The identification of discontinuous epitope in the human cystatin C - Monoclonal antibody HCC3 complex.

Human cystatin C (hCC) is a cysteine proteinase inhibitor involved in pathophysiological processes of dimerization and amyloid formation. These processes are directly associated with a number of neurodegenerative disorders such as Alzheimer disease or hereditary cystatin C amyloid angiopathy (HCCAA). One of the ideas on how to prevent amyloid formation is to use immunotherapy. HCC3 is one of a group of antibodies binding to hCC and reducing the in vitro formation of cystatin C dimers. Therefore, identification of the binding sites in the hCC-HCC3 complex may facilitate a search of effective drugs against HCCAA as well as understanding the mechanisms of neurodegenerative disorders. In this work we present epitope identification of the hCC-HCC3 complex using methods such as affinity chromatography, epitope excision and extraction MS approach, enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry (HDX MS). Comprehensive analysis of the obtained results allowed us to identify the epitope sequence with the key fragment covering hCC L1 loop and two potential epitopic fragments - α-helical part, hCC (17-28) and ß4 strand in C-terminal part of hCC. The presence of the L1 loop in the epitope sequence accounts for the significant reduction of hCC dimer formation in the presence of HCC3 antibody. SIGNIFICANCE OF THE STUDY: Deciphering the mechanism of the cystatin C aggregation process and detailed analysis of the interactions between hCC, or its pathogenic variant, and monoclonal antibodies, potentially constituting aggregation inhibitors, might be of great value as there still is a complete lack of any kind of efficient therapy for young people with the pathogenic mutation of hCC.

High order kinoforms as a broadband achromatic diffractive optics for terahertz beams.

We discuss thin optical structures that allow chromatic aberrations to be avoided in the THz domain. The paper contains the theoretical considerations, computer modeling and experimental evaluation of the high order kinoform diffractive elements in the THz range. According to the obtained results application of the high order kinoforms enables broadband operation in the THz range.

Nanomechanical sensing of the endothelial cell response to anti-inflammatory action of 1-methylnicotinamide chloride.

BACKGROUND: There is increasing evidence that cell elastic properties should change considerably in response to chemical agents affecting the physiological state of the endothelium. In this work, a novel assay for testing prospective endothelium-targeted agents in vitro is presented. MATERIALS AND METHODS: The proposed methodology is based on nanoindentation spectroscopy using an atomic force microscope tip, which allows for quantitative evaluation of cell stiffness. As an example, we chose a pyridine derivative, 1-methylnicotinamide chloride (MNA), known to have antithrombotic and anti-inflammatory properties, as reported in recent in vivo experiments. RESULTS: First, we determined a concentration range of MNA in which physiological parameters of the endothelial cells in vitro are not affected. Then, cell dysfunction was induced by incubation with tumor necrosis factor-alpha (TNF-α) and the cellular response to MNA treatment after TNF-α incubation was studied. In parallel to the nanoindentation spectroscopy, the endothelium phenotype was characterized using a fluorescence spectroscopy with F-actin labeling, and biochemical methods, such as secretion measurements of both nitric oxide (NO), and prostacyclin (PGI2) regulatory agents. CONCLUSION: We found that MNA could reverse the dysfunction of the endothelium caused by inflammation, if applied in the proper time and to the concentration scheme established in our investigations. A surprisingly close correlation was found between effective Young's modulus of the cells and actin polymerization/depolymerization processes in the endothelium cortical cytoskeleton, as well as NO and PGI2 levels. These results allow us to construct the physiological model of sequential intracellular pathways activated in the endothelium by MNA.

Altered histone deacetylase activity and iNOS expression in cells isolated from induced sputum of COPD patients treated with tiotropium.

Chronic obstructive pulmonary disease (COPD) is the only major disease with increasing death rate. In COPD, progressive reduction in quality of life is closely related to the increasing limitation of airflow due to chronic bronchitis, cell hyperplasia, fibrosis, and irreversible lung damage. Signaling pathways involved in inflammatory processes in COPD and inflammatory response to therapy are unknown. Our aim was to isolate cells from induced sputum of COPD patients treated with formoterol or formoterol + tiotropium and assess enzymatic activity of histone deacetylases (HDACs) acetylated histone 4 (AcH4) and expression of inducible nitric oxide synthase (iNOS). HDACs are important in signal transduction and inflammation. iNOS is generating nitric oxide (NO) relevant to blood pressure regulation, inflammation and infections. Thirty stable COPD patients (21 males and 9 females, mean age 67 years) receiving 12 µg b.i.d. formoterol were assayed before and after 3 months add-on therapy consisting of 18 µg q.i.d. tiotropium. In all patients, spirometry, lung volumes, and DLCO were performed before and after tiotropium therapy and all patients were subjected to sputum induction. Sputum cells were isolated and processed to obtain cytosolic and nuclear fractions. HDAC activity was measured in nuclear fraction using colorimetric assay. Expression AcH4 and iNOS was quantified using Western blot. In patients receiving both drugs, FEV1 and lung volumes significantly improved compared with formoterol-only treated patients. Mean HDAC activity was slightly decreased (P < 0.05), while AcH4 levels and iNOS expression were significantly elevated in tiotropium-treated patients (increase by about 65 %; P < 0.01 and 77 %; P < 0.01 respectively). Our data show that beneficial effects of tiotropium in add-on therapy to formoterol may be related to altered histone signaling and increased iNOS expression.

Tiotropium increases PPARγ and decreases CREB in cells isolated from induced sputum of COPD patients.

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation and chronic inflammation of airways and lung parenchyma. Our aim was to assess two important elements of intracellular signaling involved in regulation of inflammation in COPD in patients subjected to long-acting beta2-agonist or long-acting beta2-agonist plus long-acting antimuscarinic: peroxisome proliferator-activated receptor gamma (PPARγ) protein, which has antiinflammatory and immunomodulatory properties and cAMP response element binding protein (CREB) and activated (CREB-P) protein which has histone acetyltransferase activity and increases histone acetylation and transcriptional activation of chromatin. Twenty one stable COPD patients (18 males and 3 females, mean age 65 years) receiving 12 µg B.I.D formoterol were assayed before and after 3 month add-on therapy, consisting of 18 µg Q.D. tiotropium. In all patients, sputum induction, spirometry, lung volumes, and DLCO were performed before and after therapy. Sputum cells were isolated and processed to isolate cytosolic and nuclear fractions. PPARγ, CREB, or CREB-P proteins were quantified in subcellular fractions using Western blot. Tiotropium add-on therapy improved respiratory parameters: FEV1 and lung volumes. After therapy mean expression of PPARγ in cell nuclei was significantly increased by about 180%, while CREB and phosphorylated CREB levels in cytosol and nuclei were decreased by about 30%. Our data show that the mechanism whereby tiotropium reduces exacerbations may be associated not only with persistent increase in airway functions and reduced hyperinflation mediated by muscarinic receptors, but also with possible anti-inflammatory effects of the drug, involving increased PPARγ and decreased CREB signaling.

Modeling of the optical system illuminated by quasi-monochromatic spatially incoherent light: new numerical approach.

This Letter presents a new method for modeling of complex optical setups illuminated by quasi monochromatic spatially incoherent light. The algorithm provides better performance and quality than other modeling methods both for isoplanatic and nonisoplanatic systems. The algorithm maintains energy relations, image orientation, and magnification of the system. Computer modeling and experimental results are presented.

Effective therapy of insulinoma by using long-acting somatostatin analogue. A case report and literature review.

We are reporting a case of 68-year-old woman with insulinoma, after a non-successful tumor surgery and a long-term diazoxide treatment. She had a lot of hypoglycemia cases, and a weight gain of 50 kg. An abdominal CT scan demonstrated a tumor 28 mm in the diameter, in the head of the pancreas. The patient did not agree for the repeated insulinoma surgery. Furthermore, we found a lesion in the left adrenal gland (14 mm in the diameter) and in the right lung (8 mm in the diameter). Pheochromocytoma was diagnosed on the basis of hypertension, elevated levels of normetanephrine in the 24-h urine collection, and an elevated level of norepinephrine in a plasma sample. After the left adrenal gland removal we observed lower blood pressure. Since we had revealed the presence of somatostatin receptors by the somatostatin receptors scintigraphy, we decided to control hypoglycemia by a monthly subcutaneous administration of the long-acting lanreotide. Because of higher glucose levels (300-400 mg/dl) we started an intense insulin therapy. Nowadays, the patient feels better, she has lost 20 kg of her body weight, and we have observed normal blood glucose levels during the long-term lanreotide treatment. We have noticed neither side effects nor hypoglycemic episodes and we have reduced the dose of insulin. The presented case can be an evidence of the effective treatment of the pancreatic neuroendocrine tumor of insulinoma type, with somatostatin analogue.

Strehl ratios characterizing optical elements designed for presbyopia compensation.

We present results of numerical analysis of the Strehl ratio characteristics for the light sword optical element (LSOE). For comparison there were analyzed other optical imaging elements proposed for compensation of presbyopia such as the bifocal lens, the trifocal lens, the stenopeic contact lens, and elements with extended depth of focus (EDOF), such as the logarithmic and quartic axicons. The simulations were based on a human eye's model being a simplified version of the Gullstrand model. The results obtained allow to state that the LSOE exhibits much more uniform characteristics of the Strehl ratio comparing with other known hitherto elements and therefore it could be a promising aid to compensate for the insufficient accommodation range of the human eye.

Imaging properties of the light sword optical element used as a contact lens in a presbyopic eye model.

The paper analyzes the imaging properties of the light sword optical element (LSOE) applied as a contact lens to the presbyopic human eye. We performed our studies with a human eye model based on the Gullstrand parameterization. In order to quantify the discussion concerning imaging with extended depth of focus, we introduced quantitative parameters characterizing output images of optotypes obtained in numerical simulations. The quality of the images formed by the LSOE were compared with those created by a presbyopic human eye, reading glasses and a quartic inverse axicon. Then we complemented the numerical results by an experiment where a 3D scene was imaged by means of the refractive LSOE correcting an artificial eye based on the Gullstrand model. According to performed simulations and experiments the LSOE exhibits abilities for presbyopia correction in a wide range of functional vision distances.

Tiotropium increases cytosolic muscarinic M3 receptors and acetylated H3 histone proteins in induced sputum cells of COPD patients.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible progressive airflow limitation related to tobacco smoking. This limitation is caused by chronic inflammation of the airways and lung parenchyma and is associated with increased activity of parasympathetic system. The most effective bronchodilators in COPD are muscarinic receptor antagonists (MRA), which reverse, at least in part, compromised respiratory function. MRA also contribute to control inflammatory processes via interactions with inflammatory signaling molecules. The use of the long-acting cholinolytic bronchodilatator - tiotropium, with high affinity to M3 receptors, is suggested as a first line maintenance treatment in COPD patients. MATERIAL AND METHODS: In this study we assessed M3 receptor protein expression in induced sputum of 27 stable COPD patients before and after therapy consisting of 18 µg once daily tiotropium for 12 weeks. Lung function tests including spirometry, lung volumes, and DLCO were performed before and after therapy in all COPD patients. The patients were subjected to the sputum induction procedures before and after therapy. Sputum cells were isolated, sample-specific cell profiles were characterized, and the cells were processed to isolate pure cytosolic fractions. Cytosolic M3 protein and HDAC2 levels and nuclear acetylated histone H3 (AcH3) expression was quantified using specific antibodies against human proteins and Western blot with enhanced luminescence detection. RESULTS: Therapy significantly increased the mean forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) volume (P<0.05). The mean expression of M3 protein was higher by 37% after therapy (P<0.05), HDAC2 expression was not altered, while AcH3 level was increased by about 90% (P<0.01), compared with the corresponding data before therapy. HDAC2 expression before therapy was positively correlated with AcH3 expression (r = 0.74), while after therapy no correlation was detected. FEV1, FCV, and cytosolic M3 protein expression did not correlate with other biochemical parameters tested. CONCLUSIONS: Twelve weeks of tiotropium therapy in COPD patients improves clinical indices of lung function and involves alterations in sputum cell chromatin acetylation and also increased cholinergic M3 receptor internalization.

Propofol protects rat astroglial cells against tert-butyl hydroperoxide-induced cytotoxicity; the effect on histone and cAMP-response-element-binding protein (CREB) signalling.

Propofol can be potentially beneficial in oxidative stress related malignancies as neurodegenerative diseases and traumatic brain injury but its signalling pathways are poorly understood. In this study effect of propofol on astroglial signalling in oxidative stress was evaluated. Ten days old cultures of rat astroglial cells were treated for 1 hour with t-butyl hydroperoxide (tBHP) to induce oxidative stress following by 1 hour propofol. We measured cytotoxicity, changes in cell growth and apoptosis as well as alterations in expression and acetylation of chromatin core H3 and H4 histone proteins and changes in native and phosphorylated cAMP-response-element-binding protein (CREB). tBHP induced limited cytotoxicity, increased apoptosis, decreased glutamine synthetase and enolase activities, decreased nuclear CREB, CREB-P and histone proteins but unchanged cytosolic CREB and histone acetyltransferase (HDAC) expression. Propofol clearly protected the cells against tBPH-induced toxicity, normalized alterations in cell growth, restored to some extent glial enzyme activities and reduced apoptotic cell numbers. Also, propofol restored H3 but not H4 expression/activation, but was without effect on decreased nuclear CREB expression/activation. These data show that oxidative stress in cultured astroglia significantly affects nuclear CREB and histone proteins and point to the protective role of propofol.

Increased FKBP51 in induced sputum cells of chronic obstructive pulmonary disease patients after therapy.

OBJECTIVE: Immunophilin FKBP51 assists polypeptide folding, participates in glucocorticoid actions and may play a role in glucocorticoid resistance. FKBP51 is altered in patients with asthma, but its role in chronic obstructive pulmonary disease (COPD) characterized by dysregulation of several pro/antiinflammatory genes is less clear. METHODS: We assessed changes in nuclear/cytosolic FKBP51 protein using SDS-PAGE/WB and FKBP51 mRNA by qRT-PCR in cells isolated from induced sputum of stable COPD patients treated with formoterol/budesonide or formoterol/budesonide/theo?phylline for 4 wk. RESULTS: Expression of FKBP51 was higher in formoterol/ budesonide/theophylline-treated patients, compared with formoterol/budesonide group in both cytosolic and nuclear fractions by about 57% and 31%, respectively (P<0.001, P<0.01). FKBP51 mRNA was only slightly, but not significantly, higher in patients on formoterol/ budesonide/theophylline. CONCLUSIONS: Increased FKBP51 in COPD patients treated with formoterol/ budesonide/theophylline may be important in altering signaling from corticosteroid receptors.

Imaging with extended focal depth by means of the refractive light sword optical element.

The paper presents first experiments with a refractive light sword optical element (LSOE). A refractive version of the LSOE was prepared in photoresist by gray scale photolithography. Then we examined chromatic aberrations of the produced element and compared them with those corresponding to two different lenses. For this purpose we performed two experiments, the first one where white light illumination was used and the latter one by the help of monochromatic illumination with three different wavelengths. The obtained results lead to the conclusion that the refractive LSOE does not exhibit significant chromatic aberrations and can be successfully used for imaging with extended depth of focus in polychromatic illumination.

Imaging with extended focal depth by means of lenses with radial and angular modulation.

The paper presents imaging properties of modified lenses with the radial and the angular modulation. We analyze three following optical elements with moderate numerical apertures: the forward logarithmic axicon and the axilens representing the radial modulation as well as the light sword optical element being a counterpart of the axilens with the angular modulation. The abilities of the elements for imaging with extended depth of focus are discussed in detail with the help of structures of output images and modulation transfer functions corresponding to them. According to the obtained results only the angular modulation of the lens makes possible to maintain the acceptable resolution, contrast and brightness of the output images for a wide range of defocusing. Therefore optical elements with angular modulations and moderate numerical apertures seem to be especially suitable for imaging with extended focal depth.

Synthesis of analogues of anthraquinones linked to tuftsin or retro-tuftsin residues as potential topoisomerase inhibitors.

A novel group of [(4-, 5- or 8)-hydroxy-9,10-anthraquinone-1-yl]-(tuftsin or retro-tuftsin) acids and methyl esters has been synthesized as potential anticancer compounds. The corresponding protected tuftsin or retro-tuftsin derivatives were also synthesized. We hope that combining compounds of different mechanisms of action will improve their clinical properties, and that our new analogues will be much more effective against multidrug-resistant tumour cell lines.

A TL-based anthropomorphic benchmark for verifying 3-D dose distributions from external electron beams calculated by radiotherapy treatment planning systems.

Initial results are reported of a Polish-Finnish project to verify electron dose distributions calculated by treatment planning systems (TPSs), CadPlan v.6.3.2 and Theraplan v.3.5, which use different electron beam dose distribution algorithms. Treatment of gross tumour volumes representing lung and parotid cancer was simulated in an Alderson anthropomorphic phantom with thermoluminescent detectors (TLDs) (Li(2)B(4)O(7):Mn,Si) placed at selected measurement points inside its volume. The observed discrepancy between relative values of dose calculated and measured by TLDs at each of the measurement points and those calculated by the different TPSs at the same points is discussed.

Synthesis and biological activity of tuftsin, its analogue and conjugates containing muramyl dipeptides or nor-muramyl dipeptides.

Several conjugates of muramyl dipeptide (MDP) or nor-muramyl dipeptide (nor-MDP) with tuftsin were synthesized. Conjugates 8a-f were prepared by acylation of protected tuftsin with the isoglutamine carboxyl group of MDP or nor-MDP 2a-f. Also tuftsin analogue 6 (H-Thr-Lys-Pro-Arg(NO2)-OH) was obtained. All synthesized compounds were investigated at the Medical University of Gdansk. The biological activity of the examined compounds was estimated using in vitro cultures of human monocytes and lymphocytes. The substances displayed cytotoxic effects, as was revealed in the viability tests performed. The effects were most probably mediated by the induction of an oxidative burst in monocytes and the stimulation of redox enzymes in lymphocytes. In addition, the analogues turned out to be efficient stimulators of TNFalpha and IL6 secretion by monocytes and lymphocytes. Nevertheless, the secretion of cytokines did not affect the viability of the leukocyte population used in the experiments.The beneficial properties of the compounds examined (mainly 6, 3, 8a and 8c), which implies their usefulness as potential therapeutic agents, are connected with their rapid start of action and more efficient effects compared with tuftsin alone. An in vivo assay on animal models will be performed.

Synthesis and antitumor activity of conjugates of muramyldipeptide, normuramyldipeptide, and desmuramylpeptides with acridine/acridone derivatives.

The synthesis of two groups (Chart 1, types A and B) of conjugates of MDP (muramyldipeptide) and nor-MDP (normuramyldipeptide) with acridine/acridone derivatives and the synthesis of analogues of desmuramylpeptides (Chart 1, types C and D) containing acridine/ acridone derivatives have been described. In type A conjugates, the hydroxyl group at C6 of the sugar moiety was acylated with acridine/acridone N-substituted omega-aminoalkanocarboxylic acids (Scheme 1), whereas the conjugates of type B (Table 2) and three analogues of type C or D (Scheme 2) have an amide bond formed between the carboxylic group of isoglutamine and the amine function of the respective acridine/acridone derivatives. The preliminary screening data indicate that the analogues of groups A, C, and D exhibit small cytotoxic activity, whereas several analogues of type B, 4b, 4c, 4e, 4g, 4h, 4i, and 4l, exhibiting potent in vitro cytotoxic activity against a panel of human cell lines (Table 4), have been selected by the National Cancer Institute (NCI) Evaluation Committee for further testing. Analogues 4b and 4h were active in the in vivo hollow fiber assay (Table 5). Analogue 3a shows an immunostimulating effect on the cytotoxic activity of the NK cells obtained from the spleen of healthy and Ab melanoma bearing animals.

Synthesis of new chiral peptide nucleic acid (PNA) monomers.

We have synthesised a series of new chiral type I peptide nucleic acid monomers in total yields of 36-53%, derived from Val, Ile, Ser(Bzl), Pro, and Trp, employing convenient procedure.

Lens-based theory of the Lau effect.

A new theoretical model of the Lau effect is presented. The transmittance of a diffraction grating can be expressed in an equivalent form as the sum of transmittances of thin cylindrical lenses. Therefore it is possible to explain the Lau effect on the basis of the well-known imaging properties of lenses. According to the given approach, the Lau fringes are created by overlapped images of the first grating that are formed by a set of lenses corresponding to the second grating in the setup. The theory leads to an exhaustive description of the Lau-effect parameters. In particular, one can indicate the shape of the Lau fringes and localize planes of the fringes dependent on the axial distance between gratings and their periods.