Persistent imbalance, anti-apoptotic, and anti-inflammatory signature of circulating C-C chemokines and cytokines in patients with paroxysmal nocturnal hemoglobinuria.

OBJECTIVE: Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal non-malignant disease in which hematopoietic cell apoptosis may play an important pathophysiological role. Previous studies of the content of phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) indicated the possibility of remote transmission of anti-apoptotic signals between pathological and normal hematopoietic progenitors. METHODS: The study determined the plasma levels of beta chemokines and cytokines in N = 19 patients with PNH and 31 healthy controls. The research material was peripheral blood plasma (EDTA) stored at -80 °C until the test. Beta chemokine and cytokine concentrations were tested in duplicate with Bio-Plex Pro Human Cytokine Assay (Bio-Rad, Hercules, CA, USA) using a Luminex 200 flow cytometer and xPONENT software (Luminex Corporation, Austin, TX, USA). In peripheral blood CD34+ cells we tested the proportions of PI(3,4,5)P3+ and Annexin binding apoptotic phenotype using FC and phosflow. RESULTS: Compared to the control group, the PNH group showed a significant increase in the plasma concentration of some beta chemokines and cytokines, including MIP-1alpha/CCL3, eotaxin/CCL11, MCP1/CCL2, IL4 and G-CSF. In the group of PNH patients, a significant decrease in the concentration of some cytokines was also observed: RANTES/CCL5, MIP-1beta/CCL4, PDGF-BB and IL9. At the same time, the plasma concentrations of the chemokine IP-10/CXCL10 and the cytokines IFN-gamma, TNF, IL6 and IL10 showed no significant deviations from the values for the control group. Anti-apoptotic phenotype and phosphatidylinositol (3,4,5)-trisphosphate content in PNH clone of CD34+ cells were associated with the level of CCL3 and negatively associated with CCL5, CCL4, PDGF-BB and IL9. CONCLUSIONS: This data suggest the existence of apoptotic and PI(3,4,5)P3 imbalance in PNH CD34+ cells driven by anti-apoptotic cytokine biosignature in PNH. Plasma cytokines and intracellular enzymes that regulate the phosphoinositide pathways may become a therapeutic target in PNH.

Determination of Glycerophospholipids in Biological Material Using High-Performance Liquid Chromatography with Charged Aerosol Detector HPLC-CAD-A New Approach for Isolation and Quantification.

The method of using high-performance liquid chromatography with a charged aerosol detector method (HPLC-CAD) was developed for the separation and determination of phospholipids isolated from cell membranes. The established cell lines-normal and neoplastic prostate cells and normal skin fibroblasts and melanoma cells-were selected for the study. Chromatographic separation was performed in the diol stationary phase using a gradient elution based on a mixture of n-hexane, isopropanol and water with the addition of triethylamine and acetic acid as buffer additives. Taking the elements of the Folch and Bligh-Dyer methods, an improved procedure for lipid isolation from biological material was devised. Ultrasound-assisted extraction included three extraction steps and changed the composition of the extraction solvent, which led to higher recovery of the tested phospholipids. This method was validated by assessing the analytical range, precision, intermediate precision and accuracy. The analytical range was adjusted to the expected concentrations in cell extracts of various origins (from 40 µg/mL for PS up to 10 mg/mL for PC). Both precision and intermediate precision were at a similar level and ranged from 3.5% to 9.0%. The recovery for all determined phospholipids was found to be between 95% and 110%. The robustness of the method in terms of the use of equivalent columns was also confirmed. Due to the curvilinear response of CAD, the quantification was based on an internal standard method combined with a power function transformation of the normalized peak areas, allowing the linearization of the signal with an R2 greater than 0.996. The developed method was applied for the isolation and determination of glycerophospholipids from cell membranes, showing that the profile of the tested substances was characteristic of various types of cells. This method can be used to assess changes in metabolism between normal cells and neoplastic cells or cells with certain pathologies or genetic changes.

A microcomputed tomography-based comparison of root canal filling quality following different instrumentation and obturation techniques.

OBJECTIVE: With a microcomputed tomography (microCT) imaging device, we aimed to quantitatively evaluate root canal fillings after commonly used endodontic procedures and also tested the suitability of microCT for this purpose. MATERIALS AND METHODS: Eighty single roots were instrumented and obturated with gutta-percha and Tubli-Seal. They were divided into 4 groups of 20. The Hand groups were instrumented with hand files and filled with thermoplastic (Th) compaction and cold lateral (CL) condensation, i.e. Hand-Th and Hand-CL, respectively. The Rot groups, i.e. Rot-Th and Rot-CL, were instrumented with a rotary ProFile system and filled as above. The roots were scanned and 3-dimensional (3D) visualization was obtained. The number, size, percentage of volume and distribution of voids at the filling/dentine interface (i-voids) and voids surrounded by filling material (s-voids) were measured. RESULTS: Canal fillings differed significantly with regard to the size of both types of voids and the average number of i-voids. All canals presented a low volume of voids. The highest percentage (0.69%) was found for i-voids in the Hand-CL group, while the lowest volume (0.11% for s-voids and 0.14% for i-voids) was in the Hand-Th canals. Apically, in the last 3 mm, i-voids were observed mainly in the Th groups, and s-voids occurred mostly in the coronal part of the canal filling in all cases. CONCLUSION: MicroCT was a useful tool for 3D quantitative evaluations of these root canal fillings. None of the root canal instrumentation and filling methods ensured void-free obturation. CL condensation produced mainly i-voids. With Th compaction, internal s-voids were particularly common, but there were mainly i-voids in the apical part.

Application of microcomputed tomography for quantitative analysis of dental root canal obturations.

INTRODUCTION: The aim of the study was to apply microcomputed tomography to quantitative evaluation of voids and to test any specific location of voids in tooth's root canal obturations. MATERIALS AND METHODS: Twenty root canals were prepared and obturated with gutta-percha and Tubli-Seal sealer using the thermoplastic compaction method (System B+Obtura II). Roots were scanned and three-dimensional visualization was obtained. The volume and Feret's diameter of I-voids (at the filling/dentine interface) and S-voids (surrounded by filling material) were measured. RESULTS: The results revealed that none of the scanned root canal fillings were void-free. For I-voids, the volume fraction was significantly larger, but their number was lower (P=0.0007), than for S-voids. Both types of voids occurred in characteristic regions (P<0.001). I-voids occurred mainly in the apical third, while S-voids in the coronal third of the canal filling. CONCLUSIONS: Within the limitations of this study, our results indicate that microtomography, with proposed semi-automatic algorithm, is a useful tools for three-dimensional quantitative evaluation of dental root canal fillings. In canals filled with thermoplastic gutta-percha and Tubli-Seal, voids at the interface between the filling and canal dentine deserve special attention due to of their periapical location, which might promote apical microleakage. Further studies might help to elucidate the clinical relevance of these results.

Effect of KRAS codon13 mutations in patients with advanced colorectal cancer (advanced CRC) under oxaliplatin containing chemotherapy. Results from a translational study of the AIO colorectal study group.

BACKGROUND: To evaluate the value of KRAS codon 13 mutations in patients with advanced colorectal cancer (advanced CRC) treated with oxaliplatin and fluoropyrimidines. METHODS: Tumor specimens from 201 patients with advanced CRC from a randomized, phase III trial comparing oxaliplatin/5-FU vs. oxaliplatin/capecitabine were retrospectively analyzed for KRAS mutations. Mutation data were correlated to response data (Overall response rate, ORR), progression-free survival (PFS) and overall survival (OS). RESULTS: 201 patients were analysed for KRAS mutation (61.2% males; mean age 64.2 ± 8.6 years). KRAS mutations were identified in 36.3% of tumors (28.8% in codon 12, 7.4% in codon 13). The ORR in codon 13 patients compared to codon 12 and wild type patients was significantly lower (p = 0.008). There was a tendency for a better overall survival in KRAS wild type patients compared to mutants (p = 0.085). PFS in all patients was not different in the three KRAS genetic groups (p = 0.72). However, we found a marked difference in PFS between patients with codon 12 and 13 mutant tumors treated with infusional 5-FU versus capecitabine based regimens. CONCLUSIONS: Our data suggest that the type of KRAS mutation may be of clinical relevance under oxaliplatin combination chemotherapies without the addition of monoclonal antibodies in particular when overall response rates are important. TRIAL REGISTRATION NUMBER: 2002-04-017.

New approach for amino acid profiling in human plasma by selective fluorescence derivatization.

A new approach for the separation of 6-aminoquinolyl-carbamyl (AQC)-derivatized amino acids has been proposed. The chromatography used ion-pairing mechanism to increase the method selectivity. Mobile phase was based on triethylamine buffer containing N,N-dimethyloctylamine as a modifier. A number of factors, buffer composition and pH, counterion concentration, temperature and acetonitrile gradient profile, were optimized to achieve final chromatographic conditions. With the presented analytical method, the separation and identification of 34 AQC-amino acids and amino compounds present in human plasma is possible. The results of validation proved the applicability of the method for quantification of 27 amino acids in biological samples. The ultrafiltration proposed as deproteinization procedure gave repeatable and reliable results for the amino acids under investigation. This method introduced in routine testing can be a suitable tool for amino acid profiling in plasma including all aspects of clinical application.

Analysis of biologically active peptides using two-dimensional HPLC-CE.

High-performance liquid chromatography (HPLC) is a technique most frequently used for the assessment of biologically active peptides. Owing to the ionic character of these compounds, they may also be separated and assayed using capillary electrophoresis (CE), which offers very high efficiency, short analysis time and low consumption of reagents, and is used increasingly more often. The paper describes the combination of HPLC and CE in order to increase the efficiency of the separation of complex mixture of peptides (active substance and its related impurities). The developed two-dimensional HPLC-CE technique was employed for the analysis of the impurities of octreotide, a cyclic octapeptide used in therapy. Because distinct separation mechanisms are used, the two-dimensional technique ensures higher separation efficiency and a more comprehensive impurity profile of the medicinal product than either of the techniques used separately.

Adsorption of the dye Acid Blue 158 premetalized with chromium on chitin/chitosan.

Removal of the premetalized dyes from the effluents of textile and leather industries is still open. The work aims to investigate functional groups taking part in the sorption of premetalized dye Acid Blue 158:Cr on chitin/chitosan and to investigate the mechanism of the sorption. The process was spontaneous (negative value of the Gibbs function), endothermic (ΔH = 0.2679 kJ/mol) with an affinity between the AB 158:Cr and chitin (ΔS = 1.315 J/mol*K). Langmuir, Freundlich, and Dubinin-Radushkevich isotherms were used to approximate experimental data giving a good correlation. Maximal capacity was evaluated as 46.1 mg/g and 370.4 mg/g for chitin and chitosan, respectively. Kinetics was described with a pseudo-second-order model. It was shown that physical sorption and chemisorption can occur parallelly: chemisorption on amine groups via interaction with chromium and physical sorption on amine group or hydroxyl group via interaction with the azo core of the dye.

Modification of chitin structure with tailored ionic liquids.

Chitin, poly N-acetylglucosamine, has a great potential for use on an industrial scale as an enzyme carrier but it has an unfavorable particle structure that can be modified using ionic liquids (ILs). Several ionic liquids were investigated that have the same substituents on the ring (methyl- and propyl-) but differed in the type of cationic ring (pyrrolidinium, piperidinium, and piperazinium). Organic acid ions (acetic and lactic) were used as counter ions. 1-ethyl-3-methyl-imidazolium acetate and 1-ethyl-3-methyl-imidazolium lactate were used as a reference. The results confirm that the chitin particle structure or size, or both, simultaneously changes if chitin is dissolved in an IL and then precipitated. Organic acid anions and short substituents on the cationic ring of ILs influenced particle modification substantially, whereas the type of ring played a minor role. Additionally, the ionic liquids [MPpyrr][OAc], [MPpip][OAc] and [DMPpz][OAc] could be reused up to at least 4 times without losing their ability to dissolve chitin.

Kinetics of chitin deacetylase activation by the ionic liquid [Bmim][Br].

Chitin deacetylase is the only known enzyme that can deacetylate the N-acetyl-d-glucosamine units in chitin and chitosan to D-glucosamine. Unfortunately, this enzyme, originally obtained from fungi, usually has low activity. Here, we present that it is possible to enhance the activity of chitin deacetylase using the ionic liquid [Bmim][Br]. An increase in activity of up to 160% from the basal chitin deacetylase activity was observed. Kinetic investigations suggest that [Bmim][Br] is a non-essential activator.

Role of thiamine in Huntington's disease pathogenesis: In vitro studies.

BACKGROUND: Oxidative stress accompanies neurodegeneration and also causes abnormalities in thiaminedependent processes. These processes have been reported to be diminished in the brains of patients with several neurodegenerative diseases. OBJECTIVES: The aim of this work was to conduct a comparative analysis of the impact of supplemented thiamine on the viability of human B lymphocytes with CAG abnormal expanded huntingtin gene (mHTT) (GM13509) and control, B lymphocytes without mHTT (GM14467) through the following studies: determination of the supplemented thiamine concentrations, which are effective for cell growth stimulation after incubation in thiamine deficit conditions; determination of cell capability to intake the exogenous thiamine; evaluation of exogenous thiamine influence on the profile of the genes related to thiamine and energy metabolism; determination of ATP synthesis and activities of thiamine-dependent enzymes, KGDHC and BCKDHC in the intact cells and upon the exogenous thiamine. MATERIAL AND METHODS: The following methods were used: EZ4U test for cell growth analysis; HPLC for determination of thiamine intake and ATP synthesis, qRT-PCR for evaluation of the gene profiles and spectrophotometric method for KGDHC and BCKDHC activities determination. RESULTS: Maximal cell growth stimulation was observed at 2.5 mM in GM14467 up to 135% of the control culture and at 5.0 mM in GM13509 cells up to 165% of the control culture. Native levels of total ATP and KGDHC and BCKDHC activities in both cell types were comparable and did not changed upon thiamine deficit or supplementation. GM13509 cells showed more of an increase in growth stimulation upon thiamine supplementation than GM14467 cells and this effect was reflected in the increase of intracellular thiamine concentration. CONCLUSIONS: The above results and reported changes in expression of GAPDH, IDH1 and SLC19A3 genes observed upon thiamine deficit conditions suggest that intracellular thiamine status and energy metabolism can have a role in HD pathogenesis.

Sclerosing pseudovascular rhabdomyosarcoma-immunohistochemical, ultrastructural, and genetic findings indicating a distinct subtype of rhabdomyosarcoma.

Sclerosing (pseudovascular) rhabdomyosarcoma in adults has been described as a rare variant of rhabdomyosarcoma characterized by extensive hyaline fibrosis and pseudovascular growth patterns. We describe another case of this rhabdomyosarcoma subtype including ultrastructural and genetic findings-the lesion presented in a 62-year-old male patient in the left lower leg. The tumor was located within the deep soft tissue with maximum diameter of 11.8 cm and skin ulceration. Ultrastructural analysis revealed irregularly distributed disorganized filaments without clear evidence of Z-bands and a richly collagenized matrix. Using comparative genomic hybridization, a sharply delineated loss of chromosomal region 10q22, loss of chromosome Y, and a gain of chromosome 18 (trisomy) were detected. Reciprocal translocations t(1;13) and t(2;13)(q35;q14) which are characteristic of alveolar rhabdomyosarcoma could be excluded. These findings, while showing a relation to other rhabdomyosarcoma subtypes, represent a relatively circumscribed genetic defect pattern in sclerosing (pseudovascular) rhabdomyosarcoma that is somewhat different from patterns described in most other rhabdomyosarcoma subtypes. Six months after tumor resection, the patient presented with metastatic disease. Further studies should concentrate on the identification of genes especially on chromosomal region 10q22 to elucidate more aspects in the pathogenesis of this rhabdomyosarcoma subtype.

Separation of synthetic food colourants in the mixed micellar system. Application to pharmaceutical analysis.

The paper presents a rapid method for the determination of commonly used synthetic food dyes by micellar electrokinetic capillary chromatography. Detection and separation conditions allowing complete resolution of 15 synthetic food colourants were investigated. The effect of different surfactants on the analytes mobility in relation to their structure was tested. After optimization procedure a dual micellar system was selected. All food dyes were separated in less then 20 min using a fused silica capillary in the borate/dodecylsulfate/deoxycholate buffer containing acetonitrile as organic modifier. The detection wavelength was set at 210nm. The method was successfully validated by determination of linearity ranges, detection limits, precision and repeatability for all colourants tested. In order to apply the method for pharmaceutical analysis a sample pretreatment procedures were found. Liquid pharmaceuticals were used as it or just after dilution with water. From tablets or capsules the colourants were isolated by adsorption on acidic aluminium oxide. The method was used for identification and if possible for quantification the synthetic food dyes in pharmaceuticals. The analytes are detectable at a concentration level 0.3-0.8 microg ml(-1).

Study of O/W micro- and nano-emulsions based on propylene glycol diester as a vehicle for geranic acid.

Nano- and microemulsions containing as the oil phase caprylic/capric propylene glycol diesters (Crodamol PC) were investigated as potential vehicle for controlled release of geranic acid. The influence of emulsifiers and co-surfactants on stability of the emulsions was investigated. Different kind of polysorbates (ethoxylated esters of sorbitan and fatty acids) were applied as the emulsifiers. The short-chain alcohols (ethanol, 1-propanol, 1-butanol) were used as co-surfactants. The emulsions were prepared at ambient temperature (25°C), by the phase inversion composition method (PIC). The stable O/W high dispersed emulsion systems based on Crodamol PC, of mean droplets size less than 200 nm, were prepared. Microemulsions stabilized by the mixture of Polisorbat 80 and 1-butanol were characterized by the largest degree of dispersion (137 nm) and the lowest PDI value (0.094), at surfactant/co-surfactant: oil weight ratio 90:10. The stable nano-emulsion (mean droplet size of 33 nm) was obtained for surfactant: oil (S:O) weight ratio 90:10, without co-surfactant addition. This nano-emulsion was chosen to release studies. The obtained results showed that the prepared stable nano-emulsion can be used as a carrier for controlled release of geranic acid. The active substance release from the nano-emulsion and the oil solution, after 24 hours was 22%.

Mutational analysis of the nf2 tumour suppressor gene in three subtypes of primary human malignant mesotheliomas.

Fourteen primary human malignant mesothelioma (HMM) samples obtained from 14 patients were screened for point mutations and microdeletions/microinsertions in exons 1-16 of the chromosome 22q-located tumour suppressor gene neurofibromin 2 (nf2) by single strand conformation polymorphism (SSCP) analysis. In one tumour (7%) a 10 basepair microdeletion of exon 10 was detected by SSCP and subsequently characterised in detail by sequencing. Deletion of the second nf2 allele in laser-microdissected regions of the 10 bp mutation-harbouring tumour was demonstrated by denaturing gradient gel electrophoresis (DGGE) analysis. Simultaneous comparative genomic hybridisation (CGH) analysis also showed losses at chromosome 22q. Our data indicate that functional loss of the NF2 protein may be involved in the formation of a subset of HMMs.

Pathological anatomy and molecular pathology.

The incidence of malignant mesotheliomas in Germany has increased since about the mid 1980s, and a further increase is expected until about 2020 due to the peak in asbestos processing in Germany between 1965 and 1980. About 90% of the mesotheliomas recorded in the files of the German Mesothelioma Registry in Bochum are asbestos-related and therefore possibly due to an occupational exposure. In 2003, 717 mesotheliomas were newly diagnosed at the German Mesothelioma Registry. Mesotheliomas are very heterogeneous in terms of histological appearances and of prognosis. At present, the diagnostic gold standard is conventional histology in combination with additional immunohistochemical analysis. We were not able to confirm a promising report that described telomerase reverse transcriptase catalytic subunit (TERT) for the differentiation between reactive and neoplastic mesothelial lesions, which can be extremely difficult. DNA cytometric analysis may also help differentiate between reactive and neoplastic mesothelial lesions. There are some characteristic patterns of chromosomal imbalances as detectable by comparative genomic hybridization (CGH), but at present, specific chromosomal or genetic defects that give rise to a mesothelioma are not known. A reliable pathological diagnosis is the basis for therapeutic, prognostic, and medicolegal consequences. In general, it can be achieved by thoracoscopic inspection with specifically directed biopsy. Furthermore, a description of the peculiarities of each mesothelioma by the pathologist might be the key to a more individual therapy in the future.

Development of a capillary electrophoretic method for the analysis of amino acids containing tablets.

Ketosteril is an enteral medicinal product indicated for prevention and therapy in chronic renal insufficiency in connection with a low protein diet. Tablets of Ketosteril contain five essential amino acids like: Lys, His, Thr, Trp, Tyr and another five amino acids in the form of their hydroxy and keto analogues as calcium salts, that are: alpha-ketoleucine, alpha-ketoisoleucine, alpha-ketovaline, alpha-ketophenylalanine and alpha-hydroxymethionine. The composition of Ketosteril tablets is routinely tested with three LC methods. Capillary electrophoretic method seems to be a good alternative for amino acids and their analogues determination in multicomponent pharmaceuticals because of short analysis time and the possibility to assay all components during a single run without any pretreatment. Electrophoresis was performed in 50 microm I.D. fused-silica capillaries with 65 cm distance to the detector. Capillaries were installed in Waters Quanta 4000 electrophoretic equipment with a positive power supply and on-line UV detection at 214 nm. Separations were done in a buffer containing 40 mM Tris and 160 mM boric acid titrated with NaOH to pH 10. The method developed allows the separation of all investigated analytes with an efficiency of n = 230,000 and 20 min analysis time. The method was applied for determination of all components of Ketosteril in commercial tablets.

Water leaching of titanium from ore flotation residue.

Copper ore tailings were tested for the stability of titanium submitted to water leaching in three different reactor systems (agitated vessel, bioreactor and percolated fixed-bed column). For each of these systems, titanium extraction did not exceed 1% of the available metal. Biomass removed from ore residue adsorbed a small part of the titanium with sorption capacities below 20-30 mg g(-1), but most of this biomass was sequestered in the ore residue. Oxygen and carbon dioxide concentrations were monitored and changes in concentration correlated with bacteria development at the initial stage of the process and to fungal development in the latter stages.

Capillary electrophoresis with a diamine EOF modifier as an approach for glutamine dipeptide containing formulations.

Currently, glutamine is thought to be conditionally indispensable amino acid. However, the use of glutamine in clinical practice encounters serious problems related to its limited solubility and instability in water solutions. Therefore, glutamine-containing dipeptides, which have better solubility and stability in the solution, have been introduced into the medical practice. High concentration of L-alanyl-L-glutamine in the medicinal product and thermal sterilization during the manufacturing process are the reasons of many impurities formation--the products of deamidation, racemization, cyclization etc.. The purpose of the study was to develop a single step CE method that could be applied in the purity control of L-alanyl-L-glutamine containing parenteral infusions instead of three routinely used LC methods. After systematic optimization separation conditions were selected: 100 mM borate buffer at pH 8.5 with 0.4 mM diaminopropane as EOF modifier. The method developed allows the separation and determination of the main component and the related impurities. The results show that the method is suitable for quality control of glutamine dipeptide solutions offering some advantages over routinely used LC methods.

Plasma citrulline level as a biomarker for cancer therapy-induced small bowel mucosal damage.

Regimen-related mucosal toxicity is extremely common following cytotoxic chemotherapy and radiotherapy. Mucositis is as an important determinant of the inflammatory response and infectious complications in cancer treated patients. Most assessment scales for mucosal damage are focussed on oral mucositis, since it is easy to evaluate. Measuring gastrointestinal musocal damage objectively remains difficult because it cannot be seen directly or readily detected. One of potential non-invasive biomarkers of gastrointestinal mucosal damage is plasma citrulline level. Citrulline is an amino acid produced by small bowel enterocytes. Low concentration of free circulating citrulline signifies severe intestinal mucosal damage in humans with nonmalignant disorders, such as villous atrophy-associated diseases, short bowel syndrome, Crohn's disease, and is used in follow-up after small bowel transplantation. The plasma citrulline level is a reliable and objective biochemical marker of enterocyte mass and function in humans, and therefore can be used to monitor enterocyte toxicity resulting from chemotherapy and radiotherapy during anticancer therapy in patients with severely disturbed gut integrity.