Advanced Glycation End-Products in Blood Serum-Novel Ischemic Stroke Risk Factors? Implication for Diabetic Patients.

New predictors of ischemic incidents are constantly sought since they raise the awareness of patients and their doctors of stroke occurrence. The goal was to verify whether Advanced Glycation End Products (AGEs), in particular AGE10, could be one of them. The AGE10 measurement was conducted using a non-commercial ELISA assay in the blood serum of neurological patients without cerebrovascular event (n = 24), those with transient brain attack (TIA) (n = 17), and severe ischemic stroke (n = 35). Twice as many of the people with TIA or severe stroke presented high AGE10 serum concentrations compared to the patients with other neurological conditions (χ2 = 8.2, p = 0.004; χ2 = 8.0, p = 0.005, respectively). The risk of ischemic incident was significantly risen in people with higher levels of AGE10 (OR = 6.5, CI95%: 1.7-24.8; OR = 4.7, CI95%: 1.5-14.5 for TIA and stroke subjects, respectively). We observed a positive correlation (r = 0.40) between high AGE10 levels and diabetes. Moreover, all the diabetic patients that had a high AGE10 content experienced either a severe ischemic stroke or TIA. The patients with high levels of AGE10 exhibited higher grades of disability assessed by the NIHSS scale (r = 0.35). AGE10 can be considered a new biomarker of ischemic stroke risk. Patients with diabetes presenting high AGE10 levels are particularly prone to the occurrence of cerebrovascular incidents.

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-ß-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-ß-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.

Should Nano-Particles be Weighed or Counted? Technical Considerations to In Vitro Testing Originated from Corpuscular Nature of Nano-Particles.

The abundance of nanoparticles introduced to household products created the great expectations towards the application of nanotechnology in biology and medicine. That calls for cost-effective preliminary assessment of its cytotoxicity and biological activity. There are many attempts for creating proper guidance and standards for performing studies regarding nanoparticles. But still some important aspects crucial for in vitro testing of nanomaterials need more attention. Particulate nature is an obvious and widely unappreciated property of nanoparticles. In the context of in vitro studies, this property is critical, and it should be, but rarely is, considered when designing, performing, describing or interpreting the experiments involving the solid nanoparticles. First, we should be aware of relatively small and limited number of nanoparticles in the experimental setup. Even crude estimation of its number will be useful for proper interpretation of results. Second, we should not presume even distribution of particles in the solution, moreover we should expect that sedimentation and aggregation play an important role in interactions of nanoparticles with cells. In that case, expressing the dose in mass/volume units may lead as astray. Finally, the relation of size, weight, and number of nanoparticles makes comparisons of activity of nanoparticles of different sizes very complex. Estimations of number of nanoparticles in the dose should be an integral part of experiment design, its validation and interpretation.

Synthesis, Physicochemical Characteristics and Plausible Mechanism of Action of an Immunosuppressive Isoxazolo[5,4-e]-1,2,4-Triazepine Derivative (RM33).

Previous studies demonstrated strong anti-inflammatory properties of isoxazolo[5,4-e]-1,2,4-triazepine (RM33) in vivo. The aim of this investigation was to describe synthesis, determine physicochemical characteristics, evaluate biological activities in murine and human in vitro models, as well as to propose mechanism of action of the compound. The compound was devoid of cell toxicity up to 100 µg/mL against a reference A549 cell line. Likewise, RM33 did not induce apoptosis in these cells. The compound stimulated concanavalin A (ConA)-induced splenocyte proliferation but did not change the secondary humoral immune response in vitro to sheep erythrocytes. Nevertheless, a low suppressive effect was registered on lipopolysaccharide (LPS)-induced splenocyte proliferation and a stronger one on tumor necrosis factor alpha (TNFα) production by rat peritoneal cells. The analysis of signaling pathways elicited by RM33 in nonstimulated resident cells and cell lines revealed changes associated with cell activation. Most importantly, we demonstrated that RM33 enhanced production of cyclooxygenase 2 in LPS-stimulated splenocytes. Based on the previous and herein presented results, we conclude that RM33 is an efficient, nontoxic immune suppressor with prevailing anti-inflammatory action. Additionally, structural studies were carried out with the use of appropriate spectral techniques in order to unequivocally confirm the structure of the RM33 molecule. Unambiguous assignment of NMR chemical shifts of carbon atoms of RM33 was conducted thanks to full detailed analysis of 1H, 13C NMR spectra and their two-dimensional (2D) variants. Comparison between theoretically predicted chemical shifts and experimental ones was also carried out. Additionally, N-deuterated isotopologue of RM33 was synthesized to eliminate potentially disturbing frequencies (such as NH, NH2 deformation vibrations) in the carbonyl region of the IR (infrared) spectrum to confirm the presence of the carbonyl group.

Biotin-Containing Third Generation Glucoheptoamidated Polyamidoamine Dendrimer for 5-Aminolevulinic Acid Delivery System.

Polyamidoamine PAMAM dendrimer generation 3 (G3) was modified by attachment of biotin via amide bond and glucoheptoamidated by addition of α-D-glucoheptono-1,4-lacton to obtain a series of conjugates with a variable number of biotin residues. The composition of conjugates was determined by detailed 1-D and 2-D NMR spectroscopy to reveal the number of biotin residues, which were 1, 2, 4, 6, or 8, while the number of glucoheptoamide residues substituted most of the remaining primary amine groups of PAMAM G3. The conjugates were then used as host molecules to encapsulate the 5-aminolevulinic acid. The solubility of 5-aminolevulinic acid increased twice in the presence of the 5-mM guest in water. The interaction between host and guest was accompanied by deprotonation of the carboxylic group of 5-aminolevulinic acid and proton transfer into internal ternary nitrogen atoms of the guest as evidenced by a characteristic chemical shift of resonances in the 1H NMR spectrum of associates. The guest molecules were most likely encapsulated inside inner shell voids of the host. The number of guest molecules depended on the number of biotin residues of the host, which was 15 for non-biotin-containing glucoheptoamidated G3 down to 6 for glucoheptoamidated G3 with 8 biotin residues on the host surface. The encapsulates were not cytotoxic against Caco-2 cells up to 200-µM concentration in the dark. All encapsulates were able to deliver 5-aminolevulinic acid to cells but aqueous encapsulates were more active in this regard. Simultaneously, the reactive oxygen species were detected by staining with H2DCFDA in Caco-2 cells incubated with encapsulates. The amount of PpIX was sufficient for induction of reactive oxygen species upon 30-s illumination with a 655-nm laser beam.

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.

Antibody CD133 Biofunctionalization of Ammonium Acryloyldimethyltaurate and Vinylpyrrolidone Co-Polymer-Based Coating of the Vascular Implants.

Current vascular stents, such as drug eluting stents (DES), have some serious drawbacks, like in stent restenosis and thrombosis. Therefore, other solutions are sought to overcome these post-implantations complications. These include the strategy of biofunctionalization of the stent surface with antibodies that facilitate adhesion of endothelial cells (ECs) or endothelial progenitor cells (EPCs). Rapid re-endothelialization of the surface minimizes the risk of possible complications. In this study, we proposed ammonium acryloyldimethyltaurate/vinylpyrrolidone co-polymer-based surface (AVC), which was mercaptosilanized in order to expose free thiol groups. The presence of free thiol groups allowed for the covalent attachment of CD133 antibodies by disulfide bridges formation between mercaptosilanized surface and cysteine of the protein molecule thiol groups. Various examinations were performed in order to validate the procedure, including attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and Fourier transform Raman spectroscopy (FT-Raman), atomic force microscopy (AFM) and scanning electron microscopy (SEM). By means of ATR-FTIR spectroscopy presence of the CD133 antibody within coating was confirmed. In vitro studies proved good biocompatibility for blood cells without induction of hemolytic response. Thus, proposed biofunctionalized CD133 antibody AVC surface has shown sufficient stability for adapting as cardiovascular implant coating and biocompatibility. According to conducted in vitro studies, the modified surface can be further tested for applications in various biological systems.

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

A cyclic tetrapeptide Pro-Pro-Pheß3ho-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.

Profound Nanoscale Structural and Biomechanical Changes in DNA Helix upon Treatment with Anthracycline Drugs.

In our study, we describe the outcomes of the intercalation of different anthracycline antibiotics in double-stranded DNA at the nanoscale and single molecule level. Atomic force microscopy analysis revealed that intercalation results in significant elongation and thinning of dsDNA molecules. Additionally, using optical tweezers, we have shown that intercalation decreases the stiffness of DNA molecules, that results in greater susceptibility of dsDNA to break. Using DNA molecules with different GC/AT ratios, we checked whether anthracycline antibiotics show preference for GC-rich or AT-rich DNA fragments. We found that elongation, decrease in height and decrease in stiffness of dsDNA molecules was highest in GC-rich dsDNA, suggesting the preference of anthracycline antibiotics for GC pairs and GC-rich regions of DNA. This is important because such regions of genomes are enriched in DNA regulatory elements. By using three different anthracycline antibiotics, namely doxorubicin (DOX), epirubicin (EPI) and daunorubicin (DAU), we could compare their detrimental effects on DNA. Despite their analogical structure, anthracyclines differ in their effects on DNA molecules and GC-rich region preference. DOX had the strongest overall effect on the DNA topology, causing the largest elongation and decrease in height. On the other hand, EPI has the lowest preference for GC-rich dsDNA. Moreover, we demonstrated that the nanoscale perturbations in dsDNA topology are reflected by changes in the microscale properties of the cell, as even short exposition to doxorubicin resulted in an increase in nuclei stiffness, which can be due to aberration of the chromatin organization, upon intercalation of doxorubicin molecules.

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.

Photoactive Pore Matrix for In Situ Delivery of a Photosensitizer in Vascular Smooth Muscle Cells Selective PDT.

In this study we present the porous silica-based material that can be used for in situ drug delivery, offering effective supply of active compounds regardless its water solubility. To demonstrate usability of this new material, three silica-based materials with different pore size distribution as a matrix for doping with Photolon (Ph) and Protoporphyrin IX (PPIX) photosensitizers, were prepared. These matrices can be used for coating cardiovascular stents used for treatment of the coronary artery disease and enable intravascular photodynamic therapy (PDT), which can modulate the vascular response to injury caused by stent implantation-procedure that should be thought as an alternative for drug eluting stent. The FTIR spectroscopic analysis confirmed that all studied matrices have been successfully functionalized with the target photosensitizers. Atomic force microscopy revealed that resulting photoactive matrices were very smooth, which can limit the implantation damage and reduce the risk of restenosis. No viability loss of human peripheral blood lymphocytes and no erythrocyte hemolysis upon prolonged incubations on matrices indicated good biocompatibility of designed materials. The suitability of photoactive surfaces for PDT was tested in two cell lines relevant to stent implantation: vascular endothelial cells (HUVECs) and vascular smooth muscle cells (VSMC). It was demonstrated that 2 h incubation on the silica matrices was sufficient for uptake of the encapsulated photosensitizers. Moreover, the amount of the absorbed photosensitizer was sufficient for induction of a phototoxic reaction as shown by a rise of the reactive oxygen species in photosensitized VSMC. On the other hand, limited reactive oxygen species (ROS) induction in HUVECs in our experimental set up suggests that the proposed method of PDT may be less harmful for the endothelial cells and may decrease a risk of the restenosis. Presented data clearly demonstrate that porous silica-based matrices are capable of in situ delivery of photosensitizer for PDT of VSMC.

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.

Photoactive Liposomal Formulation of PVP-Conjugated Chlorin e6 for Photodynamic Reduction of Atherosclerotic Plaque.

BACKGROUND: Liposomes serve as delivery systems for biologically active compounds. Existing technologies inefficiently encapsulate large hydrophilic macromolecules, such as PVP-conjugated chlorin e6 (Photolon). This photoactive drug has been widely tested for therapeutic applications, including photodynamic reduction of atherosclerotic plaque. METHODS: A novel formulation of Photolon was produced using "gel hydration technology". Its pharmacokinetics was tested in Sus scrofa f. domestica. Its cellular uptake, cytotoxicity, and ability to induce a phototoxic reaction were demonstrated in J774A.1, RAW264.7 macrophages, and vascular smooth muscle (T/G HA-VSMC) as well as in vascular endothelial (HUVEC) cells. RESULTS: Developed liposomes had an average diameter of 124.7 ± 0.6 nm (polydispersity index (PDI) = 0.055) and contained >80% of Photolon). The half-life of formulation in S. scrofa was 20 min with area under the curve (AUC) equal to 14.7. The formulation was noncytotoxic in vitro and was rapidly (10 min) and efficiently accumulated by macrophages, but not T/G HA-VSMC or HUVEC. The accumulated quantity of photosensitizer was sufficient for induction of phototoxicity in J774A.1, but not in T/G HA-VSMC. CONCLUSIONS: Due to the excellent physical and pharmacokinetic properties and selectivity for macrophages, the novel liposomal formulation of Photolon is a promising therapeutic candidate for use in arteriosclerosis treatment when targeting macrophages but not accompanying vascular tissue is critical for effective and safe therapy.

Towards biocompatible NIR-II nanoprobes - transfer of hydrophobic Ag2S quantum dots to aqueous solutions using phase transfer catalysed hydrolysis of poly(maleic anhydride-alt-1-octadecene).

Development of protocols for transferring of hydrophobic quantum dots (QDs) into aqueous solution is of special importance for their biomedical applications. Particularly, hydrophilization of Ag2S without quenching of photoluminescence is a great challenge. Application of standard protocol for amphiphilic polymer coating of hydrophobic Ag2S nanocrystals failed, whereas ligand exchange and direct synthesis of Ag2S in aqueous solution leads to poorly emitting materials. In this paper we present the facile method for transferring of hydrophobic Ag2S QDs into aqueous solution employing the phase transfer catalysed hydrolysis of a commercially available polymer - poly(maleic anhydride-alt-1-octadecene), (PMAO) in the presence of tetramethylammonium hydroxide. Because the original surface ligands are retained and we do not use solvents (like THF) detrimental for Ag2S emission, the modification does not deteriorate photoluminescence properties and quantum yield of modified QDs in aqueous solution reaches 60% of that for hydrophobic QDs in chloroform. The hydrodynamic diameter of modified, water soluble Ag2S QDs is about 10 nm and is only slightly larger than their original size. Moreover, the polymer coated nanocrystals are not aggregated and are stable for months. Surface characterization of QDs by NMR and IR spectroscopy indicates that polymer chains intercalate alkyl chains of dodecanethiol (DDT) bound to the surface of Ag2S. The cytotoxicity studies indicate that the presented method should be regarded as a notable progress towards the biocompatible Ag2S NIR-II emitting nanoprobes.

Dithiocarbamates: Reliable Surface Ligands for NIR-Emitting Quantum Dots.

In this paper, we report a reliable method for transferring hydrophobic, NIR-emitting PbS/CdS core-shell quantum dots (QDs) to water solutions using synthesized in situ dithiocarbamate derivatives of amino acids as stabilizing ligands. Such ligands offer apparent advantages over dihydrolipoic acid (DHLA) derivatives commonly used as ligands stabilizing quantum dots. The most effective phase transfer and the best long-term stability of water dispersions of amino acid-DTC-capped QDs were achieved using lysine dithiocarbamate. In this case, the phase transfer of PbS/CdS nanoparticles from organic to aqueous phase can be completed in 6-8 h. Prepared amino acid-DTC-capped QDs nanoparticles have narrow size distributions, and their hydrodynamic diameter remains below 9 nm. After transferring to water, lysine-DTC-capped PbS/CdS nanoparticles retain their spectroscopic properties for at least 48 h. Moreover, they are not toxic up to concentration corresponding to about 7 µg/cm3 of PbS/CdS, which is sufficient for their application in biological systems. In addition, lysine-DTC-capped PbS/CdS QDs can be straightforwardly modified by layer-by-layer polyelectrolyte coating.

Prolongation of skin graft survival in mice by an azaphenothiazine derivative.

Azaphenothiazines are predominantly immunosuppressive compounds. We evaluated the efficacy of an azaphenothiazine derivative, 6-chloroethylureidoethyldiquino[3,2-b;2',3'-e][1,4]thiazine (DQT) in prolongation of survival of skin allografts between BALB/c and C57Bl/6 mice. The mice were treated intraperitoneally (i.p.) with 100 µg of DQT on alternate days, on days 1-13 of the experiment (7 doses). The effect of DQT on a two-way mixed lymphocyte reaction (MLR) in the human model, as well as its effect on production of TNF α and IL-10 in a whole blood cell culture, stimulated by lipopolysaccharide (LPS), were evaluated. In addition, DQT effects were investigated regarding the proportion of T cell subsets in human peripheral blood lymphocytes (PBMC) by flow cytometry. Lastly, the effect of DQT on expression of signaling molecules involved in pro apoptotic pathways was determined by RT PCR. The results showed that DQT significantly extended skin graft survival. The compound also strongly suppressed two-way MLR in the human model at a concentration range of 2.5-5.0 µM. In addition, DQT inhibited LPS-inducible TNF α, but not IL-10 production. The compound preferentially caused a loss of the CD3-CD8+CD11b + PBMC cell subset, and transformed CD3+CD8+high into CD3+CD8+low cells. Lastly, we demonstrated significant increases in expression of caspases (in particular caspase 8) and of p53 in a culture of Jurkat T cells. We conclude that the immunosuppressive actions of the compound in allograft rejection may be predominantly associated with induction of cell apoptosis and inhibition of TNF α production. The apoptosis could be predominantly selective for the CD3-CD8+CD11b + cell phenotype.

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.

Functionalized CD133 antibody coated stent surface simultaneously promotes EPCs adhesion and inhibits smooth muscle cell proliferation-A novel approach to prevent in-stent restenosis.

We report a multistep strategy of biochemical surface modifications that resulted in the synthesis of new, effective and biocompatible intravascular implants coating with immobilized anti-CD133 antibodies, that proved to be the most effective in endothelial progenitor cells capture and reduced smooth muscle cells growth. Biomolecules were immobilized on differently functionalized surfaces. The distribution, nanostructural characteristics and intramolecular interactions of anti-CD133 molecules as well as their ability to bind EPCs was evaluated. We also tempted to build a molecular model of the CD133 protein to study antigen-antibody interactions. CD133 protein is expressed in endothelial progenitor cells (EPCs). Absence of preferential interaction site on CD133, but rather a presence of a small binding area, may be the specificity of reconnaissance sequence, thus importantly increasing the probability of CD133 protein binding. After all, regarding our molecular model, we are convinced that specific, and large enough interactions between anti-CD133 coating stent surface and CD133 present on EPCs will reduce risk of restenosis by favoring the endothelial growth. Additionally, the safety study of the vivo performance of modified titania based surface was performed using small animal models. No allergological or toxical local or systemic adverse effects of the developed coatings were noted.

An Anti-Inflammatory Azaphenothiazine Inhibits Interferon ß Expression and CXCL10 Production in KERTr Cells.

An azaphenothiazine derivative, 6-chloroethylureidoethyldiquino[3,2-b;2',3'-e][1,4]thiazine (DQT), has recently been shown to exhibit immunosuppressive activities in mouse models. It also inhibited the expression of CXCL10 at the protein level, at non-toxic concentrations, in the culture of KERTr cells treated with double-stranded RNA, poly(I:C). In this report, we demonstrated that DQT inhibits the transcription of the CXCL10 gene. Although CXCL10 is an IFNγ-inducible protein, we found that the CXCL10 protein was induced without the detectable release of IFNγ or IκB degradation. Hence, we concluded that IFNγ or NFκB was not involved in the regulation of the CXCL10 gene in KERTr cells transfected with poly(I:C), nor in the inhibitory activity of DQT. On the other hand, we found that IFNß was induced under the same conditions and that its expression was inhibited by DQT. Kinetic analysis showed that an increase in IFNß concentrations occurred 4⁻8 h after poly(I:C) treatment, while the concentration of CXCL10 was undetectable at that time and started to increase later, when IFNß reached high levels. Therefore, DQT may be regarded as a new promising inhibitor of IFNß expression and IFNß-dependent downstream genes and proteins, e.g., CXCL10 chemokine, which is implicated in the pathogenesis of autoimmune diseases.

Topically applied azaphenothiazines inhibit experimental psoriasis in mice.

The therapeutic efficacy of topically applied azaphenothiazine derivatives: 9-chloro-6-acetylaminobutylquinobenzo[3,2-b][1,4]thiazine (compound 4) and 6-chloroethylureidoethyldiquino[3,2-b;2';3'-e][1,4]thiazine (compound 5) in the amelioration of inflammatory symptoms of imiquimod-induced psoriasis in mice was investigated. Clobederm®, containing clobetasol propioniate, served as a reference drug. The application of the compounds led to thinning of the epidermis and reduction of the cell layers. The suppressive actions of the compounds were even stronger with regard to pathological changes of the dermis. The compounds also exerted generalized, anti-inflammatory effects by decreasing the number of circulating leukocytes, lowering subiliac lymph node weight and partially normalizing an altered blood cell composition. The changes in the composition of main cell types in the epidermis and dermis were less affected by the compounds. In addition, both compounds inhibited to a similar degree production of tumor necrosis factor α (TNF α) in human whole blood cell culture. Whereas compound 5 strongly inhibited IL-8 and CXCL10 chemokines in human keratinocytes - KERTr cell line, transfected with poly(I:C), the suppressive action of compound 4 in this model was weak. In addition, compound 5, but not compound 4, exhibited at low doses proapoptotic properties with regard to colonic cell lines. In summary, we demonstrated the therapeutic potential of two selected azaphenotiazines in the amelioration of the skin pathology elicited in a mouse experimental model of psoriasis.