Endotoxin-induced aggravation of preservation-reperfusion injury of rat liver and its modulation

BACKGROUND/AIMS: In clinical transplantation, exposure of donors to gut-derived endotoxin occurs frequently and may adversely affect liver transplantation therapy. The aim of this study was to investigate: 1) whether brief exposure of rats to endotoxin before liver procurement aggravates the early phase of reperfusion injury of hepatic explants; and if so 2) whether Kupffer cell activation is a contributing factor to liver injury; and 3) whether heparin and pentoxifylline could minimize this effect. METHODS: Male Wistar rats were injected with 0.2-4.0 mg/kg of Escherichia coli lipopolysaccharide 2 h prior to liver harvest. After preservation in University of Wisconsin cold-storage solution, the livers were reperfused using a blood-free perfusion model. To inactivate Kupffer cells, some rats were pretreated with gadolinium chloride or liposome-encapsulated dichloromethylene-diphosphonate before lipopolysaccharide administration. The other rats received lipopolysaccharide with heparin or pentoxifylline. RESULTS: In a dose-independent fashion, lipopolysaccharide impaired portal flow during graft reperfusion. In a dose-dependent way, lipopolysaccharide increased lactate dehydrogenase release into the perfusate and decreased bile flow and bromosulfophthalein excretion. Gadolinium chloride, liposomal dichloromethylene-diphosphonate, heparin, and pentoxifylline reduced lactate dehydrogenase release by 34%, 43%, 59%, and 64%, respectively, and improved functional parameters of the liver. A 52-fold increased neutrophil infiltration in the liver sinusoids after lipopolysaccharide exposure was not affected significantly by the drugs studied; however, heparin reduced markedly neutrophil activation. CONCLUSIONS: The results of this investigation provide direct evidence that aggravation of preservation-reperfusion injury of rat liver by endotoxin is mediated by Kupffer cell-dependent mechanism(s) and it can be minimized by heparin and pentoxifylline.     

Loss of the gene encoding mannose 6-phosphate/insulin-like growth factor II receptor is an early event in liver carcinogenesis

This report shows that loss of heterozygosity at the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) locus occurred in 5/8 (63%) dysplastic liver lesions and 11/18 (61%) hepatocellular carcinomas (HCCs) associated with the high risk factors of hepatitis virus infection and liver cirrhosis. Mutations in the remaining allele were detected in 6/11 (55%) HCCs, including deletions in a polydeoxyguanosine region known to be a target of microsatellite instability. M6P/IGF2R allele loss was also found in cirrhotic tissue of clonal origin adjacent to these dysplastic lesions and HCCs, demonstrating that M6P/IGF2R inactivation occurs early in liver carcinogenesis. In conclusion, HCCs frequently develop from clonal expansions of phenotypically normal, M6P/IGF2R-mutated hepatocytes, providing further support for the idea that M6P/IGF2R functions as a liver tumor-suppressor gene.     

Calpain activity promotes the sealing of severed giant axons

A barrier (seal) must form at the cut ends of a severed axon if a neuron is to survive and eventually regenerate. Following severance of crayfish medial giant axons in physiological saline, vesicles accumulate at the cut end and form a barrier (seal) to ion and dye diffusion. In contrast, squid giant axons do not seal, even though injury-induced vesicles form after axonal transection and accumulate at cut axonal ends. Neither axon seals in Ca²⁺-free salines. The addition of calpain to the bath saline induces the sealing of squid giant axons, whereas the addition of inhibitors of calpain activity inhibits the sealing of crayfish medial giant axons. These complementary effects involving calpain in two different axons suggest that endogenous calpain activity promotes plasmalemmal repair by vesicles or other membranes which form a plug or a continuous membrane barrier to seal cut axonal ends.     

Impairment of mitochondrial oxidative phosphorylation in rat fatty liver exposed to preservation-reperfusion injury

BACKGROUND/AIMS: As the impairment of the cellular energy metabolism contributes to the failure of fatty liver grafts after transplantation, we aimed to determine whether steatosis affects the oxidative phosphorylation activity during preservation. METHODS: Rat normal and fatty livers were preserved for 18 h and then reperfused with warm oxygenated solution. The oxidative phosphorylation, the F(0)F(1)-ATPase and the Complex I activities were assessed in isolated mitochondria before and after preservation, and during reperfusion. The ALT release and portal pressure were monitored during reperfusion. RESULTS: The baseline phosphorylation activity was similar in normal and steatotic mitochondria. After cold preservation, the respiratory control index and state 3 respiration decreased significantly only in steatotic livers. Reperfusion induced a further deterioration in either group. Contrary to normal liver, uncoupling of fatty liver mitochondria allowed the recovery of the maximal respiration rate only using succinate (Complex II-dependent substrate), but not glutamate-malate (Complex I-dependent). Complex I dysfunction was confirmed spectrophotometrically. The ATPase activity was also significantly lower in fatty livers. Finally, ALT release and portal pressure were greater in steatotic livers. CONCLUSIONS: The alteration of the oxidative phosphorylation activity during preservation is greatly exacerbated by fatty infiltration likely resulting from damage of the respiratory chain Complex I and of the F(0)F(1)-ATP synthase.     

Nitric oxide: a mediator in rat tubular hypoxia/reoxygenation injury.

Nitric oxide (NO), among several other functions,may play a role in hypoxia and reoxygenation injury due to its free radicalnature and high reactivity with the superoxide radical to yield peroxynitrite,an oxidant molecule. The present study was undertaken to evaluate a potentialrole for NO, either endogenous or exogenous, in a model of hypoxia/reoxygenation(H/R) in freshly isolated rat proximal tubules. NO synthase activity, asassessed by conversion of L-[3H]arginine to L-[3H]citrulline, was detected innormoxic tubules. This activity could be inhibited by N-nitro-L-arginine methylester (L-NAME), a NO synthase inhibitor, and was stimulated by 15 min ofhypoxia. The injury in proximal tubules caused by 15 min of hypoxia followed by35 min of reoxygenation was completely prevented by L-NAME as assessed byrelease of lactate dehydrogenase, whereas D-NAME, which does not inhibit NOsynthase, had no effect. In contrast, L-arginine (NO substrate) enhanced the H/Rinjury. These effects were paralleled by nitrite/nitrate production. In separateexperiments, the addition of sodium nitroprusside, a NO donor, to proximaltubules enhanced the H/R injury; this effect could be blocked by hemoglobin, aNO scavenger. Also, addition of nitroprusside reversed L-NAME protection againstH/R injury. These results demonstrate that NO is synthesized in rat proximaltubules and participates as one of the mediators in rat tubular H/Rinjury.     

The early phase of engraftment after murine blood cell transplantation is mediated by hematopoietic stem cells

Blood cell transplantation is largely replacing bone marrow transplantation because engraftment is more rapid. This accelerated engraftment is thought to be mediated by relatively mature committed hematopoietic progenitor cells. Herein, we have used a modified rhodamine (Rho) staining procedure to identify and purify Rho^+/++ (dull/bright) and Rho⁻ (negative) subpopulations of hematopoietic progenitor cells in murine cytokine-mobilized blood. The Rho^+/++ cell population contained >99% of committed progenitor cells with in vitro colony-forming ability. The Rho⁻ cell population contained the majority of hematopoietic stem cells with in vivo marrow repopulating ability. The rate of hematopoietic reconstitution was identical in recipients of grafts containing only purified Rho⁻ stem cells or purified Rho⁻ stem cells in combination with large numbers of Rho^+/++ committed progenitor cells. In contrast, transplantation of 3-fold more hematopoietic stem cells resulted in accelerated reconstitution, indicating that the reconstitution rate was determined by the absolute numbers of Rho⁻ stem cells in the graft. In addition, we observed a 5- to 8-fold reduced frequency of the subset of hematopoietic stem cells with long-term repopulating ability in cytokine-mobilized blood in comparison to steady-state bone marrow. Our results indicate that hematopoietic stem cells and not committed progenitor cells mediate early hematopoietic reconstitution after blood cell transplantation and that relative to bone marrow, the frequency of stem cells with long-term repopulating ability is reduced in mobilized blood.     

Molecular cloning of a high-affinity receptor for the growth factor-like lipid mediator lysophosphatidic acid from Xenopus oocytes

Lysophosphatidic acid (1-acyl-2-lyso-sn-glycero-3-phosphate, LPA) is a multifunctional lipid mediator found in a variety of organisms that span the phylogenetic tree from humans to plants. Although its physiological function is not clearly understood, LPA is a potent regulator of mammalian cell proliferation; it is one of the major mitogens found in blood serum. In Xenopus laevis oocytes, LPA elicits oscillatory Cl⁻ currents. This current, like other effects of LPA, is consistent with a plasma membrane receptor-mediated activation of G protein-linked signal transduction pathways. Herein we report the identification of a complementary DNA from Xenopus that encodes a functional high-affinity LPA receptor. The predicted structure of this protein of 372 amino acids contains features common to members of the seven transmembrane receptor superfamily with a predicted extracellular amino and intracellular carboxyl terminus. An antisense oligonucleotide derived from the first 5–11 predicted amino acids, selectively inhibited the expression of the endogenous high-affinity LPA receptors in Xenopus oocytes, whereas the same oligonucleotide did not affect the low-affinity LPA receptor. Expression of the full-length cRNA in oocytes led to an increase in maximal Cl⁻ current due to increased expression of the high-affinity LPA receptor, but activation of the low-affinity receptor was, again, unaffected. Oocytes expressing cRNA prepared from this clone showed no response to other lipid mediators including prostaglandins, leukotrienes, sphingosine 1-phosphate, sphingosylphosphorylcholine, and platelet-activating factor, suggesting that the receptor is highly selective for LPA.     

Lipochitooligosaccharide-induced tobacco cells release a peptide as mediator of the glycolipid signal

Lipochitooligosaccharides (LCOs) are plant growth regulators that promote at subfemtomolar concentrations cell division in tobacco protoplasts. In response to LCO treatment, tobacco cells release a second growth factor that fully mediates the growth-promoting activities of the initial extracellular LCO stimulus. This diffusible growth factor was isolated from the protoplasts’ culture filtrate and shown to be a peptide. We report that the LCO-induced mitogen released by tobacco cells and a synthetic heptadecapeptide derived from region 2 of the tobacco homolog of the early nodulin gene ENOD40 are antigenically related and qualitatively indistinguishable in their ability to stimulate cell division.     

Interferon-inducible protein-10 identified as a mediator of tumor necrosis in vivo

Human Burkitt lymphoma cell lines give rise to progressively growing subcutaneous tumors in athymic mice. These tumors are induced to regress by inoculation of Epstein–Barr virus-immortalized normal human lymphocytes. In the present study, analysis of profiles of murine cytokine/chemokine gene expression in Burkitt tumor tissues excised from the nude mice showed that expression of the murine α-chemokine interferon-inducible protein-10 (IP-10) was higher in the regressing than in the progressive Burkitt tumors. We tested the effects of IP-10 on Burkitt tumor growth in nude mice. Inoculation of established Burkitt tumors either with crude preparations of murine IP-10 or with purified human IP-10 caused visible tumor necrosis in a proportion of the animals, although no complete tumor regressions were observed. Constitutive expression of murine IP-10 in Burkitt cells reduced their ability to grow as subcutaneous tumors, and caused visible tumor necrosis in a proportion of the animals. Histologically, IP-10-treated and IP-10-expressing Burkitt tumors had widespread evidence of tumor tissue necrosis and of capillary damage, including intimal thickening and vascular thrombosis. Thus, IP-10 is an antitumor agent that promotes damage in established tumor vasculature and causes tissue necrosis in human Burkitt lymphomas established subcutaneously in athymic mice.     

From differentiation to proliferation: The secretory amyloid precursor protein as a local mediator of growth in thyroid epithelial cells

In various species, thyrotropin (TSH) is known to stimulate both differentiation and proliferation of thyroid follicle cells. This cell type has also been shown to express members of the Alzheimer amyloid precursor (APP) protein family and to release the secretory N-terminal domain of APP (sAPP) in a TSH-dependent fashion. In this study on binding to the cell surfaces, exogenously added recombinant sAPP stimulated phosphorylation mediated by mitogen-activated protein kinase and effectively evoked proliferation in the rat thyroid epithelial cell line FRTL-5. To see whether this proliverative effect of sAPP is of physiological relevance, we used antisense techniques to selectively inhibit the expression of APP and the proteolytic release of sAPP by cells grown in the presence of TSH. The antisense-induced inhibition was detected by immunoblot, immunoprecipitation, and immunocytochemical analyses. After the reduced APP expression and sAPP secretion, we observed a strong suppression of the TSH-induced cell proliferation down to 35%. Recombinant sAPP but not TSH was able to overcome this antisense effect and to completely restore cell proliferation, indicating that sAPP acts downstream of TSH, in that it is released from thyroid epithelial cells during TSH-induced differentiation. We propose that sAPP operates as an autocrine growth factor mediating the proliferative effect of TSH on neighboring thyroid epithelial cells.     

Insulin-like growth factor I restores motor coordination in a rat model of cerebellar ataxia

We tested the potential of insulin-like growth factor I (IGF-I) to induce functional recovery in an animal model of cerebellar ataxia because this motor impairment is accompanied in humans and rodents by distinct changes in several components of the IGF-I trophic system. Rats rendered ataxic by deafferentation of the cerebellar cortex with 3-acetylpyridine recovered motor function after IGF-I was administered, as determined by behavioral and electrophysiological tests. When treated with IGF-I, inferior olive neurons, the targets of the neurotoxin, were rescued to various degrees (from 92 to 27% of surviving neurons), depending on the time that treatment with IGF-I was initiated. Furthermore, full recovery was obtained regardless of the route by which the trophic factor was administered (intraventricular or subcutaneous) even in rats with severe neuronal loss. These results suggest that human ataxia could be treated with IGF-I by a simple procedure.     

Working underground: Respiratory adaptations in the blind mole rat

Mole rats (Spalax ehrenbergi superspecies) perform the heavy work of digging their subterranean burrows in Israel under highly hypoxic/hypercapnic conditions. Unlike most other mammals, they can achieve high levels of metabolic rate under these conditions, while their metabolic rate at low work rates is depressed. We explored, by comparing mole rats with white rats, whether and how this is related to adaptations in the design of the respiratory system, which determines the transfer of O₂ from the lung to muscle mitochondria. At the same body mass, mole rats were found to have a significantly smaller total skeletal muscle mass than ordinary white rats (−22%). In contrast, the fractional volume of muscle mitochondria was larger by 46%. As a consequence, both species had the same total amount of mitochondria and achieved, under normoxia, the same _O2max. Whereas the O₂ transport capacity of the blood was not different, we found a larger capillary density (+31%) in the mole rat muscle, resulting in a reduced diffusion distance to mitochondria. The structural pulmonary diffusing capacity for O₂ was greater in the mole rat (+44%), thus facilitating O₂ uptake in hypoxia. We conclude that structural adaptations in lung and muscle tissue improve O₂ diffusion conditions and serve to maintain high metabolic rates in hypoxia but have no consequences for achieving _O2max under normoxic conditions.     

Structural protein 4.1 is located in mammalian centrosomes

Structural protein 4.1 was first characterized as an important 80-kDa protein in the mature red cell membrane skeleton. It is now known to be a member of a family of protein isoforms detected at diverse intracellular sites in many nucleated mammalian cells. We recently reported that protein 4.1 isoforms are present at interphase in nuclear matrix and are rearranged during the cell cycle. Here we report that protein 4.1 epitopes are present in centrosomes of human and murine cells and are detected by using affinity-purified antibodies specific for 80-kDa red cell 4.1 and for 4.1 peptides. Immunofluorescence, by both conventional and confocal microscopy, showed that protein 4.1 epitopes localized in the pericentriolar region. Protein 4.1 epitopes remained in centrosomes after extraction of cells with detergent, salt, and DNase. Higher resolution electron microscopy of detergent-extracted cell whole mounts showed centrosomal protein 4.1 epitopes distributed along centriolar cylinders and on pericentriolar fibers, at least some of which constitute the filamentous network surrounding each centriole. Double-label electron microscopy showed that protein 4.1 epitopes were predominately localized in regions also occupied by epitopes for centrosome-specific autoimmune serum 5051 but were not found on microtubules. Our results suggest that protein 4.1 is an integral component of centrosome structure, in which it may play an important role in centrosome function during cell division and organization of cellular architecture.     

ActA is a dimer

ActA, a surface protein of Listeria monocytogenes, is able to induce continuous actin polymerization at the rear of the bacterium, in the cytosol of the infected cells. Its N-terminal domain is sufficient to induce actin tail formation and movement. Here, we demonstrate, using the yeast two-hybrid system, that the N-terminal domain of ActA may form homodimers. By using chemical cross-linking to explore the possibility that ActA could be a multimer on the surface of the bacteria, we show that ActA is a dimer. Cross-linking experiments on various L. monocytogenes strains expressing different ActA variants demonstrated that the region spanning amino acids 97–126, and previously identified as critical for actin tail formation, is also critical for dimer formation. A model of actin polymerization by L. monocytogenes, involving the ActA dimer, is presented.     

Combined liver–kidney transplantation in patients infected with human immunodeficiency virus

F. Di Benedetto, G. D'Amico, N. De Ruvo, S. Cocchi, R. Montalti, N. Cautero, G.P. Guerrini, R. Ballarin, M. Spaggiari, G. Tarantino, B. Baisi, G. Cappelli, M. Codeluppi, G.E. Gerunda. Combined liver–kidney transplantation in patients infected with human immunodeficiency virus.
Transpl Infect Dis 2011: 13: 501–506. All rights reserved Abstract: Although human immunodeficiency virus (HIV) infection has been a major global health problem for almost 3 decades, with the introduction of highly active antiretroviral therapy in 1996 and effective prophylaxis and management of opportunistic infections, mortality from acquired immunodeficiency syndrome has decreased markedly. In developed countries, this condition is now being treated as a chronic condition. As a result, rates of morbidity and mortality from other medical conditions leading to end‐stage liver, kidney, and heart disease are steadily increasing in individuals with HIV. Because the definitive treatment for end‐stage organ failure is transplantation, the demand for it has increased among HIV‐infected patients. For these reasons, many transplant centers have eliminated HIV infection as a contraindication to transplantation, as a result of better patient management and demand.     

Evidence that eukaryotic triosephosphate isomerase is of alpha-proteobacterial origin

We have cloned and sequenced genes for triosephosphate isomerase (TPI) from the gamma-proteobacterium Francisella tularensis, the green non-sulfur bacterium Chloroflexus aurantiacus, and the alpha-proteobacterium Rhizobium etli and used these in phylogenetic analysis with TPI sequences from other members of the Bacteria, Archaea, and Eukarya. These analyses show that eukaryotic TPI genes are most closely related to the homologue from the alpha-proteobacterium and most distantly related to archaebacterial homologues. This relationship suggests that the TPI genes present in modern eukaryotic genomes were derived from an alpha-proteobacterial genome (possibly that of the protomitochondrial endosymbiont) after the divergence of Archaea and Eukarya. Among these eukaryotic genes are some from deeply branching, amitochondrial eukaryotes (namely Giardia), which further suggests that this event took place quite early in eukaryotic evolution.