Darko Ciric

Large graphene quantum dots alleviate immune-mediated liver damage.

We investigated the effect of large (40 nm) graphene quantum dots (GQDs) in concanavalin A (Con A; 12 mg/kg i.v.)-induced mouse hepatitis, a T cell-mediated liver injury resembling fulminant hepatitis in humans. Intravenously injected GQDs (50 mg/kg) accumulated in liver and reduced Con A-mediated liver damage, as demonstrated by histopathological analysis and a decrease in liver lipid peroxidation and serum levels of liver transaminases. The cleavage of apoptotic markers caspase-3/PARP and mRNA levels of proapoptotic mediators Puma, Noxa, Bax, Bak1, Bim, Apaf1, and p21, as well as LC3-I conversion to autophagosome-associated LC3-II and expression of autophagy-related (Atg) genes Atg4b, Atg7, Atg12, and beclin-1, were attenuated by GQDs, indicating a decrease in both apoptosis and autophagy in the liver tissue. This was associated with the reduced liver infiltration of immune cells, particularly the T cells producing proinflammatory cytokine IFN-γ, and a decrease in IFN-γ serum levels. In the spleen of GQD-exposed mice, mRNA expression of IFN-γ and its transcription factor T-bet was reduced, while that of the IL-33 ligand ST2 was increased. The hepatoprotective effect of GQDs was less pronounced in ST2-deficient mice, indicating that it might depend on ST2 upregulation. In vitro, GQDs inhibited splenocyte IFN-γ production, reduced the activation of extracellular signal-regulated kinase in macrophage and T cell lines, inhibited macrophage production of the free radical nitric oxide, and reduced its cytotoxicity toward hepatocyte cell line HepG2. Therefore, GQDs alleviate immune-mediated fulminant hepatitis by interfering with T cell and macrophage activation and possibly by exerting a direct hepatoprotective effect.

Autophagy inhibition uncovers the neurotoxic action of the antipsychotic drug olanzapine

We investigated the role of autophagy, a controlled cellular self-digestion process, in regulating survival of neurons exposed to atypical antipsychotic olanzapine. Olanzapine induced autophagy in human SH-SY5Y neuronal cell line, as confirmed by the increase in autophagic flux and presence of autophagic vesicles, fusion of autophagosomes with lysosomes, and increase in the expression of autophagy-related (ATG) genes ATG4B, ATG5, and ATG7. The production of reactive oxygen species, but not modulation of the main autophagy repressor MTOR or its upstream regulators AMP-activated protein kinase and AKT1, was responsible for olanzapine-triggered autophagy. Olanzapine-mediated oxidative stress also induced mitochondrial depolarization and damage, and the autophagic clearance of dysfunctional mitochondria was confirmed by electron microscopy, colocalization of autophagosome-associated MAP1LC3B (LC3B henceforth) and mitochondria, and mitochondrial association with the autophagic cargo receptor SQSTM1/p62. While olanzapine-triggered mitochondrial damage was not overtly toxic to SH-SY5Y cells, their death was readily initiated upon the inhibition of autophagy with pharmacological inhibitors, RNA interference knockdown of BECN1 and LC3B, or biological free radical nitric oxide. The treatment of mice with olanzapine for 14 d increased the brain levels of autophagosome-associated LC3B-II and mRNA encoding Atg4b, Atg5, Atg7, Atg12, Gabarap, and Becn1. The administration of the autophagy inhibitor chloroquine significantly increased the expression of proapoptotic genes (Trp53, Bax, Bak1, Pmaip1, Bcl2l11, Cdkn1a, and Cdkn1b) and DNA fragmentation in the frontal brain region of olanzapine-exposed animals. These data indicate that olanzapine-triggered autophagy protects neurons from otherwise fatal mitochondrial damage, and that inhibition of autophagy might unmask the neurotoxic action of the drug.

Short-term Exposure to UV-A, UV-B, and UV-C Irradiation Induces Alteration in Cytoskeleton and Autophagy in Human Keratinocytes

Abstract Ultraviolet radiation (UV) induces a series of morphological and ultrastructural alterations in human epidermis. Alterations observed in irradiated keratinocytes in morphological studies done before were cell retraction with loss of intercellular interactions, surface blebbing, and eventually cell death by apoptosis. The aim of this study was to investigate effect of UV-A, UV-B, and UV-C irradiation on the cytoskeleton of human keratinocytes. Keratinocytes were obtained by exfoliative scrubbing procedure from buccal mucosa of healthy individuals, and treated with UV-A, UV-B, and UV-C radiation. Afterward, treated cell were labeled with anti-alfa-tubulin and anti-human-cytokeratin and analyzed by light and confocal microscopy. The intensity of the cytokeratin labeling was found to be much higher in all irradiated cells than in control cells as observed by light microscope and measured with the Image J program. This measurement showed that with the decrease in the wavelengths of UV irradiation the intensity of the labeling of cells increases. Although the authors expected to find the collapse of microtubules toward the cell nucleus or their rearrangement in UV-treated cells, these alterations were not verified on cell smears labeled with anti-alfa tubulin observed by confocal microscope. When they used electron microscopy to examine in more detail the morphology of irradiated cells, they did not find apoptotic cells, but found features of autophagy in UV-treated keratinocytes. The authors assume that autophagy found as a result of UV radiation of human keratinocytes acts as a cytoprotective mechanism against UV-induced apoptosis.

Intraspinal Delivery of Polyethylene Glycol-coated Gold Nanoparticles Promotes Functional Recovery After Spinal Cord Injury

Failure of the mammalian central nervous system (CNS) to regenerate effectively after injury leads to mostly irreversible functional impairment. Gold nanoparticles (AuNPs) are promising candidates for drug delivery in combination with tissue-compatible reagents, such as polyethylene glycol (PEG). PEG administration in CNS injury models has received interest for potential therapy, but toxicity and low bioavailability prevents clinical application. Here we show that intraspinal delivery of PEG-functionalized 40-nm-AuNPs at early stages after mouse spinal cord injury is beneficial for recovery. Positive outcome of hind limb motor function was accompanied by attenuated inflammatory response, enhanced motor neuron survival, and increased myelination of spared or regrown/sprouted axons. No adverse effects, such as body weight loss, ill health, or increased mortality were observed. We propose that PEG-AuNPs represent a favorable drug-delivery platform with therapeutic potential that could be further enhanced if PEG-AuNPs are used as carriers of regeneration-promoting molecules.

Neuroprotective arylpiperazine dopaminergic/serotonergic ligands suppress experimental autoimmune encephalomyelitis in rats.

Arylpiperazine‐based dopaminergic/serotonergic ligands exert neuroprotective activity. We examined the effect of arylpiperazine D2/5‐HT1A ligands, N‐{4‐[2‐(4‐phenyl‐piperazin‐1‐yl)‐ethyl}‐phenyl]‐picolinamide (6a) and N‐{3‐[2‐(4‐phenyl‐piperazin‐1‐yl)‐ethyl]‐phenyl}‐picolinamide (6b), in experimental autoimmune encephalomyelitis (EAE), a model of neuroinflammation. Both compounds (10 mg/kg i.p.) reduced EAE clinical signs in spinal cord homogenate‐immunized Dark Agouti rats. Compound 6b was more efficient in delaying the disease onset and reducing the maximal clinical score, which correlated with its higher affinity for D2 and 5‐HT1A receptors. The protection was retained if treatment was limited to the effector (from day 8 onwards), but not the induction phase (day 0–7) of EAE. Compound 6b reduced CNS immune infiltration and expression of mRNA encoding the proinflammatory cytokines tumor necrosis factor, IL‐6, IL‐1, and GM‐CSF, TH1 cytokine IFN‐γ, TH17 cytokine IL‐17, as well as the signature transcription factors of TH1 (T‐bet) and TH17 (RORγt) cells. Arylpiperazine treatment reduced apoptosis and increased the activation of anti‐apoptotic mediators Akt and p70S6 kinase in the CNS of EAE animals. The in vitro treatment with 6b protected oligodendrocyte cell line OLN‐93 and neuronal cell line PC12 from mitogen‐activated normal T cells or myelin basic protein‐activated encephalitogenic T cells. In conclusion, arylpiperazine dopaminergic/serotonergic ligands suppress EAE through a direct neuroprotective action and decrease in CNS inflammation.

Unusual shape and structure of lymphocyte nuclei is linked to hyperglycemia in type 2 diabetes patients

Type 2 diabetes is an endocrine disorder characterized with hyperglycemia, hyperinsulinemia and insulin resistance. Morphological changes in cell nuclei in diabetes were recently detected. The aim of this study was to compare electron microscopic features of lymphocyte nuclei in type 2 diabetes and healthy individuals using conventional computer assisted methods, fractal analysis and gray level co-occurrence matrix (GLCM) analysis of nuclear chromatin. Mononuclear cells taken from the peripheral blood of newly diagnosed type 2 diabetes patients, metformin treated type 2 diabetes patients and healthy individuals were analyzed with transmission electron microscope. Irregular nuclear contours and lower amount of heterochromatin in lymphocytes were detected with conventional computer assisted methods in type 2 diabetes. Fractal analysis of chromatin structures and GLCM angular second moment (ASM) analysis detected differences in nuclear structure between metformin treated type 2 diabetes and two other groups. Irregularities in lymphocyte nuclei correlated with blood glucose, but not with cholesterol and triglyceride levels. Decrease in fractal dimension, indicating lower level of complexity, increase in GLCM ASM, indicating higher texture uniformity, and higher amount of euchromatin that we found in metformin treated type 2 diabetes could be indicators of higher transcriptional activity in these cells.

Metformin exacerbates and simvastatin attenuates myelin damage in high fat diet-fed C57BL/6 J mice: Simvastatin attenuates myelin damage

Diabetic neuropathy is one of the most deleterious complications of diabetes mellitus in humans. High fat diet (HFD)‐fed C57BL/6 J mice are a widely used animal model for type 2 diabetes mellitus and metabolic syndrome. We investigated the effects of metformin and simvastatin on the ultrastructural characteristics of sciatic nerve fibers in these mice. Metformin treatment increased the number of structural defects of the myelin sheet surrounding these fibers in already affected nerves of HFD fed mice, and simvastatin treatment reduced these numbers to the levels seen in control mice. These results warrant further research on the effects of metformin and statins in patients developing diabetic neuropathy and advise caution when deciding about optimal treatment modalities in these patients.

Aggressive human neuroblastomas show a massive increase in the numbers of autophagic vacuoles and damaged mitochondria.

ABSTRACT Autophagy is activated in cancer cells in response to multiple stresses and has been demonstrated to promote tumor cell survival and drug resistance in neuroblastoma (NB). This study was conducted to analyze the ultrastructural features of peripheral neuroblastic tumors (pNTs) and identify the relation of the types of NTs, the proliferation rate, and MYCN gene amplification with a number of autophagic vacuoles. Our results indicate that aggressive human NBs show a massive increase in the number of autophagic vacuoles associated with proliferation rate and that alteration of the mitochondria might be an important factor for the induction of autophagy in NTs.

Aberrant Tissue Positioning of Metallophilic Macrophages in the Thymus of XCL1-Deficient Mice

Metallophilic macrophages hold a strategic position within the thymic tissue and play a considerable function in thymic physiology. The development and positioning of these cells within thymic tissue are regulated by complex molecular mechanisms involving different cytokine/chemokine axes. Herein, we studied the role of XCL1 signaling in these processes. We show that in the XCL1‐deficient thymus numerous metallophilic macrophages are aberrantly positioned in the thymic cortex, instead of their normal location in the cortico‐medullary zone. Still, these cells retain their normal appearance: very large size with prominent, ramifying cytoplasmic prolongations. This shows that XCL1 signaling is not involved in morphological development, but rather in correct positioning of metallophilic macrophages within the thymic tissue. In contrast to thymic metallophilic macrophages, the positioning of splenic marginal metallophilic macrophages is not affected by XCL1‐deficiency. Anat Rec, 297:1472–1477, 2014.