Gliosis induction on locus coeruleus in a living liver donor experimental model: A brief review.

Living Donor Liver Transplantation (LDLT) is a promising approach to treating end-stage liver diseases, however, some post-operatory complications such as pneumonia, bacteremia, urinary tract infections, and hepatic dysfunction have been reported. In murine models using partial hepatectomy (PHx), a model that emulates LDLT, it has been determined that the synthesis of hepatic cell proliferation factors that are associated with noradrenaline synthesis are produced in locus coeruleus (LC). In addition, studies have shown that PHx decreases GABA and 5-HT2A receptors, promotes loss of dendritic spines, and favors microgliosis in rat hippocampus. The GABA and serotonin-altered circuits suggest that catecholaminergic neurons such as dopamine and noradrenaline neurons, which are highly susceptible to cellular stress, can also be damaged. To understand post-transplant affections and to perform well-controlled studies it is necessary to know the potential causes that explain as a liver surgical procedure can produce brain damage. In this paper, we review several cellular processes that could induce gliosis in LC after rat PHx.

BrdU does not induce hepatocellular damage in experimental Wistar rats.

Bromodeoxyuridine (BrdU) is used in studies related to cell proliferation and neurogenesis. The multiple intraperitoneal injections of this molecule could favor liver function profile changes. In this study, we evaluate the systemic and hepatocellular impact of BrdU in male adult Wistar rats in 30 %-partial hepatectomy (PHx) model. The rats received BrdU 50 mg/Kg by intraperitoneal injection at 0.5, 1, 2, 3, 6, 9 and 16 days after 30 %-PH. The rats were distributed into four groups as follows, control, sham, PHx/BrdU(-) and PHx/BrdU(+). On day 16, we evaluated hepatocellular nuclei and analyzed histopathological features by haematoxylin-eosin stain and apoptotic profile was qualified by caspase-3 presence. The systemic effect was evaluated by liver markers such as alanine transferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (AP), bilirubin, total proteins and serum albumin content. The statistical analysis consisted of a student t-test and one-way ANOVA. BrdU did not induce apoptosis or hepatocellular damage in male rats. Multiple administrations of BrdU in male rats did not induce significant decrease body weight, but increased serum ALT and LDH levels were found. Our results show that the BrdU does not produce hepatocellular damage.

Enhanced antitumor activity induced by a DNA vaccine encoding E7 antigen fused to an ERAD-targeting sequence.

The endoplasmic reticulum (ER) is a key organelle in cell homeostasis and cell health through antigen presentation to immune cells. Thus, the ER has become a therapeutic target to induce cellular immune responses. We previously reported the antitumor effect of a DNA vaccine that expresses the E7 antigen fused to the cyclooxygenase-2 (COX-2) protein. This inflammation-related enzyme contains a degradation cassette associated with the endoplasmic reticulum-associated degradation (ERAD) pathway. To avoid the use of full-length COX-2 and any risk of adverse effects due to the activity of its catalytic site, we designed new versions of the fusion protein. These new constructs encode the E7 antigen fused to the signal peptide and the ERAD sequence of COX-2 with or without the membrane-binding domain (MBD) as well as deletion of the catalytic site. We evaluated the antigen-specific antitumor effect of these DNA constructs in murine prophylactic and therapeutic cancer models. These assays showed that the ERAD cassette is the minimum sequence in the COX-2 protein that induces an antitumor effect when fused to the E7 antigen with the advantage of eliminating any potential adverse effects from the use of full-length COX-2.

Plasmid DNA for Therapeutic Applications in Cancer.

Recently, the interest in using nucleic acids for therapeutic applications has been increasing. DNA molecules can be manipulated to express a gene of interest for gene therapy applications or vaccine development. Plasmid DNA can be developed to treat different diseases, such as infections and cancer. In most cancers, the immune system is limited or suppressed, allowing cancer cells to grow. DNA vaccination has demonstrated its capacity to stimulate the immune system to fight against cancer cells. Furthermore, plasmids for cancer gene therapy can direct the expression of proteins with different functions, such as enzymes, toxins, and cytotoxic or proapoptotic proteins, to directly kill cancer cells. The progress and promising results reported in animal models in recent years have led to interesting clinical results. These DNA strategies are expected to be approved for cancer treatment in the near future. This review discusses the main strategies, challenges, and future perspectives of using plasmid DNA for cancer treatment.

Targeting E7 antigen to the endoplasmic reticulum degradation pathway promotes a potent therapeutic antitumor effect.

It has been previously reported that targeting and retaining antigens in the endoplasmic reticulum (ER) can induce an ER stress response. In this study, we evaluated the antitumor effect of E7 antigen fused to an ERresident protein, cyclooxygenase-2, which possesses a 19-aminoacid cassette that directs it to the endoplasmic reticulum-associated protein degradation (ERAD) pathway. The featured DNA constructs, COX2-E7 and COX2-E7ΔERAD, with a deletion in the 19-aminoacid cassette, were used to evaluate the importance of this sequence. In vitro analysis of protein expression and ER localisation were verified. We observed that both constructs induced an ER stress response. This finding correlated with the antitumor effect in mice injected with TC-1 cells and treated with different DNA constructs by biolistic vaccination. Immunisation with COX2-E7 and COX2-E7ΔERAD DNA constructs induced a significant antitumor effect in mice, without a significant difference between them, although the COX2-E7 construct induced a significant E7-specific immune response. These results demonstrate that targeting the E7 antigen to the ERAD pathway promotes a potent therapeutic antitumor effect. This strategy could be useful for the design of other antigen-specific therapies.

Caspase-3-related apoptosis prevents pathological regeneration in a living liver donor rat model.

PURPOSE: The main goal of this study was to determine the relationship of cleaved-caspase-3 (C3)-related apoptosis and hepatic proliferation, during the liver repopulation in a living liver donor rat model. MATERIAL/METHODS: Thirty-three animals were randomized into eleven groups and evaluated on postoperative from 3 â€‹h until 384 â€‹h after 30%-partial hepatectomy (30%-PHx). Liver sections (5 â€‹µm) were processed by hematoxylin-eosin, and immunostaining for C3, accompanied by hepatic function test. C3 content and the hepatic lobule enlargement were analyzed by optical density, followed by cell counting. RESULTS: Transient variations of alanine transferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were found. Significant increase in the C3 levels, and cell nuclei number, were detected at 12 â€‹h and 48 â€‹h after 30%-PHx, evidencing a correlation of p â€‹= â€‹-0.3679. CONCLUSION: In the 30%-PHx rat model, C3-related apoptosis prevents proliferative pathological conditions during the hepatic lobule re-modeling.

Antitumor effect of adenoviruses expressing mutant non-oncogenic E7 versions from HPV-16 fused to calreticulin.

PURPOSE: To compare the antitumor effect of adenoviruses that express mutant variants of the protein E7 from HPV-16 fused to calreticulin. METHODS: Recombinant adenoviruses were generated to express calreticulin fused to mutant versions of E7 (CRT/E7m and CRT/E7dm). Western blot and immunofluorescence assays were made to demonstrate protein expression. Antitumor assays were performed in C57BL6 mice injected with TC-1 cell line. RESULTS: When HEK293 cells were infected with these adenoviruses, we detected that all the recombinant proteins were expressed at endoplasmic reticulum, as expected. Next, the antitumor effect was tested on a murine tumor model established by inoculation of TC-1 cell line. We detected that both Ad CRT/E7m and Ad CRT/E7dm were capable of reducing the antitumor volume when compared to Ad LacZ, which was used as negative control. No significant difference was observed when compared to Ad CRT/E7, a positive control. CONCLUSIONS: Here we demonstrated that the mutant versions of E7 HPV-16 fused to calreticulin generate similar antitumor effect than the wild type version.

The mRVG-9R peptide as a potential therapeutic vector to the central nervous system cells.

Our research group has developed a cell-penetrating peptide-based delivery system that includes the Asn194Lys mutation in the rabies virus glycoprotein-9R peptide (mRVG-9R). This system has the capacity to deliver DNA in astrocytes and SH-SY5Y cells. The aim of this study was to evaluate the ability of the mRVG-9R peptide to deliver DNA molecules to murine brain cells. The mRVG-9R peptide, a karyophilic peptide (KP) and a plasmid encoding green fluorescent protein (GFP) were bound by electrostatic charges to form the mRVG-9R complex. mRVG-9R complex was injected into the cerebral cortex, striatum and hippocampus of C57BL/6 mice by stereotactic surgery. After 2, 4, and 20 days, the animals were sacrificed and their brains were prepared for quantitative reverse-transcription polymerase chain reaction and histological analysis. We detected the GFP expression in neurons and glial cells in the cerebral cortex, striatum, and hippocampus of the murine brain. The results suggest that the mRVG-9R peptide has the ability to deliver DNA molecules to murine brain cells. Also, the expression of the reporter gene is maintained at least up to 20 days after injection in neurons, astrocytes, oligodendrocytes, and microglia cells. Thus, the in vivo transfection ability of the mRVG-9R peptide, makes it a promising candidate as a therapeutic gene delivery vector to the central nervous system cells.

Four main therapeutic keys for Parkinson's disease: A mini review.

OBJECTIVES: Parkinson's disease (PD) is characterized by motor and cognitive dysfunctions. The progressive degeneration of dopamine-producing neurons that are present in the substantia nigra pars compacta (SNpc) has been the main focus of study and PD therapies since ages. MATERIALS AND METHODS: In this manuscript, a systematic revision of experimental and clinical evidence of PD-associated cell process was conducted. RESULTS: Classically, the damage in the dopaminergic neuronal circuits of SNpc is favored by reactive oxidative/nitrosative stress, leading to cell death. Interestingly, the therapy for PD has only focused on avoiding the symptom progression but not in finding a complete reversion of the disease. Recent evidence suggests that the renin-angiotensin system imbalance and neuroinflammation are the main keys in the progression of experimental PD. CONCLUSION: The progression of neurodegeneration in SNpc is due to the complex interaction of multiple processes. In this review, we analyzed the main contribution of four cellular processes and discussed in the perspective of novel experimental approaches.

Asn194Lys mutation in RVG29 peptide increases GFP transgene delivery by endocytosis to neuroblastoma and astrocyte cells.

OBJECTIVES: A cell-penetrating peptide-based delivery system could target specific types of cells for therapeutic genes delivery. To increase the gene delivery efficiency into neuronal phenotype cells, we introduced an Asn194Lys mutation to RVG29 peptide derived from rabies virus glycoprotein and added a nuclear localization signal to enhance its nuclear import. METHODS: Mutant RVG or wild-type RVG peptide, a karyophilic peptide (KP) and a plasmid encoding green fluorescent protein (pGL) were bound by electrostatic charges to form four different kinds of RVG complexes. Immunofluorescence was used to assess the gene transfection efficiency into astrocytes, oligodendrocyte precursor cells (OPCs), SH-SY5Y, HeLa and NIH/3T3 cells. The cellular uptake mechanism of RVG29 complexes was examined using endocytosis inhibitors. KEY FINDINGS: The mRVG29 peptide has the ability to enhance the nuclear import of plasmids. The Asn194Lys mutation in RVG29 peptide of the pGL-mRVG29 complex and the addition of KP to the pGL-RVG29-KP complex increased the capacity to deliver DNA by endocytosis in astrocytes and SH-SY5Y cells. CONCLUSIONS: The complexes pGL-mRVG29 and pGL-RVG29-KP have specificity for transfecting astrocytes and SH-SY5Y cells. The karyophilic capacity of this new mRVG peptide render it promising candidate to act as gene delivery vector into the brain cells.

Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways.

Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson's disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of wild type (WT) or mutant A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic and yeast cells in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways.

Enhancement of Ad-CRT/E7-mediated antitumor effect by preimmunization with L. lactis expressing HPV-16 E7.

Although current polyvalent vaccines can prevent development of cervical cancer, they cannot be used to treat patients who already have the disease. Adenovirus expressing calreticulin-E7 (Ad-CRT-E7) has shown promising results in the cervical cancer murine model. We also demonstrated that immunization with Lactococcus lactis encoding HPV-16 E7 (Ll-E7) anchored to its surface induces significant HPV-16 E7-specific immune response. Here, we assessed the combination of both approaches in the treatment of a cervical cancer animal model. Intranasal preimmunization of Ll-E7, followed by a single Ad-CRT/E7 application, induced ∼80% of tumor suppression in comparison with controls. Mice treated with a combination of Ll-E7 and Ad-CRT/E7 resulted in a 70% survival rate 300 days post-treatment, whereas 100% of the mice in the control groups died by 50 days. Significant CD8+ cytotoxic T-lymphocytes infiltration was detected in the tumors of mice treated with Ll-E7+Ad-CRT/E7. Tumors with regression showed a greater number of positive cells for in situ TUNEL staining than controls. Our results suggest that preimmunization with Ll-E7 enhances the Ad-CRT/E7-mediated antitumor effect. This treatment provides an enormous advantage over repeated applications of Ad-CRT/E7 by maintaining the effectiveness of the three-dose application of Ad-CRT/E7, but avoiding the high systemic toxicities associated with such repeat treatments.

Alterations in energy/redox metabolism induced by mitochondrial and environmental toxins: a specific role for glucose-6-phosphate-dehydrogenase and the pentose phosphate pathway in paraquat toxicity.

Parkinson's disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat "hijacks" the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations in energy metabolism, which are specific for distinct mitochondiral/environmental toxins, are not bystanders to energy failure but also contribute significant to cell death progression.

Antioxidant gene therapy against neuronal cell death.

Oxidative stress is a common hallmark of neuronal cell death associated with neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, as well as brain stroke/ischemia and traumatic brain injury. Increased accumulation of reactive species of both oxygen (ROS) and nitrogen (RNS) has been implicated in mitochondrial dysfunction, energy impairment, alterations in metal homeostasis and accumulation of aggregated proteins observed in neurodegenerative disorders, which lead to the activation/modulation of cell death mechanisms that include apoptotic, necrotic and autophagic pathways. Thus, the design of novel antioxidant strategies to selectively target oxidative stress and redox imbalance might represent important therapeutic approaches against neurological disorders. This work reviews the evidence demonstrating the ability of genetically encoded antioxidant systems to selectively counteract neuronal cell loss in neurodegenerative diseases and ischemic brain damage. Because gene therapy approaches to treat inherited and acquired disorders offer many unique advantages over conventional therapeutic approaches, we discussed basic research/clinical evidence and the potential of virus-mediated gene delivery techniques for antioxidant gene therapy.

Glutaredoxin 1 protects dopaminergic cells by increased protein glutathionylation in experimental Parkinson's disease.

AIMS: Chronic exposure to environmental toxicants, such as paraquat, has been suggested as a risk factor for Parkinson's disease (PD). Although dopaminergic cell death in PD is associated with oxidative damage, the molecular mechanisms involved remain elusive. Glutaredoxins (GRXs) utilize the reducing power of glutathione to modulate redox-dependent signaling pathways by protein glutathionylation. We aimed to determine the role of GRX1 and protein glutathionylation in dopaminergic cell death. RESULTS: In dopaminergic cells, toxicity induced by paraquat or 6-hydroxydopamine (6-OHDA) was inhibited by GRX1 overexpression, while its knock-down sensitized cells to paraquat-induced cell death. Dopaminergic cell death was paralleled by protein deglutathionylation, and this was reversed by GRX1. Mass spectrometry analysis of immunoprecipitated glutathionylated proteins identified the actin binding flightless-1 homolog protein (FLI-I) and the RalBP1-associated Eps domain-containing protein 2 (REPS2/POB1) as targets of glutathionylation in dopaminergic cells. Paraquat induced the degradation of FLI-I and REPS2 proteins, which corresponded with the activation of caspase 3 and cell death progression. GRX1 overexpression reduced both the degradation and deglutathionylation of FLI-I and REPS2, while stable overexpression of REPS2 reduced paraquat toxicity. A decrease in glutathionylated proteins and REPS2 levels was also observed in the substantia nigra of mice treated with paraquat. INNOVATION: We have identified novel protein targets of glutathionylation in dopaminergic cells and demonstrated the protective role of GRX1-mediated protein glutathionylation against paraquat-induced toxicity. CONCLUSIONS: These results demonstrate a protective role for GRX1 and increased protein glutathionylation in dopaminergic cell death induced by paraquat, and identify a novel protective role for REPS2.

Thiol-redox signaling, dopaminergic cell death, and Parkinson's disease.

SIGNIFICANCE: Parkinson's disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta, which has been widely associated with oxidative stress. However, the mechanisms by which redox signaling regulates cell death progression remain elusive. RECENT ADVANCES: Early studies demonstrated that depletion of glutathione (GSH), the most abundant low-molecular-weight thiol and major antioxidant defense in cells, is one of the earliest biochemical events associated with PD, prompting researchers to determine the role of oxidative stress in dopaminergic cell death. Since then, the concept of oxidative stress has evolved into redox signaling, and its complexity is highlighted by the discovery of a variety of thiol-based redox-dependent processes regulating not only oxidative damage, but also the activation of a myriad of signaling/enzymatic mechanisms. CRITICAL ISSUES: GSH and GSH-based antioxidant systems are important regulators of neurodegeneration associated with PD. In addition, thiol-based redox systems, such as peroxiredoxins, thioredoxins, metallothioneins, methionine sulfoxide reductases, transcription factors, as well as oxidative modifications in protein thiols (cysteines), including cysteine hydroxylation, glutathionylation, and nitrosylation, have been demonstrated to regulate dopaminergic cell loss. FUTURE DIRECTIONS: In this review, we summarize major advances in the understanding of the role of thiol-redox signaling in dopaminergic cell death in experimental PD. Future research is still required to clearly understand how integrated thiol-redox signaling regulates the activation of the cell death machinery, and the knowledge generated should open new avenues for the design of novel therapeutic approaches against PD.

Production of biologically active human lymphotactin (XCL1) by Lactococcus lactis.

Lymphotactin-XCL1 is a chemokine produced mainly by activated CD8+ T-cells and directs migration of CD4+ and CD8+ lymphocytes and natural killer (NK) cells. We expressed human lymphotactin (LTN) by the lactic-acid bacterium Lactococcus lactis. Biological activity of LTN was confirmed by chemo-attraction of human T-cells by chemotaxis demonstrating, for the first time, how this chemokine secreted by a food-grade prokaryote retains biological activity and chemoattracts T lymphocytes. This strain thus represents a feasible well-tolerated vector to deliver active LTN at a mucosal level.