Gaseous oxygen persufflation or oxygenated machine perfusion with Custodiol-N for long-term preservation of ischemic rat livers?

The aim of the present study was to evaluate the potential benefit of two different techniques for the provision of tissue aerobiosis upon cold preservation of marginal livers from non-heart beating donors using a recently developed improved preservation solution.Rat livers were harvested 30 min after cardiac arrest, flushed via the portal vein and cold-stored in HTK or modified HTK-solution (Custodiol-N) for 18 h at 4 °C. Other organs were flushed with Custodiol-N and subjected to aerobic conditions by either vascular systemic oxygen persufflation (VSOP) of the cold stored organ or hypothermic machine perfusion (HMP) with oxygenated Custodiol-N. Viability of the livers was assessed after 18 h of preservation by warm reperfusion in vitro for 120 min.Free radical mediated lipid peroxidation was significantly abrogated by the use of Custodiol-N in all groups compared with HTK. Custodiol-N improved enzyme leakage upon reperfusion and histological integrity, but had no impact on functional recovery (bile production, energetic status). However, VSOP further minimized enzyme release during the whole reperfusion period, led to a rise in hepatic bile production and enhanced recovery of energy charge (p < 0.05, resp. vs Custodiol-N). Histological appearance was concordantly improved in VSOP. During the first 45 min of reperfusion, leakage of ALT and LDH was also reduced by MP but deteriorated thereafter and became significantly higher compared to Custodiol-N at the end of the experiment. In conclusion, the results of the present study recommend the use of gaseous oxygen persufflation to improve tissue integrity and functional recovery of predamaged livers.     

Controversy of free radical hypothesis: reactive oxygen species--cause or consequence of tissue injury?

For a decade or two, the hypothesis of causality of various disorders by reactive oxygen species (ROS), due to their potentially harmful effect towards cellular constituents, is one of the most frequently cited in biomedical sciences. In fact, the ROS-mediated alterations of biomacromolecules are considered to be essential events in the etiopathogenesis of those diseases where involvement of ROS has been indicated. ROS easily react in vitro with most biological molecules, causing their degradation and destruction. This may implicitly suggest that, when excessively produced in vivo, ROS are deleterious to integral components of the cell and cause their dysfunctions. Some experimental data indicate that ROS-mediated lipid peroxidation, protein oxidation and oxidative alterations to nucleic acids are crucial events of unfavorable actions of ROS. Yet the most convincing evidence, i.e. unambiguous inhibition of tissue injury by pretreatment with antioxidants, has not been provided. On the contrary, there are quite a few papers reporting failure in applying antioxidants to heal those pathologies where the causal role of ROS was supposed. Other papers reported serious complications arising from antioxidant therapy, which is quite in contradiction to its expected effect. On the other hand, an increasing number of recent findings have provided evidence of a key role of ROS in both intracellular signaling and intercellular communication, processes involved in maintaining homeostasis. Hence, some investigators consider excessive production of ROS to be rather a "smoke after the fire" than "a deleterious fire" itself, suggesting the occurrence of overproduced ROS as being the consequence of some primary damage. The present paper aims at summarizing some pros and cons of various opinions with an attempt to help better understand the involvement of ROS in tissue injury.     

Different behavior of agmatine in liver mitochondria: Inducer of oxidative stress or scavenger of reactive oxygen species?

Agmatine, at concentrations of 10 μM or 100 μM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H₂O₂ generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H₂O₂ and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron-sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca²⁺. Instead, 1 mM agmatine generates larger amounts of H₂O₂ than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca²⁺-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.     

Different behavior of agmatine in liver mitochondria: inducer of oxidative stress or scavenger of reactive oxygen species?

Agmatine, at concentrations of 10 microM or 100 microM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H(2)O(2) generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H(2)O(2) and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron-sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca(2+). Instead, 1 mM agmatine generates larger amounts of H(2)O(2) than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca(2+)-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.     

Localized cellular responses to Ichthyophthirius multifiliis: protection or pathogenesis?

The ciliate Ichthyophthirius multifiliis is an economically important parasite in aquaculture as well as in the ornamental fish trade. The parasite has been well studied and details of life cycle, host response and pathogenesis have been characterized. Parasite development within host epithelial tissues initiates localized leukocytic infiltrations, although the relationship between these responses and host resistance is uncertain, and whether or not leukocyte responses play a role in protective immunity is unclear. Here Frank Cross outlines the character of localized cellular responses during I. multifiliis infection and discusses the contribution of such responses to protective immunity and pathogenesis.     

Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis?

Programmed cell death (apoptosis) is used by multicellular organisms during development and to maintain homeostasis within mature tissues. One of the first genes shown to regulate apoptosis was bcl-2. Subsequently, a number of Bcl-2-related proteins have been identified. Despite overwhelming evidence that Bcl-2 proteins are evolutionarily conserved regulators of apoptosis, their precise biochemical function remains controversial. Three biochemical properties of Bcl-2 proteins have been identified: their ability to localize constitutively and/or inducibly to the outer mitochondrial, outer nuclear and endoplasmic reticular membranes, their ability to form heterodimers with proteins bearing an amphipathic helical BH3 domain, and their ability to form ion-conducting channels in synthetic membranes. The discovery that mitochondria can play a key part in the induction of apoptosis has focused attention on the role that Bcl-2 proteins may have in regulating either mitochondrial physiology or mitochondria-dependent caspase activation. Here we attempt to synthesize our current understanding of the part played by mitochondria in apoptosis with a consideration of how Bcl-2 proteins might control cell death through an ability to regulate mitochondrial physiology.     

Identification of cellular isoform of oviduct-specific glycoprotein: role in oviduct tissue remodeling?

The oviduct is known to secrete mucins (MUC1 and MUC9) under the influence of ovarian steroids. The secreted form of MUC1 binds gametes in the oviduct, whereas the cellular form seen in breast cancers has been implicated in cell adhesion and morphogenesis. The secreted MUC9 or oviduct-specific glycoprotein (OGP), in addition to being a mucin, belongs to family 18 glycosylhydrolases and is known to bind gametes and embryos in the oviduct. Studies in our laboratory have identified non-muscle myosin IIA (involved in cell shape, polarity, and morphogenesis) as the protein partner to OGP in gametes. In view of the crucial role of the cortical cytoskeleton in the selective internalization of tight junctions (TJs) /adherent junctions (AJs) or apical junctional complex (AJC) in simple epithelial cells during tissue remodeling, the present study has been undertaken to evaluate the existence of a cellular form of OGP in oviductal tissue, which itself undergoes cyclic tissue remodeling. In silico analysis of the deduced amino-acid sequence of OGP has revealed the presence of several conserved motifs; these imply that OGP is a component of multi-protein complexes such as TJs. Corroborative immunoelectron-microscopic analysis in peri-ovulatory oviduct epithelia in the bonnet monkey has revealed the presence of OGP at the TJ. Co-localization studies of OGP and cadherin demonstrate that, whereas OGP is localized at the tonofilaments of the TJs, cadherin is localized at the intercellular space of the AJ. The possible role of OGP in oviductal tissue remodeling is discussed in light of the present findings and those reported in the literature. This study was supported by funds from the Indian Council for Medical Research and by senior research fellowships (to K. Kadam) from the Council for Scientific and Industrial Research.     

Swab or biopsy samples for bioburden testing of allograft musculoskeletal tissue?

Swab and biopsy samples of allograft musculoskeletal tissue are most commonly collected by tissue banks for bacterial and fungal bioburden testing. An in vitro study was performed using the National Committee for Clinical Laboratory Standards standard ‘Quality control of microbiological transport systems’ (2003) to validate and evaluate the recovery of six challenge organisms from swab and biopsy samples of allograft musculoskeletal tissue. On average, 8.4 to >100 and 7.2 to >100 % of the inoculum was recovered from swab and biopsy samples respectively. A retrospective review of donor episodes was also performed, consisting of paired swab and biopsy samples received in this laboratory during the period 2001–2012. Samples of allograft femoral heads were collected from living donors during hip operations. From the 3,859 donor episodes received, 21 paired swab and biopsy samples each recovered an isolate, 247 swab samples only and 79 biopsy samples only were culture positive. Low numbers of challenge organisms were recovered from inoculated swab and biopsy samples in the in vitro study and validated their use for bioburden testing of allograft musculoskeletal tissue. Skin commensals were the most common group of organisms isolated during a 12-year retrospective review of paired swab and biopsy samples from living donor allograft femoral heads. Paired swab and biopsy samples are a suitable representative sample of allograft musculoskeletal tissue for bioburden testing.     

Senescence: a telomeric limit to immortality or a cellular response to physiologic stresses?

Cells entering a state of senescence undergo a irreversible cell cycle arrest, associated by a set of functional and morphological changes. Senescence occurs following telomeres shortening (replicative senescence) or exposure to other acute or chronic physiologic stress signals (a phenomenon termed stasis: stress or aberrant signaling-induced senescence). In this review, I discuss the pathways of cellular senescence, the mechanisms involved and the role that these pathways have in regulating the initiation and progression of cancer. Telomere-initiated senescence or loss of telomere function trigger focal recruitement of protein sensors of the DNA double-strand breaks leading to the activation of the DNA damage checkpoint responses and the tumour suppressor gene product, p53, which in turn induces the cell-cycle inhibitor, p21(WAF1). Loss of p53 and pRb function allows continued cell division despite increasing telomere dysfunction and eventually entry into telomere crisis. Immortalisation is an essential prerequisite for the formation of a tumour cell. Therefore, a developing tumour cell must circumvent at least two proliferative barriers--cellular senescence and crisis--to achieve neoplastic transformation. These barriers are regulated by telomere shortening and by the p16(INK4a)/Rb and p53 tumour suppressor pathways. Elucidation of the genes and emerging knowledge about the regulatory mechanisms that lead to senescence and determine the pattern of gene expression in senescent cells may lead to more effective treatments for cancer.     

Nomadic or sessile: can Kupffer cells function as portals for malaria sporozoites to the liver?

The initial site of replication for Plasmodium parasites in mammalian hosts are hepatocytes, cells that offer unique advantages for the extensive parasite replication occurring prior to the erythrocytic phase of the life cycle. The liver is the metabolic centre of the body and has an unusual relationship to the immune system. However, to reach hepatocytes, sporozoites must cross the sinusoidal barrier, composed of specialized endothelia and Kupffer cells, the resident macrophages of the liver. Mounting evidence suggests that, instead of taking what would seem a safer route through endothelia, the parasites traverse Kupffer cells yet suffer no harm. Kupffer cells have a broad range of responses towards incoming microorganisms, toxins and antigens which depend on the nature of the intruder, the experimental conditions and the environmental circumstances. Kupffer cells may become activated or remain anergic, produce pro- or anti-inflammatory mediators. Consequently, outcomes are diverse and include development of immunity or tolerance, parenchymal necrosis or regeneration, chronic cirrhotic transformation or acute liver failure. Here we review data concerning the unique structural and functional characteristics of Kupffer cells and their interactions with Plasmodium sporozoites in the context of a model in which these hepatic macrophages function as the sporozoite gate to the liver.     

Reactivity of carbon nanotubes: Free radical generation or scavenging activity?

Carbon nanotubes (CNTs) currently attract intense research efforts because of their unique properties which make them suitable for many industrial applications. When inhaled, CNTs constitute a possible hazard to human health. Several studies have shown that when instilled in the lung of experimental animals, CNTs induced an inflammatory and fibrotic response similar to that caused by other toxic particles which might be the result of oxidative stress caused by particle- and/or cell-derived free radicals. There is, however, no direct experimental evidence of a capacity of carbon nanotubes to generate directly free radicals. Here we report that multiwall carbon nanotubes (MWCNT) in aqueous suspension do not generate oxygen or carbon-centered free radicals in the presence of H2O2 or formate, respectively, as detected with the spin-trapping technique. Conversely, we observed that, when in contact with an external source of hydroxyl or superoxide radicals, MWCNT exhibit a remarkable radical scavenging capacity. It is therefore possible that the inflammatory reaction reported in vivo must be ascribed to MWCNT features other than particle-derived free radical generation.     

Metabolic regulation of oxygen and redox homeostasis by p53: Lessons from evolutionary biology?

The genetic links between p53 and metabolic processes such as oxidative phosphorylation are being studied with increasing interest given that cellular metabolism seems to play an important role in tumorigenesis. This review focuses on how p53 regulation of various metabolic genes may influence redox homeostasis, as the genome is constantly susceptible to oxidative damage, a consequence of living in an aerobic environment. Because p53-like genetic sequences are also found in life forms that may not necessarily benefit from tumor suppression, an evolutionary introduction is given in an attempt to understand why p53 might regulate a basic cellular activity such as metabolism. The presented epidemiologic and experimental data suggest that one reason may be for the homeostatic regulation of oxygen, the essential substrate for reactive oxygen species generation.     

Sequestration of cellular native factors by biomolecular assemblies: Physiological or pathological?

In addition to native-state structures, biomolecules often form condensed supramolecular assemblies or cellular membraneless organelles that are critical for cell life. These biomolecular assemblies, generally including liquid-like droplets (condensates) and amyloid-like aggregates, can sequester or recruit their interacting partners, so as to either modulate various cellular behaviors or even cause disorders. This review article summarizes recent advances in the sequestration of native factors by biomolecular assemblies and discusses their potential consequences on cellular function, homeostasis, and disease pathology.     

Insulin resistance in the liver: Deficiency or excess of insulin?

In insulin-resistant states (obesity, pre-diabetes, and type 2 diabetes), hepatic production of glucose and lipid synthesis are heightened in concert, implying that insulin deficiency and insulin excess coexists in this setting. The fact that insulin may be inadequate or excessive at any one point in differing organs and tissues has many biologic ramifications. In this context the concept of metabolic compartmentalization in the liver is offered herein as one perspective of this paradox. In particular, we focus on the hypothesis that insulin resistance accentuates differences in periportal and perivenous hepatocytes, namely periportal glucose production and perivenous lipid synthesis. Subsequently, excessive production of glucose and accumulation of lipids could be expected in the livers of patients with obesity and insulin resistance. Overall, in this review, we provide our integrative perspective regarding how excessive production of glucose in periportal hepatocytes and accumulation of lipids in perivenous hepatocytes interact in insulin resistant states.     

Intriguing novel abilities of Nutlin-3A: Induction of cellular quiescence as opposed to cellular senescence—Implications for chemotherapy

Comment on: Cellular quiescence caused by the Mdm2 inhibitor nutlin-3a. Korotchkina LG, et al. Cell Cycle 2009; 8:3777-81.     

Endoplasmic reticulum stress-induced apoptosis in the development of diabetes: is there a role for adipose tissue and liver?

Diabetes mellitus (DM) is a multifactorial chronic metabolic disease characterized by hyperglycaemia. Several different mechanisms have been implicated in the development of the disease, including endoplasmic reticulum (ER) stress. ER stress is increasingly acknowledged as an important mechanism in the development of DM, not only for β-cell loss but also for insulin resistance. Accumulating evidence suggests that ER stress-induced apoptosis may be an important mode of β-cell loss and therefore important in the development of diabetes. Recent data also suggest a role of ER stress-induced apoptosis in liver and adipose tissue in relation to diabetes, but more extensive studies on human adipocyte and hepatocyte (patho)physiology and ER stress are needed to identify the exact interactions between environmental signals, ER stress and apoptosis in these organs.