Ceramides and sphingosine-1-phosphate as potential markers in diagnosis of ischaemic stroke

Background. Brain imaging in stroke diagnostics is a powerful tool, but one that can fail in more challenging cases, and one that is not particularly useful in identifying transient ischaemic attacks (TIAs). Thus, new reliable blood biomarkers of cerebral ischaemia are constantly sought. Objective. We studied the potential usefulness of sphingolipids (SFs) as biomarkers of acute ischaemic stroke and TIA. Material and methods. Levels of individual ceramide species and sphingosine-1-phosphate (Sph-1-P) in blood serum of patients with acute ischaemic stroke, TIA, and age-matched neurological patients without cerebral ischaemia, were assessed by tandem mass spectrometry liquid chromatography (LC- MS / MS). Results. We found significant increases of several sphingolipid levels, with particularly strong elevations of Cer-C20:0 in patients with acute stroke. Cer-C24:1 was the only ceramide species to decrease as a result of acute stroke. Moreover, its levels inversely correlated with the number of days after stroke onset, suggesting that Cer-C24:1 is an independent parameter related to the course of stroke. To increase the sensitivity of sphingolipid-based tests in stroke diagnostics, we calculated the values of ratios of Sph-1-P / individual ceramide species and Cer-C24:1 individual ceramide species. We found several ratios significantly changed in stroke patients. Two ratios, Sph-1-P / Cer-C24:1 and Cer-C24:0 / Cer-C24:1, presented especially strong increments in patients with acute stroke. Moreover, Sph-1-P / Cer-C24:1 values were augmented in TIA patients. Conclusion. Serum SFs could be good candidates to be ischaemic stroke biomarkers. We have identified two SF ratios, Sph-1-P / Cer-C24:1 and Cer-C24:0 / Cer-C24:1, with strong diagnostic potential in ischaemic stroke. We found Sph-1-P / Cer-C24:1 ratio to be possibly useful in TIA diagnostics, also in the long term after ischaemic incidence

In Vitro Photodynamic Therapy with Chlorin e6 Leads to Apoptosis of Human Vascular Smooth Muscle Cells

Percutaneous coronary intervention has become the most common and widely implemented method of heart revascularization. However, the development of restenosis remains the major limitation of this method. Photodynamic therapy (PDT) recently emerged as a new and promising method for the prevention of arterial restenosis. Here the efficacy of chlorin e6 in PDT was investigated in vitro using human vascular smooth muscle cells (TG/HA-VSMCs) as one of the cell types crucial in the development of restenosis. PDT-induced cell death was studied on many levels, including annexin V staining, measurement of the generation reactive oxygen species (ROS) and caspase-3 activity, and assessment of changes in mitochondrial membrane potential and fragmentation of DNA. Photosensitization of TG/HA-VSMCs with a 170 μM of chlorin e6 and subsequent illumination with the light of a 672-nm diode laser (2 J/cm2) resulted in the generation of ROS, a decrease in cell membrane polarization, caspase-3 activation, as well as DNA fragmentation. Interestingly, the latter two apoptotic events could not be observed in photosensitized and illuminated NIH3T3 fibroblasts, suggesting different outcomes of the model of PDT in various types of cells. The results obtained with human VSMCs show that chlorin e6 may be useful in the PDT of aerial restenosis, but its efficacy still needs to be established in an animal model

The mitochondrial localization of RelB and NFATx in immature T cells

In order to exert their activity, transcription factors must be transported to the nucleus. Certain transcription factors have also been found on mitochondria. Here, the localization of RelB and NFATx in the mitochondrial fractions of normal thymocytes and thymic lymphoma cells is shown for the first time. CREB was only found in the nucleus, while p50 (NFkappaB) was found in both the nucleus and the cytoplasm, but outside the mitochondria. The translocation of transcription factors to the mitochondria is differentially regulated. Unlike RelB, which is always present in the mitochondrial fraction, NFATx appeared on the mitochondria in cells treated with ionomycin together with an immunosuppressant and inhibitor of calcineurin (FK506). This data reveals that the mitochondrial localization of some transcription factors is precisely controlled by a calcium signal sensitive to FK506 in T cells

The Contrasting Delayed Effects of Transient Exposure of Colorectal Cancer Cells to Decitabine or Azacitidine

(1) Background: Decitabine and azacitidine are cytosine analogues representing the class of drugs interfering with DNA methylation. Due to their molecular homology and similar clinical application, both drugs are often regarded as interchangeable. Despite their unique mechanism of action the studies designed for observation and comparison of the prolonged activity of these drugs are rare. (2) Methods: The short-time (20–72 h) and long-term (up to 20 days) anti-cancer activity of decitabine and azacitidine has been studied in colorectal cancer cells. We observe the impact on cell culture’s viability, clonogenicity, proliferation, and expression of CDKN1A, CCND1, MDM2, MYC, CDKN2A, GLB1 genes, and activity of SA-β-galactosidase. (3) Results: Decitabine has much stronger anti-clonogenic activity than azacitidine. We show that azacitidine, despite significant immediate toxicity, has negligible long-term effects. Contrary, decitabine, which does not exert initial toxicity, profoundly worsened the condition of the cells over time. On the 13th day after treatment, the viability of cells was decreased and proliferation inhibited. These functional changes were accompanied by up-regulation of expression CDKN1A, CCND1, and CDKN2A genes and increased activation of SA-β-galactosidase, indicating cellular senescence. (4) Conclusions: Our head-to-head comparison revealed profound differences in the activities of decitabine and azacitidine important in their anti-cancer potential and clinical application. The effects of decitabine need relatively long time to develop. This property is crucial for proper design of studies and therapy concerning decitabine and undermines opinion about the similar therapeutic mechanism and interchangeability of these drugs

Toxicity Mechanism of Low Doses of NaGdF4:Yb3+,Er3+ Upconverting Nanoparticles in Activated Macrophage Cell Lines

Gadolinium-doped nanoparticles (NPs) are regarded as promising luminescent probes. In this report, we studied details of toxicity mechanism of low doses of NaGdF4-based fluorescent nanoparticles in activated RAW264.7, J774A.1 macrophages. These cell lines were specifically sensitive to the treatment with nanoparticles. Using nanoparticles of three different sizes, but with a uniform zeta potential (about −11 mV), we observed rapid uptake of NPs by the cells, resulting in the increased lysosomal compartment and subsequent superoxide induction along with a decrease in mitochondrial potential, indicating the impairment of mitochondrial homeostasis. At the molecular level, this led to upregulation of proapoptotic Bax and downregulation of anti-apoptotic Bcl-2, which triggered the apoptosis with phosphatidylserine externalization, caspase-3 activation and DNA fragmentation. We provide a time frame of the toxicity process by presenting data from different time points. These effects were present regardless of the size of nanoparticles. Moreover, despite the stability of NaGdF4 nanoparticles at low pH, we identified cell acidification as an essential prerequisite of cytotoxic reaction using acidification inhibitors (NH4Cl or Bafilomycin A1). Therefore, approaching the evaluation of the biocompatibility of such materials, one should keep in mind that toxicity could be revealed only in specific cells. On the other hand, designing gadolinium-doped NPs with increased resistance to harsh conditions of activated macrophage phagolysosomes should prevent NP decomposition, concurrent gadolinium release, and thus the elimination of its toxicity

Anti-Inflammatory Activity of a Cyclic Tetrapeptide in Mouse and Human Experimental Models

A cyclic tetrapeptide Pro-Pro-Pheβ3ho-Phe (4B8M) was tested for immunosuppressive activity and potential therapeutic utility in several in vitro and in vivo mouse and human models. The tetrapeptide was less toxic for mouse splenocytes in comparison to cyclosporine A (CsA) and a parent cyclolinopeptide (CLA). The tetrapeptide demonstrated potent anti-inflammatory properties in antigen-specific skin inflammatory reactions to oxazolone and toluene diisocyanate as well to nonspecific irritants such as salicylic acid. It also inhibited inflammatory processes in an air pouch induced by carrageenan. In addition, 4B8M proved effective in amelioration of animal models corresponding to human diseases, such as nonspecific colon inflammation induced by dextran sulfate and allergic pleurisy induced by ovalbumin (OVA) in sensitized mice. The tetrapeptide lowered expression of EP1 and EP3 but not EP2 and EP4 prostaglandin E2 (PGE2) receptors on lipopolysaccharide-stimulated Jurkat T cells and ICAM-1 expression on human peripheral blood mononuclear cells (PBMC). Its anti-inflammatory property in the carrageenan reaction was blocked by EP3 and EP4 antagonists. In addition, 4B8M induced an intracellular level of PGE2 in a human KERTr keratinocyte cell line. In conclusion, 4B8M is a low toxic and effective inhibitor of inflammatory disorders with potential therapeutic use, affecting the metabolism of prostanoid family molecules

Gallato Zirconium (IV) Phtalocyanine Complex Conjugated with SiO₂ Nanocarrier as a Photoactive Drug for Photodynamic Therapy of Atheromatic Plaque

A new conjugate of gallato zirconium (IV) phthalocyanine complexes (PcZrGallate) has been obtained from alkilamino-modified SiO2 nanocarriers (SiO2-(CH2)3-NH2NPs), which may potentially be used in photodynamic therapy of atherosclerosis. Its structure and morphology have been investigated. The photochemical properties of the composite material has been characterized. in saline environments when exposed to different light sources Reactive oxygen species (ROS) generation in DMSO suspension under near IR irradiation was evaluated. The PcZrGallate-SiO2 conjugate has been found to induce a cytotoxic effect on macrophages after IR irradiation, which did not correspond to ROS production. It was found that SiO2 as a carrier helps the photosensitizer to enter into the macrophages, a type of cells that play a key role in the development of atheroma. These properties of the novel conjugate may make it useful in the photodynamic therapy of coronary artery disease

Therapeutic effects of an azaphenothiazine derivative in mouse experimental colitis

Phenothiazines represent a class of compounds of potential therapeutic utility. In this report we evaluated therapeutic value of an azaphenothiazine derivative, 6-acetylaminobutyl-9-chloroquino[3,2- b]benzo[1,4]thiazine (QBT), given intragastrically, in the model of dextran sodium sulfate-induced colitis in C57BL/6 mice using 5-aminosalicylic acid (5-ASA) as a reference drug. Colitis symptoms such as body weight loss, diarrhea and hematochezia (blood in stool) were observed and registered and disease activity index (DAI) was calculated. In addition, weight and cell numbers in the lymphatic organs and histological parameters of the colon wall were analyzed. The effects of QBT on viability of colon epithelial cell lines were also determined. We showed that weight and cell number of draining mesenteric lymph nodes were lower in mice treated with QBT in comparison to their control counterparts. The number of thymocytes, drastically reduced in control mice, was elevated in mice treated with the compounds with a significant effect of 5-ASA. In addition, an abnormal composition of blood cell types was partially corrected in these groups. Histological analysis of the colon revealed that the pathological changes were partially normalized by QBT and even to a higher degree by 5-ASA. In conclusion we demonstrated a therapeutic efficacy of the compound in amelioration of local and systemic pathological changes associated with chemically-induced colitis in mice. A possible mechanism of action of the compound is discussed