Timing-dependent protection of hypertonic saline solution administration in experimental liver ischemia/reperfusion injury

Background. Diving liver ischemia, the decrease in mitochondrial energy causes cellular damage that is aggravated after reperfusion. This injury can trigger a systemic inflammatory syndrome, also producing remote organ damage. Several substances have been employed to decrease this inflammatory response during liver transplantation, liver resections, and hypovolemic shock. The aim of this study was to evaluate the effects of hypertonic saline solution and the best timing of administration to prevent organ injury during experimental liver ischemia/reperfusion. Methods. Rats underwent 1 hr of warm liver ischemia followed by reperfusion. Eighty-four rats Were allocated into 6 groups: sham group, control of ischemia group) (C), pre-ischemia treated NaCl 0.9% (ISS) and NaCl 7.5% (HTS) groups, pre-repefusion ISS, and HTS groups. Blood and tissue samples were collected 4 hr after reperfusion. Results. HTS showed beneficial effects in prevention of live ischemia/reperfusion injury. HTS groups developed increases in AST and ALT levels that were significantly less than ISS groups; however, the HTS pre-reperfusion group showed levels significantly less than the HTS pre-ischemia group. No differences in IL-6 and IL-10 levels, were observed. A significant decrease in mitochondrial dysfunction as well as hepatic edema was observed in the HTS pre-reperfusion group. Pulmonary vascular permeability Was significantly less in the pre-reperfusion HTS group compared to the ISS group. No differences in myeloperoxidase activity were observed. The liver histologic score was significantly less in the pre-reperfusion HTS group compared to the pre-ischemia I-ITS group. Conclusion. HTS ameliorated local and systemic injuries in experimental liver ischemia/reperfusion. Infusion of HTS in the pre-reperfusion period may be an important adjunct to accomplish the best results. (Surgery 2010;147:415-23.)

DNA damage and repair in leukocytes of melanoma patients exposed in vitro to cisplatin

Resistance to chemotherapeutic drugs can be an obstacle to a successful treatment of cancer patients in part associated with individual response and differences in the DNA repair system. The Comet assay is an informative test to investigate DNA damage and repair in cells in response to a variety of DNA-damaging agents, including chemotherapeutic drugs. The aim of this study was to assess leukocytes damage after in-vitro cisplatin treatment and DNA repair action using the Comet assay in 20 patients with melanoma and 20 cancer-free individuals. Leukocytes` DNA damage before and after cisplatin treatment, in three different concentrations, was analyzed. The DNA repair capability was investigated after 1-5 h of in-vitro cells growing without cisplatin. The Comet score of the patients` basal DNA damage was higher than that observed in controls, but the difference was not statistically significant (P=0.85). Although both groups had similar Comet scores to all cisplatin concentrations tested and the DNA repair times, the basal DNA damage (P < 0.001) and cisplatin damages (P < 0.005) were statistically lower than the different repair times investigated. Considering the progressive increase in the Comet score due to repair time, the negative results here observed could be associated with the reduced cell culture incubation that should be better evaluated. Considering the mutagenic action of cisplatin on tumor cells and the importance of individual DNA repair mechanisms in the chemotherapeutic melanoma treatment, the peripheral leukocytes could be particularly useful as a tool for DNA repair response identified by the Comet assay. Melanoma Res 21:99-105 (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

A rodent model of NASH with cirrhosis, oval cell proliferation and hepatocellular carcinoma

Background/Aims: Hepatocellular carcinoma (HCC) is a well recognized complication of advanced NASH (non-alcoholic steatohepatitis). We sought to produce a rat model of NASH, cirrhosis and HCC. Methods: Adult Sprague-Dawley rats, weighing 250-300 g, were fed a choline-deficient, high trans-fat diet and exposed to DEN in drinking water. After 16 weeks, the animals underwent liver ultrasound (US), sacrifice and assessment by microscopy, immunohistochemistry and transmission electron microscopy (TEM). Results: US revealed steatosis and focal lesions in 6 of 7. All had steatohepatitis defined as inflammation, advanced fibrosis and ballooning with Mallory-Denk bodies (MDB) with frank cirrhosis in 6. Areas of more severe injury were associated with anti-CK19 positive ductular reaction. HCC, present in all, were macro-trabecullar or solid with polyhedral cells with foci of steatosis and ballooned cells. CK19 was positive in single or solid nests of oval cells and in neoplastic hepatocytes. TEM showed ballooning with small droplet fat, dilated endoplasmic reticulum and MDB in non-neoplastic hepatocytes and small droplet steatosis in some cancer cells. Conclusions: This model replicated many features of NASH including steatohepatitis with ballooning, fibrosis, cirrhosis and hepatocellular carcinoma. Oval cell proliferation was evident and the presence anti-CK 19 positivity in the cancer suggests oval cell origin of the malignancy. (C) 2008 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Immunohistochemical evaluation of macrophage activity and its relationship with apoptotic cell death in the polar forms of leprosy

The objective of the present study was to investigate the correlation between macrophage activity and apoptosis in the polar forms of leprosy because the immunopathological phenomena involved in these forms are still poorly understood For this purpose, 29 skin biopsy samples obtained from patients with the polar forms of leprosy were analyzed. Macrophage activity and apoptosis were evaluated by immunohistochemistry using lysozyme, CD68, iNOS and caspase 3 as markers The nonparametric Mann-Whitney test and Spearman`s linear correlation test were used for statistical analysis The results suggest that the apoptosis rate is under the direct influence of macrophage activity in lesions of patients with the tuberculoid form In contrast, in lepromatous lesions other factors seem to induce programmed cell death, possibly TGF-beta. Further studies are necessary to identify additional factors involved in the immunopathogenesis of leprosy. (C) 2010 Elsevier Ltd. All rights reserved

Can LASSBio 596 and dexamethasone treat acute lung and liver inflammation induced by microcystin-LR?

The treatment of microcystin-LR (MCYST-LR)-induced lung inflammation has never been reported Hence. LASSBio 596, an anti-Inflammatory drug candidate, designed as symbiotic agent that modulates TNF-alpha levels and inhibits phosphodiesterase types 4 and 5, or dexamethasone were tested in this condition Swiss mice were intraperitoneally (i p) injected with 60 mu l of saline (CTRL) or a sub-lethal dose of MCYST-LR (40 mu g/kg). 6 h later they were treated (i p.) with saline (TOX), LASSB10 596 (10 mg/kg, L596), or dexamethasone (1 mg/kg, 0.1 mL, DEXA). 8 h after MCYST-LR injection, pulmonary mechanics were determined, and lungs and livers prepared for histopathology, biochemical analysis and quantification of MCYST-LR. TOX showed significantly higher lung impedance than CTRL and L596, which were similar. DEXA could only partially block the mechanical alterations. In both TOX and DEXA alveolar collapse and inflammatory cell influx were higher than in CTRL and L596, being LASSB10 596 more effective than dexamethasone. TOX showed oxidative stress that was not present in an and L596, while DEXA was partially efficient. MCYST-LR was detected in the livers of all mice receiving MCYST-LR and no recovery was apparent In conclusion, LASSBio 596 was more efficient than dexamethasone in reducing the pulmonary functional impairment induced by MCYST-LR. (C) 2010 Elsevier Ltd. All rights reserved

Selective Inner Hair Cell Loss in Prematurity: A Temporal Bone Study of Infants from a Neonatal Intensive Care Unit

Premature birth is a well-known risk factor for sensorineural hearing loss in general and auditory neuropathy in particular. However, relatively little is known about the underlying causes, in part because there are so few relevant histopathological studies. Here, we report on the analysis of hair cell loss patterns in 54 temporal bones from premature infants and a control group of 46 bones from full-term infants, all of whom spent time in the neonatal intensive care unit at the Hospital de Nios in San Jose, Costa Rica, between 1977 and 1993. The prevalence of significant hair cell loss was higher in the preterm group than the full-term group (41% vs. 28%, respectively). The most striking finding was the frequency of selective inner hair cell loss, an extremely rare histopathological pattern, in the preterm vs. the full-term babies (27% vs. 3%, respectively). The findings suggest that a common cause of non-genetic auditory neuropathy is selective loss of inner hair cells rather than primary damage to the cochlear nerve.

Pkd1 Haploinsufficiency Increases Renal Damage and Induces Microcyst Formation following Ischemia/Reperfusion

Mutations in PKD1 cause the majority of cases of autosomal dominant polycystic kidney disease (ADPKD). Because polycystin 1 modulates cell proliferation, cell differentiation, and apoptosis, its lower biologic activity observed in ADPKD might influence the degree of injury after renal ischemia/reperfusion. We induced renal ischemia/reperfusion in 10- to 12-wk-old male noncystic Pkd1(+/-) and wild-type mice. Compared with wild-type mice, heterozygous mice had higher fractional excretions of sodium and potassium and higher serum creatinine after 48 h. In addition, in heterozygous mice, also cortical damage, rates of apoptosis, and inflammatory infiltration into the interstitium at time points out to 14 d after injury all increased, as well as cell proliferation at 48 h and 7 d. The mRNA and protein expression of p21 was lower in heterozygous mice than wild-type mice at 48 h. After 6 wk, we observed dilated tubules, microcysts, and increased renal fibrosis in heterozygotes. The early mortality of heterozygotes was significantly higher than that of wild-type mice when we extended the duration of ischemia from 32 to 35 min. In conclusion, ischemia/reperfusion induces a more severe injury in kidneys of Pkd1-haploin-sufficient mice, a process that apparently depends on a relative deficiency of p2l activity, tubular dilation, and microcyst formation. These data suggest the possibility that humans with ADPKD from PKD1 mutations may be at greater risk for damage from renal ischemia/reperfusion injury.

Pathogenic role of anti-endothelial cell antibodies in autoimmune rheumatic diseases

Anti-endothelial cells antibodies have been detected in numerous autoimmune and inflammatory diseases, including systemic lupus erythematous, rheumatoid arthritis, vasculitis and sarcoidosis. Anti-endothelial cells antibodies bind to endothelial cell antigens and induce endothelial damage. Their effects on the endothelial cell have been considered responsible, at least in part, by the vascular injury which occurs in these pathological conditions. Lupus (2009) 18, 1233-1238.

EFFECTS OF A POTENT PEROXYNITRITE DECOMPOSITION CATALYST IN MURINE MODELS OF ENDOTOXEMIA AND SEPSIS

Excessive free-radical production due to various bacterial components released during bacterial infection has been linked to cell death and tissue injury. Peroxynitrite is a highly reactive oxidant produced by the combination of nitric oxide (NO) and superoxide anion, which has been implicated in cell death and tissue injury in various forms of critical illness. Pharmacological decomposition of peroxynitrite may represent a potential therapeutic approach in diseases associated with the overproduction of NO and superoxide. In the present study, we tested the effect of a potent peroxynitrite decomposition catalyst in murine models of endotoxemia and sepsis. Mice were injected i.p. with LPS 40 mg/kg with or without FP15 [Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether) pyridyl porphyrin] (0.1, 0.3, 1, 3, or 10 mg/kg per hour). Mice were killed 12 h later, followed by the harvesting of samples from the lung, liver, and gut for malondialdehyde and myeloperoxidase measurements. In other subsets of animals, blood samples were obtained by cardiac puncture at 1.5, 4, and 8 h after LPS administration for cytokine (TNF-alpha, IL-1 beta, and IL-10), nitrite/nitrate, alanine aminotransferase, and blood urea nitrogen measurements. Endotoxemic animals showed an increase in survival from 25% to 80% at the FP15 doses of 0.3 and 1 mg/kg per hour. The same dose of FP15 had no effect on plasma levels of nitrite/nitrate. There was a reduction in liver and lung malondialdehyde in the endotoxemic animals pretreated with FP15, as well as in hepatic myeloperoxidase and biochemical markers of liver and kidney damage (alanine aminotransferase and blood urea nitrogen). In a bacterial model of sepsis induced by cecal ligation and puncture, FP15 treatment (0.3 mg/kg per day) significantly protected against mortality. The current data support the view that peroxynitrite is a critical factor mediating liver, gut, and lung injury in endotoxemia and septic shock: its pharmacological neutralization may be of therapeutic benefit.

Antigenotoxicity and antimutagenicity of lycopene in HepG2 cell line evaluated by the comet assay and micronucleus test

Epidemiological studies have provided evidence that high consumption of tomatoes effectively reduces the risk of reactive oxygen species (ROS)-mediated diseases such as cancer. Tomatoes are rich sources of lycopene, a potent singlet oxygen-quenching carotenoid. In addition to its antioxidant properties, lycopene shows an array of biological effects including antimutagenic and anticarcinogenic activities. In the present study, the chemopreventive action of lycopene was examined on DNA damage and clastogenic or aneugenic effects of H2O2 and n-nitrosodiethylamine (DEN) in the metabolically competent human hepatoma cell line (HepG2 cells). Lycopene at concentrations of 10. 25, and 50 mu M, was tested under three protocols: before, simultaneously, and after treatment with the mutagen, using the comet and micronucleus assays. Lycopene significantly reduced the genotoxicity and mutagenicity of H2O2 in all of the conditions tested. For DEN, significant reductions of primary DNA damage (comet assay) were detected when the carotenoid (all of the doses) was added in the cell culture medium before or simultaneously with the mutagen. In the micronucleus test, the protective effect of lycopene was observed only when added prior to DEN treatment. In conclusion, our results suggest that lycopene is a suitable agent for preventing chemically-induced DNA and chromosome damage. (C) 2007 Elsevier Ltd. All rights reserved.

Evidences of a Role for Eukaryotic Translation Initiation Factor 5A (eIF5A) in Mouse Embryogenesis and Cell Differentiation

Eukaryotic translation initiation factor 5A (eIF5A) has a unique character: the presence of an unusual amino acid, hypusine, which is formed by post-translational modifications. Even before the identification of hypusination in eIF5A, the correlation between hypusine formation and protein synthesis, shifting cell proliferation rates, had already been observed. Embryogenesis is a complex process in which cellular proliferation and differentiation are intense. In spite of the fact that many studies have described possible functions for eIF5A, its precise role is under investigation, and to date nothing has been reported about its participation in embryonic development. In this study we show that eIF5A is expressed at all mouse embryonic post-implantation stages with increase in eIF5A mRNA and protein expression levels between embryonic days E10.5 and E13.5. Immunohistochemistry revealed the ubiquitous presence of eIF5A in embryonic tissues and organs at E13.5 day. Interestingly, stronger immunoreactivity to eIF5A was observed in the stomodeum, liver, ectoderm, heart, and eye, and the central nervous system; regions which are known to undergo active differentiation at this stage, suggesting a role of eIF5A in differentiation events. Expression analyses of MyoD, a myogenic transcription factor, revealed a significantly higher expression from day E12.5 on, both at the mRNA and the protein levels suggesting a possible correlation to eIF5A. Accordingly, we next evidenced that inhibiting eIF5A hypusination in mouse myoblast C2C12 cells impairs their differentiation into myotubes and decreases MyoD transcript levels. Those results point to a new functional role for eIF5A, relating it to embryogenesis, development, and cell differentiation. J. Cell. Physiol. 225: 500-505, 2010. (C) 2010 Wiley-Liss, Inc.

Brown spider venom toxins interact with cell surface and are endocytosed by rabbit endothelial cells

Bites from the Loxosceles genus (brown spiders) cause severe clinical symptoms, Including dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of dermonecrotic lesions in animals exposed to Loxosceles intermedia venom show numerous vascular alterations Study of the hemorrhagic consequences of the venom in endothelial cells has demonstrated that the degeneration of blood vessels results not only from degradation of the extracellular matrix molecule or massive leukocyte infiltration, but also from a direct and primary activity of the venom on endothelial cells. Exposure of an endothelial cell line in vitro to L. intermedia venom induce morphological alterations, such as cell retraction and disadhesion to the extracellular matrix. The aim of the present study was to investigate the interaction between the venom toxins and the endothelial cell surface and their possible internalization, in order to illuminate the information about the deleterious effect triggered by venom After treating endothelial cells with venom toxins, we observed that the venom Interacts with cell surface. Venom treatment also can cause a reduction of cell surface glycoconjugates When cells were permeabilized, it was possible to verify that some venom toxins were internalized by the endothelial cells The venom internalization involves endocytic vesicles and the venom was detected in the lysosomes. However, no damage to lysosomal integrity was observed, suggesting that the cytotoxic effect evoked by L interned:a venom on endothelial cells is not mediated by venom internalization (C) 2010 Elsevier Ltd. All rights reserved

Pore Formation Triggered by Legionella spp. Is an Nlrc4 Inflammasome-Dependent Host Cell Response That Precedes Pyroptosis

Legionella pneumophila, the etiological agent of Legionnaires disease, is known to trigger pore formation in bone marrow-derived macrophages (BMMs) by mechanisms dependent on the type IVB secretion system known as Dot/Icm. Here, we used several mutants of L. pneumophila in combination with knockout mice to assess the host and bacterial factors involved in pore formation in BMMs. We found that regardless of Dot/Icm activity, pore formation does not occur in BMMs deficient in caspase-1 and Nlrc4/Ipaf. Pore formation was temporally associated with interleukin-1 beta secretion and preceded host cell lysis and pyroptosis. Pore-forming ability was dependent on bacterial Dot/Icm but independent of several effector proteins, multiplication, and de novo protein synthesis. Flagellin, which is known to trigger the Nlrc4 inflammasome, was required for pore formation as flaA mutant bacteria failed to induce cell permeabilization. Accordingly, transfection of purified flagellin was sufficient to trigger pore formation independent of infection. By using 11 different Legionella species, we found robust pore formation in response to L. micdadei, L. bozemanii, L. gratiana, L. jordanis, and L. rubrilucens, and this trait correlated with flagellin expression by these species. Together, the results suggest that pore formation is neither L. pneumophila specific nor the result of membrane damage induced by Dot/Icm activity; instead, it is a highly coordinated host cell response dependent on host Nlrc4 and caspase-1 and on bacterial flagellin and type IV secretion system.

In Vitro Phototoxicity of Liposomes and Nanocapsules Containing Chloroaluminum Phthalocyanine on Human Melanoma Cell Line

In this study, the photodynamic action of liposomes (LP) and nanocapsules (NC) containing Chloroaluminum phthalocyanine (CIAIPc), on the human melanoma cell (WM 1552C), was assessed. The light source was setup at 672 nm, which corresponds to the maximum absorption wavelength of the CIAIPc. Both colloidal carriers presented size in nanometric scale as well as negative zeta potential. The cellular damage was light dose dependent ranging from 30% of cell death at 70 mJ.cm(-2) to 90% of death at 700 mJ.cm(-2). However, the photocytotoxic effect of LP at 70 mJ.cm(-2) was slightly more efficient to induce cellular death than NC formulation. At 140 mJ.cm(-2), and 700 mJ.cm(-2) both nanocarriers were equally efficient to induce cellular damage. Therefore, in the present work, the maximum phototoxic effect was obtained with 700 mJ.cm(-2) of light dose, in combination with 0.29 mu g.mL(-1) of CIAIPc encapsulated into LP and NC. The cells were also positive to annexin V, after the PDT treatment with LP and NC, showing that one of the mechanisms of cellular death involved is apoptosis. In summary, the potential of LP and NC as a drug delivery system, in Photodynamic Therapy (PDT) against melanoma, has been confirmed using a lower concentration of the photosensitizer and lower light doses than that applied in current protocols. This is an innovative proposal to treat melanoma cell lines that until now have not received the benefit of the PDT protocol for treatment.

The Hus1 homologue of Leishmania major encodes a nuclear protein that participates in DNA damage response

The protozoan parasite Leishmania presents a dynamic and plastic genome in which gene amplification and chromosome translocations are common phenomena. Such plasticity hints at the necessity of dependable genome maintenance pathways. Eukaryotic cells have evolved checkpoint control systems that recognize altered DNA structures and halt cell cycle progression allowing DNA repair to take place. In these cells, the PCNA-related heterotrimeric complex formed by the proteins Hus1, Rad9, and Rad1 is known to participate in the early steps of replicative stress sensing and signaling. Here we show that the Hus1 homolog of Leishmania major is a nuclear protein that improves the cell capability to cope with replicative stress. Overexpression of LmHus1 confers resistance to the genotoxic drugs hydroxyurea (HU) and methyl methanesulfonate (MMS) and resistance to HU correlates to reduced net DNA damage upon LmHus1 expression. (C) 2011 Elsevier B.V. All rights reserved.