Inhibition of Drug-Induced Liver Injury in Mice Using a Positively Charged Peptide That Binds DNA.

Hepatic cell death occurs in response to diverse stimuli such as chemical and physical damage. The exposure of intracellular contents such as DNA during necrosis induces a severe inflammatory response that has yet to be fully explored therapeutically. Here, we sought means to neutralize the ability of extracellular DNA to induce deleterious tissue inflammation when drug-induced liver injury had already ensued. DNA exposure and inflammation were investigated in vivo in drug-induced liver injury using intravital microscopy. The necrotic DNA debris was studied in murine livers in vivo and in DNA debris models in vitro by using a positively charged chemokine-derived peptide (MIG30; CXCL9[74-103]). Acetaminophen-induced liver necrosis was associated with massive DNA accumulation, production of CXC chemokines, and neutrophil activation inside the injured tissue. The MIG30 peptide bound the healthy liver vasculature and, to a much greater extent, to DNA-rich necrotic tissue. Moreover, MIG30 bound extracellular DNA directly in vivo in a charge-dependent manner and independently of glycosaminoglycans and chemokines. Post-treatment of mice with MIG30 reduced mortality, liver damage, and inflammation significantly. These effects were not observed with a control peptide that does not bind DNA. Mechanistically, MIG30 inhibited the interaction between DNA and histones, and promoted the dissociation of histones from necrotic debris. MIG30 also inhibited the pro-inflammatory effect of CpG DNA, as measured by a reduction in CXCL8 production, indicating that MIG30 disturbs the ability of DNA to induce hepatic inflammation. Conclusion: The use of DNA-binding peptides reduces necrotic liver injury and inflammation, even at late timepoints.

Mastoparan-induced programmed cell death in the unicellular alga Chlamydomonas reinhardtii.

BACKGROUND AND AIMS: Under stress-promoting conditions unicellular algae can undergo programmed cell death (PCD) but the mechanisms of algal cellular suicide are still poorly understood. In this work, the involvement of caspase-like proteases, DNA cleavage and the morphological occurrence of cell death in wasp venom mastoparan (MP)-treated Chlamydomonas reinhardtii were studied. METHODS: Algal cells were exposed to MP and cell death was analysed over time. Specific caspase inhibitors were employed to elucidate the possible role of caspase-like proteases. YVADase activity (presumably a vacuolar processing enzyme) was assayed by using a fluorogenic caspase-1 substrate. DNA breakdown was evaluated by DNA laddering and Comet analysis. Cellular morphology was examined by confocal laser scanning microscopy. KEY RESULTS: MP-treated C. reinhardtii cells expressed several features of necrosis (protoplast shrinkage) and vacuolar cell death (lytic vesicles, vacuolization, empty cell-walled corpse-containing remains of digested protoplast) sometimes within one single cell and in different individual cells. Nucleus compaction and DNA fragmentation were detected. YVADase activity was rapidly stimulated in response to MP but the early cell death was not inhibited by caspase inhibitors. At later time points, however, the caspase inhibitors were effective in cell-death suppression. Conditioned medium from MP-treated cells offered protection against MP-induced cell death. CONCLUSIONS: In C. reinhardtii MP triggered PCD of atypical phenotype comprising features of vacuolar and necrotic cell deaths, reminiscent of the modality of hypersensitive response. It was assumed that depending on the physiological state and sensitivity of the cells to MP, the early cell-death phase might be not mediated by caspase-like enzymes, whereas later cell death may involve caspase-like-dependent proteolysis. The findings substantiate the hypothesis that, depending on the mode of induction and sensitivity of the cells, algal PCD may take different forms and proceed through different pathways.

Powerplex® ES versus Powerplex® S5--casework testing of the new screening kit.

The new Powerplex® S5 Mini STR-System from Promega with the four provided STR loci D18S51, D8S1179, TH01 and FGA as well as the Amelogenin marker (PCR products ranging from 80 to 220 bp not considering the longer FGA fragments) is designed as a screening tool especially in difficult casework samples. To test its suitability we amplified highly degraded DNA from casework samples, which had shown no or only poor results in analyses with the Powerplex® ES kit, as well as artificially degraded DNA or DNA samples containing PCR inhibitors. Despite a tendency for allelic drop-ins in the amplification of highly degraded DNA the Powerplex® S5 kit was a reliable tool for the analysis of casework samples with degraded DNA which gave better results than the Powerplex® ES kit in 64% of analysed swabs. Furthermore, it was especially suitable for the investigation of formalin fixed tissue, tissue samples showing advanced putrefaction or telogen hair samples. However, there was no strict relation between positive Powerplex® S5 results and amplification success with the Powerplex® ES kit.

Effect of presumptive tests reagents on human blood confirmatory tests and DNA analysis using real time polymerase chain reaction.

Bloodstains often constitute the major physical evidence in crime investigation. Diluted blood invisible to the naked eye can be detected through presumptive tests however such tests can damage samples and prevent further processing such as DNA analysis. In this study, we compared the effects of luminol (prepared according to Weber [15]), Luminol 16(®), Bluestar(®) and benzidine for inhibition in the human antiglobulin test and the human hemoglobin immunochromatographic test and on the total human DNA concentration up to 120 days after sample treatment. Treatment with both luminol solutions and Bluestar(®) still allowed positive results for the immunologic tests, indicating non-interference with human blood confirmatory tests. However, samples treated with benzidine could not be further analyzed by serological tests. Also, DNA quantification showed that 48h after benzidine treatment, but not luminol or Bluestar solution application, sample DNA was degraded. Luminol 16(®) caused DNA degradation already at 30 days post-application. At 120 days post treatment, all samples treated with any of the agents but not untreated samples had DNA degradation.

Developmental cell death in the liver and newborn lethality of Ku86 deficient mice suppressed by antioxidant N-acetyl-cysteine.

Repair of DNA double-strand breaks (DSBs) is essential for genome integrity and cell survival. Ku86 is involved in the repair of DNA DSBs by non-homologous end joining (NHEJ). Mice deficient in Ku86 show growth retardation, dwarfism, premature aging, and immunodeficiency. In this study, we observed severely compromised survival of Ku86(-/-) mice, such that most Ku86(-/-) mice died within the first postnatal weeks and only 1.5% of the expected 25% from heterozygous crosses survived for 1 month. Since post-mortem analysis was not possible due to parental cannibalism, histopathological examination was performed on Ku86(-/-) fetuses to assess possible causes of newborn death. Eighty percent and 75% of Ku86(-/-) fetuses exhibited apoptosis and necrosis in the liver, while only 20% and 10% of Ku86(+/+) littermates had apoptosis and necrosis, respectively. In addition, the severity of liver damage was significantly higher in Ku86(-/-) fetuses. Developmental liver damage may have led to postnatal lethality because the fetal liver with pre-existing injury may not be able to undergo transformation from a lymphohematopoietic to an indispensable metabolic organ. Free radicals can cause chromosomal breaks and lead to cell death. We postulated that endogenous oxidative stress might be involved in the resulting liver damage and animal lethality in Ku86(-/-) mice deficient in DNA DSB repair. This hypothesis was tested by treating Ku86(-/-) mice with the well known free radical scavenger, thiol antioxidant N-acetyl-cysteine (NAC), during embryonic development. We found that a significantly higher percentage, 7.7% of NAC treated Ku86(-/-) offspring versus 1.5% untreated Ku86(-/-) mice were alive at 1 month of age. In addition, the incidence of liver necrosis decreased by 21% and the severity of necrosis significantly reduced. Thus, Ku86 deficiency results in severe developmental liver damage and newborn lethality associated with oxidative stress.