPase) were interpreted as possible defensive mechanisms for tolerating high dosage of the compounds.

Subnormothermic machine perfusion protects against rat liver preservation injury: a comparative evaluation with conventional cold storage.

UNLABELLED: Hypothermic machine perfusion (MP) of the liver has been reported to improve graft function reclaiming marginal livers, such as those from non-heart-beating donors. Livers from obese donors often have fatty infiltrates and are more susceptible to hypothermic conditions. No data exist about MP at temperatures >4 degrees C. This study evaluated liver function after organ preservation by comparing MP at 20 degrees C with conventional cold storage. METHODS: For MP, rat livers were perfused for 6 hours using an oxygenated Krebs-Henseleit (KH) solution at 20 degrees C (pH 7.4). For cold storage, livers were perfused in situ and preserved with Celsior solution at 4 degrees C for 6 hours. The reperfusion period with KH (2 hours at 37 degrees C) was performed under the same conditions both among livers preserved by MP or cold storage. Hepatic enzyme release (aspartate aminotransferase [AST], alanine aminotransferase [ALT], lactate dehydrogenase [LDH], and gamma-glutamyl transferase [GGT]), bile production, and ATP levels were measured during MP and reperfusion. RESULTS: At the end of reperfusion, livers preserved by MP showed significantly decreased liver damage compared with cold storage: AST, 18 +/- 4 vs. 45 +/- 6 mU/mL (P < .01); ALT, 1.5 +/- .07 vs. 6 +/- 0.5 mU/mL (P < .01); and LDH, 82 +/- 2 vs. 135 +/- 29 mU/mL (P < .05). No difference was observed between bile production between MP and cold storage. High levels of biliary GGT and LDH were found in cold preserved livers. ATP levels were higher in livers preserved with MP compared with those preserved by cold storage. CONCLUSIONS: MP at 20 degrees C resulted in a better quality of liver preservation, improving hepatocyte survival, compared with conventional cold storage. This may provide a new method for successful utilization of marginal livers, in particular fatty livers.

Morphofunctional evidence of changes in principal and mitochondria-rich cells in the epidermis of the frog Rana kl. esculenta living in a polluted habitat.

The epidermis of vertebrates is the body's principal barrier against environment and its possible contaminants. The presence of keratins, as well as specific detoxifying molecules or enzyme activities, in the various epidermis layers is believed to be involved in providing protection from harmful environmental influences. Anuran integument is poorly hornified and thus permeable to some endogenous and exogenous compounds and thus serves as a good bioindicator of overall environmental conditions. In the present investigation, we studied the epidermis of Rana kl. esculenta adult specimens collected at two different rice fields, relatively unpolluted and heavily polluted, respectively. Environmental pollution was assayed by chemical analysis performed on both sediments and animals. We evaluated the structural aspects of the epidermis at both light and electron microscopy levels and the pattern of keratinization by immunohistochemistry. Furthermore, we studied the activities of some enzymes (acid and alkaline phosphatase, nitric oxide synthase-related nicotinamide adenine dinucleotide phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, catalase, nonspecific esterases, and succinic dehydrogenase) involved mainly in membrane transport, xenobiotics, and oxidative metabolism. Compared with controls, in polluted animals we found the following results: (1) an increase in pollutant levels (i.e., cadmium, mercury, and lead); (2) less keratinized superficial cells in the epidermis; and (3) changes in most enzyme activities in keratinocytes and mitochondria-rich cells (particularly glucose-6-phosphate dehydrogenase and esterases, both important to counteract oxidative and toxic stress). Taken as a whole, the present data indicate the morphofunctional plasticity of the frog epidermis in response to environmental contamination.

Little Egrets (Egretta garzetta) and trace-metal contamination in wetlands of China.

The rapidly developing Asian countries may face threatening environmental contamination, that however remains largely unassessed. We studied persistent trace elements in three wetlands, Poyang Lake, relatively unpolluted, and Tai Lake, and Pearl River Delta, selected as hotspots of pollution in Central China. We adopted as indicator the Little Egret, a widespread colonial waterbird, and during 1999 and 2000 we collected and analysed the same samples as for a parallel study we conducted in Pakistan, e.g. eggs, body feathers of chicks, prey spontaneously regurgitated by the chicks, and sediments at the areas most used by foraging egrets. The levels of trace-metals at our three study areas were similar, or within the range, of those found in the few other studies for East Asia, with few exceptions. The concentrations of the various elements were below the threshold that may affect the survival or reproduction of the birds, and even the highest concentrations relative to background, i.e. Se in eggs, and Hg in feathers at Pearl Delta, do not pose toxic hazards. In sediments, the levels of trace elements were lower than the critical levels assumed for contaminated soil, except for alarming high levels of As at Poyang. These results do not confirm our expectation, that Poyang was relatively uncontaminated, while Tai and Pearl were polluted. Although trace metal concentration differed significantly among the three study areas, these differences were minor and were not consistent among elements and samples. The bioaccumulation ratios from sediments through prey, feather and egg, were consistent with our previous findings for Pakistan. Only Hg exhibited high bioaccumulation, while Se and Zn had low accumulation, and the other elements no accumulation. This reasserts that feathers of predators such as egrets, may be more sensitive indicators of environmental contamination for the elements subject to bioaccumulation, whereas the sediments or the organisms low in the food chain are better indicators for the other elements.

Egrets as monitors of trace-metal contamination in wetlands of Pakistan.

Environments in Pakistan are subject to increasing pollution, but previous studies were very scanty. During 1999 and 2000, we assessed trace element contamination at three wetlands, Karachi Harbour (with presumed industrial-urban pollution), Taunsa Barrage (agricultural pollution), and Haleji Lake (relatively unpolluted), using as indicators the eggs and the feathers of colonial waterbirds, particularly Little Egrets, their prey, and the sediments collected within their foraging areas. The concentrations of As, Cd, Cr, Pb, Hg, Mn, Se, and Zn were generally within the normal background level, and mostly below the threshold that may affect bird survival or reproduction. However, somewhat high concentrations were found in fish from Karachi, for Pb that was at levels that may harm fish reproduction, and for Hg that was at limit concentration for human consumption. Alarming concentrations were found for Cr and Se in sediments from Karachi, that were above the critical levels for contaminated soil, and Se in eggs, that may affect egret reproduction. The differences among the three wetlands were less marked than hypothesized. The egret species within the same area differed in the concentration of certain elements in their eggs, possibly because females may have foraged in different habitats before breeding, whereas no interspecies difference was found in chick feathers, presumably because their food had been collected in similar habitats around the colony. High bioaccumulation from sediments to organic samples occurred for Hg, while Cd, Se, and Zn exhibited low accumulation; for all these elements, feathers of predatory birds such as the egrets are the best indicators of environmental contamination. On the other hand, As and Cr did not bioaccumulate, and the sediments, or the organisms low in the food chain, like fish or crustaceans, are better indicators of their presence in the environment than predatory birds.

Persistent organic pollutants in little egret eggs from selected wetlands in Pakistan.

The main goal of the present work has been to study the use of egret eggs to assess environmental pollution by POPs (HCB, HCHs, cyclodienes, DDTs and PCBs) in three Pakistani wetlands that are presumed to be affected by different types of pollution. Taunsa Barrage, affected mainly by agricultural pollutants; Karachi Harbor because of the supposed exposure to industrial activity-related POPs; and Haleji Lake as a relatively pristine area because of its location in a stony desert. Taunsa Barrage and Haleji Lake are wetlands of international importance according to the Ramsar Convention, while the Karachi Harbor is of interest because of the large human population living there. Eggs of the white ( Egretta garzetta garzetta) and dark ( Egretta garzetta gularis) morphs of Little Egrets were used as monitoring tools. Concentrations were also determined in several prey in this species' diet and in the sediments collected in their foraging areas. Differences in egg pollutant content among the three localities were significant for all the compounds. Overall, the eggs from Haleji Lake and Karachi showed, respectively, the lowest and highest percentages of detection and organochlorine concentrations. Biomagnification from sediments to prey and then to eggs has been documented in the three areas studied and is accompanied by higher percentages of detection of different compounds through the compartments. Differences in the biomagnification factor among the areas were small, even when differences in pollutant concentrations were high, suggesting that eggs are reliable indicators of POPs in the environment. The values found were generally lower than those reported for the eggs of large herons from North America or the Mediterranean basin, and are about the same order of magnitude that those of other medium-sized egrets from other parts of the world.

Characterization of a Snorhizobium meliloti ATP-binding cassette histidine transporter also involved in betaine and proline uptake.

The symbiotic soil bacterium Sinorhizobium meliloti uses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli (ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S. meliloti. This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine was used as a nitrogen source, a 30% inhibition of growth was observed in hut mutants (hutX and hutH2). Expression analysis of the hut operon determined using a hutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.

Occurrence of choline and glycine betaine uptake and metabolism in the family rhizobiaceae and their roles in osmoprotection

The role of glycine betaine and choline in osmoprotection of various Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, and Bradyrhizobium reference strains which display a large variation in salt tolerance was investigated. When externally provided, both compounds enhanced the growth of Rhizobium tropici, Sinorhizobium meliloti, Sinorhizobium fredii, Rhizobium galegae, Agrobacterium tumefaciens, Mesorhizobium loti, and Mesorhizobium huakuii, demonstrating their utilization as osmoprotectants. However, both compounds were inefficient for the most salt-sensitive strains, such as Rhizobium leguminosarum (all biovars), Agrobacterium rhizogenes, Rhizobium etli, and Bradyrhizobium japonicum. Except for B. japonicum, all strains exhibit transport activity for glycine betaine and choline. When the medium osmolarity was raised, choline uptake activity was inhibited, whereas glycine betaine uptake was either increased in R. leguminosarum and S. meliloti or, more surprisingly, reduced in R. tropici, S. fredii, and M. loti. The transport of glycine betaine was increased by growing the cells in the presence of the substrate. With the exception of B. japonicum, all strains were able to use glycine betaine and choline as sole carbon and nitrogen sources. This catabolic function, reported for only a few soil bacteria, could increase competitiveness of rhizobial species in the rhizosphere. Choline dehydrogenase and betaine-aldehyde dehydrogenase activities were present in the cells of all strains with the exception of M. huakuii and B. japonicum. The main physiological role of glycine betaine in the family Rhizobiaceae seems to be as an energy source, while its contribution to osmoprotection is restricted to certain strains.

Presence of a gene encoding choline sulfatase in Sinorhizobium meliloti bet operon: choline-O-sulfate is metabolized into glycine betaine.

Glycine betaine is a potent osmoprotectant accumulated by Sinorhizobium meliloti to cope with osmotic stress. The biosynthesis of glycine betaine from choline is encoded by an operon of four genes, betICBA, as determined by sequence and mutant analysis. The betI and betC genes are separated by an intergenic region containing a 130-bp mosaic element that also is present between the betB and betA genes. In addition to the genes encoding a presumed regulatory protein (betI), the betaine aldehyde dehydrogenase (betB), and the choline dehydrogenase (betA) enzymes also found in Escherichia coli, a new gene (betC) was identified as encoding a choline sulfatase catalyzing the conversion of choline-O-sulfate and, at a lower rate, phosphorylcholine, into choline. Choline sulfatase activity was absent from betC but not from betB mutants and was shown to be induced indifferently by choline or choline-O-sulfate as were the other enzymes of the pathway. Unlike what has been shown in other bacteria and plants, choline-O-sulfate is not used as an osmoprotectant per se in S. meliloti, but is metabolized into glycine betaine. S. meliloti also can use this compound as the sole carbon, nitrogen, and sulfur source for growth and that depends on a functional bet locus. In conclusion, choline-O-sulfate and phosphorylcholine, which are found in higher plants and fungi, appear to be substrates for glycine betaine biosynthesis in S. meliloti.

Prolapsing large aneurysm of the atrial septum simulating a right atrial mass.

An unusual case of a large, prolapsing atrial septal aneurysm in a patient with an otherwise normal heart is described. The aneurysm caused right atrial obstruction and resulted in a "tumor effect." The patient experienced debilitating symptoms for years before receiving an appropriate diagnosis and curative surgical treatment.