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A series of copper(II) complexes of 2-imino-2H-chromen-3-yl-1,3,5-triazines 2a-h, 3-(benzoxazol-2-yl)-2H-chromen-2-imines 4a-b, and 3-(benzothiazol-2-yl)-2H-chromen-2-imines 6a-c were obtained by reacting of appropriate 2-iminocoumarin ligands L1a-h, L3a-b, and L5a-c with 3-fold molar excess of copper(II) chloride. The structure of these compounds was confirmed by IR spectroscopy, elemental analysis, and single-crystal X-ray diffraction data (2f, 2g, 2h, and 6c). All the synthesized complexes were screened for their activity against five human cancer cell lines: DAN-G, A-427, LCLC-103H, SISO, and RT-4 by using a crystal violet microtiter plate assay and relationships between structure and in vitro cytotoxic activity are discussed. The coordination of 2-iminocoumarins with copper(II) ions resulted in complexes 2a-h, 4a-b, and 6a-c with significant inhibitory properties toward tested tumor cell lines with IC50 values ranging from 0.04 μM to 15.66 μM. In comparison to the free ligands L1a-h, L3a-b, and L5a-c, the newly prepared Cu(II) complexes often displayed increased activity. In the series of copper(II) complexes of 2-imino-2H-chromen-3-yl-1,3,5-triazines 2a-h the most potent compound 2g contained a 4-phenylpiperazine moiety at position 6 of the 1,3,5-triazine ring and an electron-donating diethylamino group at position 7′ of the 2-iminocoumarin scaffold. Among the Cu(II) complexes of 3-(benzoxazol-2-yl)-2H-chromen-2-imines 4a-b and 3-(benzothiazol-2-yl)-2H-chromen-2-imines 6a-c the most active was benzoxazole-2-iminocoumarin 4b that also possessed a diethylamino group at position 7′ of the 2-iminocoumarin moiety. Moreover, compound 4b was found to be the most prominent agent and displayed the higher potency than cisplatin against tested cell lines.

Introduction: Nasopharyngeal carcinoma (NPC) is a major public health problem in several countries, especially those in Southeast Asia and North Africa. In its typical poorly differentiated form, the Epstein-Barr virus (EBV) genome is present in the nuclei of all malignant cells with restricted expression of a few viral genes. The malignant phenotype of NPC cells results from the influence of these viral products in combination with cellular genetic, epigenetic and functional alterations. With regard to host/tumor interactions, NPC is a remarkable example of immune escape in the context of a hot tumor.Areas covered: This article has an emphasis on emerging therapeutic targets that are considered upstream or at an early stage of clinical application. It examines targets related to cellular oncogenic alterations, latent EBV infection and tumor interactions with the immune system.Expert opinion: There is a remarkable emergence of new agents that target EBV products. The clinical application of these agents would benefit from a systematic and comprehensive molecular classification of NPCs and from easy access to pre-clinical models in public repositories. There is a strong rationale for more investigations on the potential of immune modulators, especially those related to NK cells.

Cancers 14(5), 1261 (2022). doi:10.3390/cancers14051261, Published by MDPI, Basel

Cancer immunology immunotherapy : CII (2020). doi:10.1007/s00262-020-02681-x, Published by Springer, Heidelberg

International audience; The combination of neoadjuvant chemotherapy, radiochemotherapy, and maintenance therapy with interferon beta (IFNβ) has led to superior results in the treatment of children and adolescents with nasopharyngeal carcinoma (NPC). However, nothing is known about the mechanism of the antitumor activity of IFNβ in NPC. Here, we investigate the role of IFNβ on apoptosis in NPC cells. Six NPC cell lines, one patient-derived NPC xenograft (PDX) and one SV40-transformed nasoepithelial cell line were used. Induction of apoptosis by IFNβ was measured by flow cytometric analysis of subG1-DNA-content, Hoechst 33258 staining and activation of caspase-3. Dissection of death ligand signaling pathways included measuring surface expression of its components by flow cytometry, activation by death ligands and neutralization with specific antibodies and siRNA. IFNβ induced apoptosis at concentrations achievable in humans in five of six NPC cell lines and in PDX cells but not in nasoepithelial cells. Inhibition of caspases-3 and-8 abrogated this effect suggesting IFNβ promoted apoptosis through the extrinsic pathway. IFNβ induced surface expression of TRAIL and TRAIL-R2 and the addition of an anti-TRAIL-antibody or transfection with TRAIL-siRNA blocked IFNβ-induced apoptosis. No induction of TRAIL-expression was noted in the IFNβ-resistant cell line. In conclusion, IFNβ leads to apoptosis in NPC cells in an autocrine way via the induction of TRAIL expression and subsequent activation of the TRAIL-signaling pathway. The mechanism described could at least partly explain the clinical benefit of IFNβ in the treatment of NPC. Further studies in a mouse-xenograft model are warranted to substantiate this effect in vivo.

Cells : open access journal 10(9), 2458 (2021). doi:10.3390/cells10092458 special issue: "Special issue "Regulation of natural killer cell development and functions" / special issue editors: Subramaniam Malarkannan, guest editor; Anahid Jewett, guest editor", Published by MDPI, Basel

Nasopharyngeal carcinoma (NPC) is an EBV-associated neoplasm occurring endemically in Southeast Asia and sporadically all over the world. In children and adolescents, high cure rates have been obtained using chemotherapy, radiochemotherapy and maintenance therapy with interferon beta (IFNβ). The mechanism by which IFNβ contributes to a low systemic relapse rate has not yet been fully revealed. NK cells and serum samples from two patients with NPC were analyzed before and at different time points during IFNβ therapy, for assessment of TRAIL expression and NK cell cytotoxicity. Cytotoxicity was measured using the calcein release assay and the contribution of different death effector pathways was analyzed using specific inhibitors. Treatment with IFNβ induced TRAIL expression on patients’ NK cells and increased their cytotoxicity against NPC targets in vitro. NK cell-mediated cytotoxicity was predominately mediated via TRAIL. IFNβ also induced the production of soluble TRAIL (sTRAIL) by NK cells and its release upon contact with NPC cells. IFNβ treatment increased serum levels of sTRAIL in patients. Moreover, sTRAIL concentrated from patients’ serum samples induced apoptosis ex vivo in NPC cells from a patient-derived xenograft. Increased cytotoxicity of NK cells against NPC cells and increased serum levels of biologically active TRAIL in patients treated with IFNβ could be a means to eliminate micrometastatic disease and explain the low systemic relapse rate in this patient group.

Introduction: Smoking cigarettes is currently one of the most significant health and social issues. The consequences of smoking affect both individuals as well as entire society. Addiction to nicotine has been recognised as a major environmental factor fostering numerous diseases. Aim: The aim of this study was to identify the causes of and motives for quitting smoking among the adult inhabitants of Ostrowiec Świętokrzyski. The authors were also interested in the level of nicotine addiction. Material and methods: The study was conducted in a group of 209 inhabitants of Ostrowiec Świętokrzyski who were former or ongoing smokers. The study employed a survey technique, with the authors’ own questionnaire as a study tool. The Fagerström test determining addiction to nicotine (nicotine dependence) was used too. Results and conclusions: The study revealed that smoking is a serious social issue. The majority of respondents had quit smoking (63.1%), 19.1% had never made any attempt to quit, whereas in 17.7% of respondents the cessation was unsuccessful and they returned to smoking. All respondents were aware of health-affecting consequences of smoking, but were unable to list more than four smoking-related diseases (lung and tongue cancers, arteriosclerosis, and hypertension). Attempts to cease smoking were made by 81,0% of the survey participants, mostly for health and financial reasons (42.0% and 21.3% respectively). Cessation of smoking resulted in numerous side effects, such as irritability (36.4%), outbursts of anger (20.7%), gaining weight (20.4%) or binge eating of sweets (11.7%). The factor preventing respondents from quitting smoking was stress (29,0%).

A series of 2-imino-2H-chromen-3-yl-1,3,5-triazine compounds 5&ndash, 12, which are namely hybrids of 2,4-diamino-1,3,5-triazines and 2-imino-coumarins, was synthesized by reacting 2-(4,6-diamine-1,3,5-triazin-2-yl)acetonitriles 1&ndash, 4 with 2-hydroxybenzaldehydes. After this, upon heating in aqueous DMF, 2-imino-2H-chromen-3-yl-1,3,5-triazines 10 and 12 were converted into the corresponding 2H-chromen-3-yl-1,3,5-triazines 13 and 14, which are essentially hybrids of 2,4-diamino-1,3,5-triazines and coumarins. The in vitro anticancer activity of the newly prepared compounds was evaluated against five human cancer cell lines: DAN-G, A-427, LCLC-103H, SISO and RT-4. The greatest cytotoxic activity displayed 4-[7-(diethylamino)-2-imino-2H-chromen-3-yl]-6-(4-phenylpiperazin-1-yl)-1,3,5-triazin-2-amine (11, IC50 in the range of 1.51&ndash, 2.60 &mu, M).

Graphical abstract, Highlights • Instant structured illumination microscope: 2-fold resolution enhancement. • Fast image acquisition rate (exceeding 100 fps). • Detailed description of construction and use., A challenge in biological imaging is to capture high-resolution images at fast frame rates in live cells. The “instant structured illumination microscope” (iSIM) is a system designed for this purpose. Similarly to standard structured illumination microscopy (SIM), an iSIM provides a twofold improvement over widefield microscopy, in x, y and z, but also allows much faster image acquisition, with real-time display of super-resolution images. The assembly of an iSIM is reasonably complex, involving the combination and alignment of many optical components, including three micro-optics arrays (two lenslet arrays and an array of pinholes, all with a pitch of 222 μm) and a double-sided scanning mirror. In addition, a number of electronic components must be correctly controlled. Construction of the system is therefore not trivial, but is highly desirable, particularly for live-cell imaging. We report, and provide instructions for, the construction of an iSIM, including minor modifications to a previous design in both hardware and software. The final instrument allows us to rapidly acquire fluorescence images at rates faster than 100 fps, with approximately twofold improvement in resolution in both x–y and z; sub-diffractive biological features have an apparent size (full width at half maximum) of 145 nm (lateral) and 320 nm (axial), using a 1.49 NA objective and 488 nm excitation.

Phosphodiesterase 4 (PDE4) activity mediates cAMP-dependent smooth muscle cell (SMC) activation following vascular injury. In this study we have investigated the effects of specific PDE4 inhibition with roflumilast on SMC proliferation and inflammatory activation in vitro and neointima formation following guide wire-induced injury of the femoral artery in mice in vivo. In vitro, roflumilast did not affect SMC proliferation, but diminished TNF-α induced expression of the vascular cell adhesion molecule 1 (VCAM-1). Specific activation of the cAMP effector Epac, but not PKA activation mimicked the effects of roflumilast on VCAM-1 expression. Consistently, the reduction of VCAM-1 expression was rescued following inhibition of Epac. TNF-α induced NFκB p65 translocation and VCAM-1 promoter activity were not altered by roflumilast in SMCs. However, roflumilast treatment and Epac activation repressed the induction of the activating epigenetic histone mark H3K4me2 at the VCAM-1 promoter, while PKA activation showed no effect. Furthermore, HDAC inhibition blocked the inhibitory effect of roflumilast on VCAM-1 expression. Both, roflumilast and Epac activation reduced monocyte adhesion to SMCs in vitro. Finally, roflumilast treatment attenuated femoral artery intima-media ratio by more than 50% after 4weeks. In summary, PDE4 inhibition regulates VCAM-1 through a novel Epac-dependent mechanism, which involves regulatory epigenetic components and reduces neointima formation following vascular injury. PDE4 inhibition and Epac activation might represent novel approaches for the treatment of vascular diseases, including atherosclerosis and in-stent restenosis.

We present research on optical fiber sensors based on microstructured multi-core fiber. Elaborated sensor can be advantageously used in hard-to-reach areas by taking advantage of the fact, that optical fibers can play both the role of sensing elements and they can realize signal delivery. By using the sensor, it is possible to increase the level of the safety in the explosive endangered areas, e.g. in mine-like objects. As a base for the strain remote sensor we use dual-core fibers. The multi-core fibers possess a characteristic parameter called crosstalk, which is a measure of the amount of signal which can pass to the adjacent core. The strain-sensitive area is made by creating the tapered section, in which the level of crosstalk is changed. Due to this fact, we present broadened conception of fiber optic sensor designing. Strain measurement is realized thanks to the fact, that depending on the strain applied, the power distribution between the cores of dual-core fibers changes. Principle of operation allows realization of measurements both in wavelength and power domain.

Liquid–liquid equilibrium curves for 1-ethyl-3-methylimidazolium tetrafluoroborate -[C2MIM,BF4], 1-butyl-3-methylimidazolium tetrafluoroborate -[C4MIM,BF4] and trihexyl(tetradecyl) phosphonium tetrafluoroborate -[P6,6,6,14,BF4] with chosen dihydroxy alcohols and their deuterated analogues have been determined. All fourteen obtained phase diagrams are described by the upper critical solution temperature (UCST) type behavior. The mutual solubility of 1-ethyl-3-methylimidazolium tetrafluoroborates decreases with the increase in alkyl chain of 1,2-dihydroxy alcohols but the opposite effect was observed for the ionic liquid with phosphonium cation. The impact of the hydroxyl group position in butanediol molecule on miscibility with trihexyl(tetradecyl) phosphonium tetrafluoroborate was examined. The results lead to the conclusion that vicinal(1,2 and 2,3)-butanediols are much better miscible than more polar (1,3) and (1,4) isomers. Deuterium substitution in both hydroxyl groups of investigated diols generates downward isotope shift of UCST both for phosphonium and imidazolium based ionic liquids thus making the miscibility better in each case.

Six series of structurally different mono- and binuclear copper(II) complexes 5–10 were obtained by reacting N-(2-pyridyl)imidazolidin-2-ones (1a–l), N,N'-bis(2-pyridyl)imidazolidin-2-ones (2a,b), N-acyl-N'(2-pyridyl)imidazolodin-2-ones (3a–j) and N-(2-pyridyl)imidazolidine-2-thiones (4a–g) with copper(II) chloride at an ambient temperature. The coordination modes of the complexes obtained were established by elemental analysis, IR spectroscopic data and single crystal X-ray diffraction studies. The in vitro cytotoxic activities of both the free ligands and copper(II) complexes were evaluated using a crystal violet microtiter plate assay on five human tumor cell lines: LCLC-103H, A-427, SISO, RT-4 and DAN-G. The free ligands 1–4 at concentration attainable in cancer cells of 20 μM showed no meaningful cytotoxic effect with cell viability in the range of 88%–100%. The most potent copper(II) complex of 1-(6-ethoxy-2-pyridyl)imidazolidin-2-one (6b) exhibited selective cytotoxicity against A-427 lung cancer cell line, while the complexes of 1-(5-methyl-2-pyridyl)imidazolidine-2-thione (5h) and 1-(4-tert-butyl-2-pyridyl)imidazolidine-2-thione (5j) showed cytostatic effect against a whole panel of five human tumor cell lines. In conclusion, the only complexes that showed remarkably increased activity in comparison to the free ligands were those obtained from N-(2-pyridyl)imidazolidine-2-thiones 4c and 4e substituted with alkyl group at position 4 or 5 of pyridine ring.

Liquid–liquid miscibility temperatures as a function of composition have been determined experimentally for the binary systems formed by trihexyl(tetradecyl)phosphonium tetrafluoroborate with cyclohexane, cycloheptane, methylcyclohexane, 1,2-dimethylcyclohexane and 1,3-dimethylcyclohexane. All the measured systems present solubility curves characterized by the asymmetry with respect to the equimolar composition and represent the phase diagrams with the upper critical solution temperatures. The effect of the deuterium substitution in cyclohexane and methylcyclohexane has also been studied revealing small upward shift of the UCSTs which means slightly worse miscibility of ILs studied with selected deuterated cycloalkanes.

In this paper, we have carried out a study on miscibility of ([bmim]+, [hmim]+, [omim]+)[BF4]− ionic liquids and dihydroxy alcohols: vicinal (1,2-diol) — C2(1,2)(OH)2 to C6(1,2)(OH)2, terminal (1,ω-diol) — C2(1,2)(OH)2 to C5(1,5)(OH)2 and butanediol isomers. Phase diagrams were determined at atmospheric pressure over the temperature range (270 to 340) K using the cloud-point method. LLE data for all 18 binary systems determined show the same behavior, representing phase diagrams with the upper critical solution temperature (UCST). With increasing chain length in C-3 position of the imidazolium cation, the UCST shifts toward higher temperatures for the systems with ethane-1,2-diol. Contrary, for the systems of longer diols with hydroxyl group in 1,2-position (propane-1,2-diol, butane-1,2-diol, pentane-1,2-diol and hexane-1,2-diol) improvement of miscibility was observed. The influence of different characteristics of alcohols and ILs, including cation alkyl chain length, alcohol chain length, and relative position of both OH groups in diol was also studied.

It is found that 1,2-dihydropyridine derivatives: 6-aryl-2,3-dihydro-6aH-imidazo[1,2-a]pyrido[1,2-c][1,3,5]triazin-5(6H)-ones and 3,6,7,8a-tetrahydro-2H-diimidazo[1,2-c:1',2'-e]pyrido[1,2-a][1,3,5]triazine underwent Diels-Alder reactions with highly reactive azadienophiles: 4phenyl-1,2,4-triazoline-3,5-dione and phthalazine-1,4-dione. The structures of the products were confirmed by IR, 1 H– 13 C heterocorrelated 2D NMR spectra HSQC and HMBC, mass spectra as well as single crystal X-ray crystallography.

Hydroxylamine-O-sulfonic acid (HOSA) was used as an efficient nucleophilic amination reagent for 2-chloropyrimidines, 2-chloroquinolines, and 1-chloroisoquinoline. The newly obtained heteroaromatic hydroxylamine-O-sulfonates subjected to the reaction with acyl isothiocyanates underwent tandem nucleophilic addition–electrophilic 5-endo-trig cyclization. The mechanism of the cyclization was investigated with use of the long-range corrected hybrid density functional ωB97X-D/6-31+G∗ and SM8 (DMF) solvation model. The structures of the heteroaromatic hydroxylamine-O-sulfonates and N-(5-methoxy-2H-[1,2,4]thiadiazolo[2,3-a]pyrimidin-2-ylidene)benzamide were confirmed by single crystal X-ray analysis. N-(2H-[1,2,4]thiadiazolo[3,2-a]isoquinolin-2-ylidene)benzamide exhibited a pronounced in vitro cytostatic activity against human tumor cell lines SISO, LCLC, A-427, DAN-G, and RT-4 (IC50 in the range 1.47–2.97 μM).

Liquid−liquid miscibility temperatures as a function of composition have been determined experimentally for the binary systems formed by trihexyl(tetradecyl)phosphonium chloride with hexane, octane...

The liquid–liquid miscibility temperatures as a function of composition and deuterium substitution have been experimentally determined for the binary mixtures of 1-decyl-3-methylimidazolium bis(trifluoromethyl)sulfonyl imide, [C10MIM][NTf2], and 1-dodecyl-3-methylimidazolium bis(trifluoromethyl)sulfonyl imide, [C12MIM][NTf2] with benzene and toluene and their deuterated forms. All systems exhibit upper critical solution temperatures (UCSTs) and better miscibility for ionic liquids with longer alkyl chain. The interchange of benzene for toluene results in worse miscibility for [C10MIM][NTf2] and better for [C12MIM][NTf2]. Deuteration of the aromatics leads to better miscibility in each case. The sign of the isotope effect is in agreement with expectations and its value seems to be seriously affected by the neighborhood of the hypercritical state.

PLoS one 11(11), e0166766 (2016). doi:10.1371/journal.pone.0166766, Published by PLoS, Lawrence, Kan.

Guillain-Barré syndrome (GBS) is a monophasic inflammatory disease considered to be due to autoimmunity. In order to test the hypothesis that the disease is associated with a perturbation of the circulating lymphoid cell population, we tested the mononuclear cells from the venous blood of 21 patients with Guillain-Barré syndrome (GBS) and 20 healthy controls by flow cytometry. The proportions and numbers of B and T lymphocytes, and CD4, CD8, double negative and gammadelta T cell subsets and numbers of monocytes were not significantly different in the patients compared with the controls. However, the number and proportion of CD4+CD25+ cells were reduced in acute GBS (mean number 61.7 cells/microl, 95% CI 42.9-80.4 and mean percentage 4.6%, 95% CI 3.8-5.4) compared with controls (mean number 99.8 cells/microl, 95% CI 74.7-124.9, p=0.02, and mean percentage 6.0%, 95% CI 4.9-7.1%, p=0.037). In addition, in GBS patients, the number and proportion of CD4+ T cells expressing CD25+ and HLA-DP, DQ, DR (mean number 11.9 cells/microl, 95% CI 7.6-16.1 and mean percentage 0.8%, 95% CI 0.5-1.1%) was lower than in healthy controls (23.5 cells/microl, 95% CI 16.4-30.6, p=0.01, and mean percentage 1.4%, 95% CI 1.1-1.8%, p=0.005. Since CD4+CD25+ cells include cells with special immunoregulatory functions, further investigation of this phenomenon and its relation to possible loss of regulatory T cell function in GBS is warranted.

Although myofascial tenderness is thought to play a key role in the pathophysiology of tension-type headache, very few studies have addressed neck muscle nociception. The neuronal activation pattern following local nerve growth factor (NGF) administration into semispinal neck muscles in anaesthetized mice was investigated using Fos protein immunohistochemistry. In order to differentiate between the effects of NGF administration on c-fos expression and the effects of surgical preparation, needle insertion and intramuscular injection, the experiments were conducted in three groups. In the sham group ( n = 7) cannula needles were only inserted without any injection. In the saline ( n = 7) and NGF groups ( n = 7) 0.9% physiological saline solution or 0.8 µM NGF solution were injected in both muscles, respectively. In comparison with sham and saline conditions, NGF administration induced significantly stronger Fos immunoreactivity in the mesencephalic periaqueductal grey (PAG), the medullary lateral reticular nucleus (LRN), and superficial layers I and II of cervical spinal dorsal horns C1, C2 and C3. This activation pattern corresponds very well to central nervous system processing of deep noxious input. A knowledge of the central anatomical representation of neck muscle pain is an essential prerequisite for the investigation of neck muscle nociception in order to develop a future model of tension-type headache.

Liquid–liquid miscibility temperatures as a function of composition and deuterium substitution have been experimentally determined for binary mixtures of aniline and cyclohexane and their various deuterated forms: aniline-d2, aniline-d5, cyclohexane-d12. In all cases studied, the deuteration leads to the upward shift of the upper critical solution temperature. This effect is discussed in the frame of the theory of the condensed phase isotope effects coupled with the phenomenological gE thermodynamic model of solutions.

Densities, viscosities, and speeds of sound were measured for the binary mixtures of nitromethane and 1-pentanol and their deuterated forms in the vicinity of the upper critical solution temperature. The excess molar volumes and excess isentropic compressibilities as well as the deviations of viscosity were determined. The influence of the deuterium substitution was analyzed and discussed.

BMC cancer 15(1), 552 (2015). doi:10.1186/s12885-015-1564-7, Published by BioMed Central, London

Introduction: Activation and dedifferentiation of smooth muscle cells (SMCs) at sites of vascular injury is regulated by epigenetic mechanisms. Epigenetic histone methylation has been recognized as a dynamic mark controlling many biological processes in health and disease. We assesed the hypothesis, that specific inhibitors of histone methyltransferases and demethylases effect SMC proliferation, inflammation and dedifferentiation. Methods: We studied the effects of the following epigenetic modifiers on smooth muscle cell proliferation, MCP-1 expression and TNFα-induced dedifferentiation: chaetocin, 3-deazaneplanocin (3-DZN), AMI-1, UNC0638, 2,4-pyridinedicarboxylic acid, methylstat, daminozide and GSKJ-4. Results: SMC proliferation was inhibited by treatment with 3-DZN, AMI-1, methylstat, chaetocin and GSK-J. TNFα-induced down regulation of SM22α expression as a marker of dedifferentiation was attenuated by treatment with UNC0638 and GSK-J4. TNFα-induced MCP-1 expression was particularly sensitive to inhibition of the histone methylation machinery, as all tested compounds with the exception of methylstat and UNC0638 caused a significant reduction of MCP-1 expression. UNC0638, chaetocin and 2,4-PDCA could attenuate TNFα-induced down regulation of SM22α expression up to 72h after the initial treatment. The sustained effect of the specific G9a histone methyltransferase inhibitor UNC0638 was associated with a strong and sustained reduction of histone h3 lysine 9 dimethylation at the SM22α promoter after 24 and 72h. Conclusion: Our data hint at the potential of modulating specific histone methylation marks in SMCs to target SMC activation and phenotypes in vascular diseases and warrant further research to dissect histone methylation dependent mechanisms in SMCs.

Introduction: Upregulation of phosphodiesterase 4 (PDE4) activity causes cAMP-dependent vascular smooth muscle cell (VSMC) activation at sites of vascular inflammation and tissue remodeling. Hypothesis: PDE4 inhibition reduces VSMC activation and neointima formation following vascular injury. Methods: C57BL/6 mice (n=16) treated with roflumilast underwent guide wire-induced endothelial denudation injury of the femoral artery. Neointima formation was quantified after 4 weeks. In vitro, we analyzed the effects of roflumilast on VSMC proliferation and inflammation. Results: Roflumilast treatment attenuated neointima formation by 40% (17252 ± 2315 vs. 10119 ± 738 μm 2 , p=0,02) and femoral artery intima-media ratio by more than 50% (2,76 ± 0,32 vs. 1,35 ± 0,25 ,p=0,005). In vitro, roflumilast did not affect VSMC proliferation, but diminished TNF-α induced expression of the inflammatory marker vascular cell adhesion molecule 1 (VCAM-1) by 60% (p Conclusions: PDE4 inhibtion reduces neointimal hyperplasia after vascular injury and regulates VCAM-1 through a novel Epac-dependent mechanism, which modulates specific histone methylation patterns. PDE4 inhibition might represent a novel approach for the treatment of vascular diseases, including atherosclerosis and in-stent restenosis.

NKT cells are associated with immunological control of autoimmune disease and cancer and can recognize cell surface mCD1d without addition of exogenous antigens. Cellular antigens presented by mCD1d have not been identified, although NKT cells can recognize a synthetic glycolipid, alpha-GalCer. Here we show that after addition of a lipid extract from a tumor cell line, plate-bound mCD1d molecules stimulated an NKT cell hybridoma. This hybridoma also responded strongly to three purified phospholipids, but failed to recognize alpha-GalCer. Seven of sixteen other mCD1d restricted hybridomas also showed a response to certain purified phospholipids. These findings suggest NKT cells can recognize cellular antigens distinct from alpha-GalCer and identify phospholipids as potential self-antigens presented by mCD1d.

A specialized subpopulation of T lymphocytes is reactive to the MHC class I-like molecule CD1d. It is not clear which cells are the major antigen-presenting cells in vivo in the activation of CD1-restricted immune responses. We have characterized a subset of B lymphocytes expressing six- to eightfold higher levels of CD1 than the bulk of B cells. The cells have a surface phenotype (CD21(high), CD23(low), IgM(high), IgD(low)) found previously to characterize B cells residing in the splenic marginal zones. CD1(high) B cells localize preferentially to the spleen, and appear late in ontogeny, at 3 - 4 weeks of age. The CD1(high) B cells were present in mice lacking conventional helper T cells, ruling out an exclusive origin from T cell-dependent immune responses. Still, some CD1(high) B cells had been involved in T cell-dependent immune responses as suggested by mutations in their rearranged immunoglobulin gene regions. The population could still be found in mice with severely reduced B cell reactivity to bacterial lipopplysaccharides (C3H / HeJ mice) and in mice unable to respond to thymus-independent type 2 antigens (NFR.Xid mice), as well as in germ-free mice, indicating that bacterial antigens are not major stimuli for the induction of CD1(high) B cells. In contrast, the CD1(high) B cell population was severely reduced in CD19-deficient mice. Taken together, the results imply that the CD1(high) population is heterogenous and of mixed origin, dependent for its development or maintenance on signaling through the CD19 molecule.

The miscibility of isobutyric acid with water and the influence of the isotope substitution of oxygen (16O/18O) in water over a broad concentration range are reported. The system exhibits a phase diagram with an upper critical solution temperature (UCST) and a visible isotope effect thereon. The oxygen isotope substitution decreases the UCST, leading to better miscibility of isobutyric acid with water. The isotope shift, DeltaTc = Tc(18O) - Tc(16O), extrapolated to the 100% enrichment in 18O, is equal to -1.15 K; hence it is much smaller and opposite to that found for deuterium substitution. The origin of the observed miscibility isotope effect has been qualitatively discussed in terms of the condensed-phase isotope effect theory.

The 7th Abercrombie Meeting took place in Oxford this past summer. It was organized by The Royal Microscopical Society with the support of The British Society for Cell Biology. Michael Abercrombie was a pioneer in the field of investigating cell behavior using time-lapse microscopy. The meeting was focused on “multi-dimensional cell migration in development and disease” and it brought together many of the world's leading researchers in the area, providing an opportunity to discuss the very latest advances and possible future developments in the field. The meeting sessions included Invasive Migration, Invasive Adhesions in Migrating Cells, Signaling in Migration, Immune Cell Migration, Migrations during Morphogenesis and Migration and Disease. As with all Abercrombie meetings, the conference delegates were treated to a staggering array of live cell imaging, in vivo imaging and images generated by the latest developments in microscopy.

Liquid–liquid equilibria for hexafluorophosphate ionic liquids with 1-alkyl-3-methylimidazolium family of cations: 1-butyl-3-methylimidazolium, [bmim] + , 1-hexyl-3-methylimidazolium, [hmim] + , 1-octyl-3-methylimidazolium, [omim] + with polyhydric alcohols: 1,2-ethanediol, 1,2-propanediol, 1,2-butanediol, 1,2-pentanediol, 1,2-hexanediol, 1,3-propanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 1,5-pentanediol have been measured using the cloud point method. The influence of different characteristics of alcohols and ILs, including cation alkyl chain length, alcohol chain length, and relative position of the both OH groups in alcohol were studied. In general, the same type of the phase behavior is observed in all cases showing upper critical solution temperature but the impact of the above-mentioned factors is highly diversified.

Multiple endocrine neoplasia type 1 (MEN 1) is a rare autosomal dominant inherited endocrine disease characterized by pancreatic, parathyroid, and anterior pituitary tumours. Hypercalcaemia due to parathyroid tumours is usually the first manifestation of MEN 1. Pancreatic islet tumours occur less frequently, among them gastrinomas and insulinomas are the most prevalent. Prolactinomas are a relatively common pituitary presentation of the syndrome. The gene causing MEN 1 is localized in chromosome 11q13 and encodes a protein named menin, which interacts with various proteins involved in transcriptional regulation, cell division, and DNA repair. Various mutations in the menin gene have been described, but so far no strong correlation between genotype and phenotype has been found.We report a case of a 31-year-old man, a lawyer, who was diagnosed with MEN 1 syndrome in 1999 at the age of 21 when he was operated because of prolactinoma and hyperparathyroidism. In 2000 insulinoma was suspected and eventually multifocal lesions in the pancreas were revealed. However, the patient did not agree to be operated on. Since then he has been followed up and has been treated with diazoxid. We observed gradual progression of the disease, but the patient remains in relatively good condition.Careful screening for MEN 1 is important in young patients with pituitary tumours. Regular follow up is crucial even after surgical treatment. The presented patient developed gradual enlargement of insulinomas and reoccurrence of hyperparathyroidism as well.

Liquid-liquid miscibility temperatures as a function of composition have been determined experimentally for the binary systems formed by ionic liquids ([bmim][BF(4)], [bmim][PF(6)], [emim][Tf(2)N], [bmim][Tf(2)N], [hmim][Tf(2)N]) and polyhydric alcohols (1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 1,2,3-propanetriol, 1,2-butanediol). The impact of ionic liquid and di- or three-hydroxy alcohol characteristics focusing on the effect of the IL's anion nature, cation alkyl chain length, and alcohol structure (number of hydroxyl groups, position of the hydroxy groups in the molecule, and number of carbon atoms in the diols) is presented. It appears that all systems exhibit upper critical solution temperatures. For dihydroxy alcohols mentioned above, miscibility with 1-butyl-3-methylimidazolium ionic liquids follows the order [BF(4)](-)[Tf(2)N](-)[PF(6)](-) and is dependent on the hydrogen-bond basicity of the anion. Analysis of these findings leads us to conclude that the miscibility of ionic liquids is likely related to the hydrogen-bond acceptor strength of the anion. Comparing the miscibility of 1,2-ethanediol with 1-alkyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imides, it can be seen that surprisingly, T(c)([emim](+))T(c)([bmim](+))T(c)([hmim](+)). This arrangement of critical temperatures is opposite to that observed earlier for systems with monohydroxy alcohols. Analyzing the influence of the polyhydroxy alcohol structure, we also noticed that the miscibility of the polyhydroxy alcohols with [bmim][Tf(2)N] or [bmim][BF(4)] decreases when the polarity of the alcohol rises.

Introduction: Tension-type headache (TTH) is associated with noxious input from neck muscles. Intravenous administration of the unspecific nitric oxide synthase inhibitor L-NMMA in chronic TTH patients caused analgesia and reduction of neck muscle tenderness. Methods: The unspecific nitric oxide synthase inhibitor L-NMMA was applied in an experimental model for neck muscle nociception in anesthetized mice ( N = 25). Results: Local injection of α,β-meATP into semispinal neck muscles induced sustained facilitation of brainstem nociception as monitored by the jaw-opening reflex. Preceding intraperitoneal administration of L-NMMA (0.05, 0.1, 1 mg/kg) prevented reflex facilitation evoked by α,β-meATP in a dose-dependent manner. Intraperitoneal injection of L-NMMA subsequent to intramuscular α,β-meATP application reversed established brainstem reflex facilitation back to baseline values. Discussion: Both experiments with preceding and subsequent L-NMMA indicate the involvement of nitric oxide synthases in the induction and maintenance of facilitation. However, future experiments will have to address the involvement of various isoenzymes in order to provide for new therapeutic concepts in TTH.

Neck muscle nociception probably plays a major role in the pathophysiology of tension-type headache. Recent studies have demonstrated sustained facilitation of brainstem nociception due to noxious neck muscle input evoked by nerve growth factor (NGF) or α,β-methylene ATP (ATP) in mice. Hypothesized different afferent pathways in NGF and ATP models were addressed by local application of tetrodotoxin (TTX) in neck muscles. Brainstem nociception was monitored in 55 anaesthetized mice by the jaw-opening reflex elicited by electrical tongue stimulation. Sole administration of 100nmol/l ATP or 0.8 μmol/l NGF evoked sustained reflex facilitation for at least 95 min. Preceding TTX administration prevented ATP-induced facilitation, but was without effect on NGF. Subsequent administration of 100 nmol/l TTX reversed ATP-evoked facilitation, but was ineffective on NGF. Divergent effects of TTX suggest preferential excitation of group III muscle afferents by ATP and group IV by NGF. Thus, both models address different pathways in pericranial pain.

Background: Potential target autoantigens in the demyelinating form of Guillain–Barre syndrome (GBS) include the myelin proteins PMP22, P0 and P2. Methods: We investigated immunoreactivity to P0, P2 and PMP22 proteins in 37 patients with GBS and 32 healthy controls. Results: Antibodies to PMP22 or P0 peptides were detected at presentation in only 5 out of 37 patients. In ELISPOT assays, blood mononuclear cells from 15 out of 24 patients with GBS, but none of the control subjects, produced interleukin-10 (IL-10) in response to peptides from proteins P0, P2 or PMP22 (p = 0.0003). The cells from only two patients produced interferon-γ (IFNγ). The cells from 11 patients with GBS had increased IL-10 responses to peptides representing sequences from the extracellular domains of PMP22 before intravenous immunoglobulin (IVIg) treatment (p = 0.006). The cells from 11 patients with GBS, including 7 who responded to the extracellular domains of PMP22, had increased IL-10 responses to the intracellular domain of P0 before (p = 0.005) and those from 9 patients after they had been treated with IVIg (p = 0.01). Conclusions: Antibodies to P0 and PMP22 protein peptides do occur in GBS but are uncommon. Circulating mononuclear cell IFNγ responses to P0, P2 and PMP22 myelin protein peptides are rare, but IL-10 responses occur significantly more often than in normal subjects. They might be part of a harmful pathogenetic process or represent a regulatory response.

The liquid-liquid miscibility temperatures as a function of composition and deuterium substitution have been experimentally determined for the binary mixtures of 1-butyl-3-methylimidazolium tetrafluroborate with 1-butanol, isobutyl alcohol, 2-butanol, and tert-butyl alcohol and their deuterated forms (OH/OD substitution). All systems exhibit upper critical solution temperatures (UCSTs) with a visible effect of branching in alcohols. Deuteration of alcohols in the hydroxyl group results in a decrease of the UCST of the given system and the largest shift is observed for tert-butyl alcohol. These solvent isotope effects nicely correlate with the polarity expressed by dielectric constants or E(T)(30) parameters of alcohols. The effect of the isotope substitution on the miscibility of ILs with butanols can be rationalized by using the statistical-mechanical theory of the isotope effects coupled with a phenomenological g(E) model. Following this procedure one finds that the isotope shift of UCST is associated mainly with the zero-point energy contribution.

We have previously demonstrated that CD4+ CD25+ natural regulatory T cells (Treg cells) induce down-modulation of CD80 and CD86 (B7) molecules on dendritic cells (DCs) in vitro. In this report we show that the extent of down-modulation is functionally significant because Treg-cell conditioned DCs induced poor T-cell proliferation responses. Further, we report that down-modulation was induced rapidly and was inhibited by blocking cytotoxic T lymphocyte antigen-4 (CTLA-4), which is constitutively expressed by the Treg cells. Even though Treg cells have previously been reported to kill antigen-presenting cells, the down-modulation was not due to selective killing of DCs expressing high level of the costimulatory molecules. We propose that Treg cells down-modulate B7-molecules on DCs in a CTLA-4-dependent way, thereby enhancing suppression of T-cell activity.

Noxious input from neck muscles probably plays a key role in tension-type headache pathophysiology. ATP selectively excites group III and IV muscle afferents in vitro. Accordingly, ATP infusion into trapezius muscle induces strong pain and local tenderness in healthy man. The present study addresses the impact of ATP on neck muscle nociception in anaesthetized mice. Craniofacial nociceptive processing was tested by the jaw-opening reflex via noxious electrical tongue stimulation. Within 2 h after injection of 100 nmol/l or 1 μmol/l ATP into semispinal neck muscles, reflex integrals significantly increased by 114% or 328%, respectively. Preceding intramuscular administration of the P2X receptor antagonist PPADS (3–100 nmol/l) suppressed the ATP effect. Subsequent application of PPADS (100 nmol/l) caused a total recovery of facilitated reflex to baseline values. ATP induces sustained facilitation of craniofacial nociception by prolonged excitation of P2X receptors in neck muscles.

The acute lesions of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) consist of endoneurial foci of chemokine and chemokine receptor expression and T cell and macrophage activation. The myelin protein antigens, P2, P0, and PMP22, each induce experimental autoimmune neuritis in rodent models and might be autoantigens in CIDP. The strongest evidence incriminates P0, to which antibodies have been found in 20% of cases. Failure of regulatory T-cell mechanism is thought to underlie persistent or recurrent disease, differentiating CIDP from the acute inflammatory demyelinating polyradiculoneuropathy form of Guillain-Barre syndrome. Corticosteroids, intravenous immunoglobulin and plasma exchange each provide short term benefit but the possible long-term benefits of immunosuppressive drugs have yet to be confirmed in randomised, controlled trials.

We have taken advantage of the Cre/lox system to generate a mouse model with inducible deficiency of transforming growth factor beta receptor II (TbetaRII). Using this approach, transforming growth factor beta (TGF-beta) signaling deficiency can be restricted to the hematopoietic system by bone marrow transplantation. Mice that received transplants with TbetaRII-/- bone marrow develop a lethal inflammatory disorder closely resembling that of TGF-beta1-null mice. Previous in vitro studies have suggested multiple roles for TGF-beta in T-cell development, including proliferation, apoptosis, and differentiation. We used our transplantation model to ask whether T-cell development is normal in the absence of TGF-beta signaling. The findings show for the first time in vivo and in fetal thymus organ culture (FTOC) that TGF-beta is not required for thymocytes to differentiate along the entire pathway of thymic T-cell development, as defined by the expression patterns of CD4, CD8, CD25, and CD44. In contrast to previous investigations, no increase of thymocyte apoptosis was observed. However, TbetaRII-deficient CD8+ thymocytes displayed a 2-fold increase in proliferation rate, as determined by bromodeoxyuridine (BrdU) incorporation in vivo. These results reinforce the importance of TGF-beta as an immune regulator critical for T-cell function.

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an acquired disorder of the peripheral nervous system with a probable auto-immune pathogenesis. The nature of the responsible autoantigens is unclear in most patients. We used the Western immunoblot technique to seek antibodies to peripheral nerve protein antigens. Sera from eight of 32 (25%) CIDP patients, 12 of 37 (32%) Guillain-Barre syndrome (GBS) patients, zero of 30 (0%) chronic idiopathic axonal polyneuropathy patients and two of 39 (5%) healthy control subjects contained anti-peripheral nerve protein antibodies. The frequency of such antibodies was significantly greater in both CIDP (p = 0.04) and GBS (p = 0.003) patients than in normal control subjects. For CIDP patients, there were non-significant trends for antibodies to be more common in females and in those who responded to treatment with either intravenous immunoglobulin or plasma exchange. The commonest antibodies were directed against a band at 28 kDa, resembling that labelled by a monoclonal antibody against myelin protein zero (P0). Six CIDP and seven GBS patients' sera reacted with this band. These results support the view that antibodies to myelin proteins, and especially P0, are present in the serum of some patients with CIDP and GBS.

Invasive pituitary adenomas with extension to the cavernous sinus are rare and comprise 6-10% of all pituitary tumors. Their effective surgical treatment is very difficult. Cavernous sinus invasion increases the morbidity and mortality associated with surgical procedures. Prolonged medical treatment and radiotherapy are an alternative to the surgery.Efficiency of the extended transnasal transsphenoidal approach in the treatment of the invasive pituitary adenomas with extension to the cavernous sinus.During the past two years (from January 2000 to December 2001) authors carried out 24 surgical operation in the medial compartment of the cavernous sinus (extended transnasal transsphenoidal approach). Our experience is based on the analysis of 24 patients (19 patients with GH-secreting tumors and 5 patients with ACTH-secreting adenomas--2 with Cushing disease and 3 with Nelson's syndrome) with invasive macroadenomas expanding to the cavernous sinus. Mean age was 46.21 years (range 28-68, SD +/-9.54 years) in our group. Mean serum GH level was 43.95 microg/l (range 9-170 microg/l, SD +/-39.68 microg/l) and mean serum ACTH level in the group of Nelson's syndrome was 1180 pg/ml (range 790-1780 pg/ml, SD +/-527.35 pg/ml). Parasellar extension of the tumor was measured according to Knosp's scale. There were 16 patients in stage III and 9 patients in stage IV.Total surgical removal of invasive GH-secreting adenoma (according cure criteria: basal serum GH level below 2.5 microg/l, OGTT1 microg/l, normal sex and age-related IGF-I level) we achieved in 6 patients. Basal serum GH level was below 5 microg/l (postoperative mean serum IGF-I level was significantly lower but abnormal) in the next five patients. Remission (normalization of 24-hour urine-free cortisol (UFC) and its metabolites) was achieved in one patient with Cushing disease. Out of 3 patients with Nelson's syndrome two were cured. Their postoperative serum ACTH level was below 80 pg/ml. There were no mortality and no deterioration of cranial and optic nerves. CSF leakage occurred in one patient and was treated surgically. Deterioration of pituitary function and diabetes insipidus occurred in one patient.Extended transnasal transsphenoidal approach is the safe surgical procedure in the treatment of invasive GH or ACTH-secreting pituitary adenomas. The overall morbidity rate of this surgical intervention is rare. We suppose the extended transnasal transsphenoidal approach is an alternative for prolonged medical treatment and radiotherapy.

Tension-type headache is the most common type of primary headaches but no conclusive concept of pathophysiology exists. This may be due to a lack of an appropriate animal model. This study addressed the hypothesis that noxious neck muscle input induces central sensitization of orofacial sensorimotor processing. The effect of hypertonic saline injection into the semispinal neck muscle on the jaw-opening reflex (JOR) was investigated in anaesthetized mice ( n = 11). Hypertonic saline injection into the neck muscle facilitated the JOR for at least one hour: integral (+94.5%) and duration (+18.7%) increased, latency decreased (-7.5%). The reflex threshold decreased to 61% after injection. Isotonic saline injection into the neck muscle ( n = 11) or hypertonic saline injection into a hindpaw muscle ( n = 10) did neither change the reflex integral nor the threshold. Long-term potentiation of the JOR by noxious neck muscle input may be an appropriate model to investigate tension-type headache pathophysiology.

To evaluate efficiency of the transcranial epidural approach in the treatment of invasive GH- or ACTH-secreting pituitary adenomas with extension to the cavernous sinus.During the past two years (from January 2000 to December 2001) 14 patients with invasive GH- or ACTH-secreting pituitary adenomas extending to the cavernous sinus were operated on using the transcranial epidural approach. Our experience is based on an analysis of 12 patients with GH-secreting tumors and 2 patients with ACTH-secreting adenomas. The patients' mean age was 45.36 years (range 28-66, SD +/- 10.26 years). Parasellar extension of the tumor was measured using the Knosp scale--in all the cases there was an extension to the cavernous sinus, in stage III (4 patients) or stage IV (10 patients).In none of the cases a total surgical removal of the invasive GH-secreting adenoma was attained (according the following cure criteria: basal serum GH level below 2.5 micrograms/l, OGTT1 microgram/l, normal sex- and age-related IGF-I level). In four patients the surgery resulted in a reduction of the basal serum GH level to below 5 micrograms/l (their postoperative mean serum IGF-I level was 530 micrograms/l--significantly lower, but still abnormal, p0.05). In a single case of a female patient the basal serum GH level was below 10 micrograms/l, while in other 7 patients the GH level remained above 10 micrograms/l. Remission (normalization of 24-hour urine-free cortisol (UFC) and its metabolites) was achieved in one patient with the Cushing disease. There were no complications involving case fatality. A transient deterioration of the third cranial nerve function observed in one patient disappeared within 3 months from the surgery. There was no deterioration of pituitary function and no cases of diabetes insipidus in our group.Transcranial epidural approach is an alternative to radiotherapy and/or prolonged medication in the treatment of invasive GH- or ACTH-secreting pituitary adenomas.

Nociceptive input from neck muscles probably plays a role in the pathophysiology of tension-type headache. In order to elaborate an animal model, the impact of noxious input from neck muscles on orofacial sensorimotor processing was investigated by electrophysiological means in anesthetized mice. Group IV muscle afferents of the semispinal neck muscle were excited by local injection of nerve growth factor (NGF, 0.8 microM, 20 microl). Orofacial sensorimotor processing was monitored by the jaw-opening reflex (JOR) elicited by electric tongue stimulation. After unilateral NGF injection into the right neck muscle (n = 10), JOR integral (+89%) and duration (+9%) increased and latency decreased (-5%) for at least 1 h. Bilateral injection of NGF (n = 10) into neck muscles induced an increase of JOR integral (+111%) and duration (+20%) and a reduction of latency (-9%). This facilitation of the JOR lasted for at least 90 min without any downward drift (n = 5). Electric JOR threshold diminished after NGF injection. After intramuscular injection of isotonic saline into the right semispinal neck muscle (20 microl), the JOR remained unchanged (n = 10). Local NGF injection into neck muscles evoked noxious input to the brainstem that induced a sustained central facilitation of the JOR for more than 1 h. This long-term facilitation of orofacial sensorimotor processing by a singular NGF injection possibly reflects plastic changes of nociceptive synaptic processing that may be involved in the pathophysiology of headache.