[Selective depletion of alloreactive T cells and study of anti-tumor activity of specific T cell clones in patients with leukemia]. / Selektivní deplece aloreaktivních T lymfocytu a studium protinádorové aktivity specifických klonu T lymfocytu u pacientu s leuikémií.

BACKGROUND: Graft-versus-host disease (GVHD) is a severe complication of allogeneic transplantation of hematopoietic stem cells. Donor T cells play a major role in GVHD leading to the host tissue damage, mainly the skin, liver, and gastrointestinal tract. A selective depletion using an anti-CD25 immunotoxin can eliminate harmful alloreactive T cells while preserving other donor T cells with antileukemic and antiinfectious reactivity. PATIENTS AND METHODS: We performed 15 mixed lymphocyte reactions with clinical specimens from 12 patients with various types of leukemia (7x AML, 3x ALL, 1x CML, 1x CLL) and PBMC from 15 healthy volunteers from Transfusive station FN Brno Bohunice. RESULTS: In our experiments we have demonstrated, that antileukemic (GVL) effect of donor, especially CD4+ T cells was well preserved (7.46%), while unfavourable alloreactive (GVH) reaction of donor T cells was completely removed. The graft-versus-host (GVH) reactivation of donor cells was negligible ever after repeated stimulation with irradiated patient's PBMC. CONCLUSION: We have shown that anti-CD25 immunotoxin (IT), RFT5-SMPT-dgA, launched against alpha chain for human interleukin 2 (IL-2), led to long-term selective depletion of alloreactive donor T cell clones while their antileukemic activity was well preserved. Base on our results the clinical phase I/II study was designed. This study was initiated in year 2007 in three clinical centers in Czech Republic.

Comparison of zirconia- and silica-based reversed stationary phases for separation of enkephalins.

In this study, the separation of biologically active peptides on two zirconia-based phases, polybutadiene (PBD)-ZrO2 and polystyrene (PS)-ZrO2, and a silica-based phase C18 was compared. Basic differences in interactions on both types of phases led to quite different selectivity. The retention characteristics were investigated in detail using a variety of organic modifiers, buffers, and temperatures. These parameters affected retention, separation efficiency, resolution and symmetry of peaks. Separation systems consisting of Discovery PBD-Zr column and mobile phase composed of a mixture of acetonitrile and phosphate buffer, pH 2.0 (45:55, v/v) at 70 degrees C and Discovery PS-Zr with acetonitrile and phosphate buffer, pH 3.5 in the same (v/v) ratio at 40 degrees C were suitable for a good resolution of enkephalin related peptides. Mobile phase composed of acetonitrile and phosphate buffer, pH 5.0 (22:78, v/v) was appropriate for separation of enkephalins on Supelcosil C18 stationary phase.

Chiral separation of beta-adrenergic antagonists, profen non-steroidal anti-inflammatory drugs and chlorophenoxypropionic acid herbicides using teicoplanin as the chiral selector in capillary liquid chromatography.

Three groups of structurally diverse chiral compounds were used to study the interaction mechanism responsible for stereoselective recognition with teicoplanin as chiral selector in capillary liquid chromatography. Teicoplanin-based chiral stationary phase (CSP) was used. The effect of the variation of mobile phase composition on retention and enantioselective separation was studied. The mobile phase composition suitable for enantioresolution of the various chiral compounds differed according to the interaction forces needed for chiral recognition. Mobile phases with high buffer portion (70-90 vol.%) were preferred for separation of enantiomers of profen non-steroidal anti-inflammatory drugs and chlorophenoxypropionic acid herbicides that require hydrophobic interactions, inclusion and pi-pi interactions for stereoselective recognition with teicoplanin. Higher concentration triethylamine in the buffer (0.5-1.0%) increased resolution of these acids. On the other hand, H-bonding and electrostatic interactions are important in stereoselective interaction mechanism of beta-adrenergic antagonists with teicoplanin. These interaction types predominate in the reversed phase separation mode with high organic modifier content (95% methanol) and in polar organic mobile phases. For this reason beta-adrenergic antagonists were best enantioresolved in the polar organic mode. The mobile phase composed of methanol/acetic acid/triethylamine, 100/0.01/0.01 (v/v/v), provided enantioresolution values of all the studied beta-adrenergic antagonists in the range 1.1-1.9. Addition of teicoplanin to the mobile phase, which was suitable for enantioseparation of certain compounds on the CSP, was also investigated. This system was used to dispose of nonstereoselective interactions of analytes with silica gel support that often participate in the interaction with CSPs. Very low concentration of teicoplanin in the mobile phase (0.1 mM) resulted in enantioselective separation of 2,2- and 2,4-chlorophenoxypropionic acids.

Comparison of vancomycin-based stationary phases with different chiral selector coverage for enantioselective separation of selected drugs in high-performance liquid chromatography.

Two vancomycin-based chiral stationary phases (CSPs) with different coverage of the chiral selector vancomycin (Chirobiotic V and Chirobiotic V2) were compared. beta-Blockers and profens, as structurally diverse groups of drugs, were chosen as analytes. Retention and enantioseparation of beta-blockers were studied in reversed-phase (RP) and polar-organic (PO) separation modes. Higher retention and better enantioresolution were obtained on the CSP with higher coverage of vancomycin in the both separation modes. Baseline separation of four beta-blockers (eight enantiomers) in the PO mode was achieved on the Chirobiotic V2 column within 15 min. The enantioseparation of profens did not bring so excellent and easy to interpret results. Higher retention of profens on the Chirobiotic V2 column was not always accompanied by an improvement of their chiral separation in the RP mode. The polar-organic mode was not suitable for these derivatives at all. The most interesting result was obtained with flobufen; its chiral center is further away from the rigid part of the molecule, which mostly causes difficulties in enantioselective recognition. Nevertheless, the enantiomers of flobufen were shown to be much better (baseline) resolved on the CSP with lower coverage of the chiral selector (Chirobiotic V).

Study on the aggregation of teicoplanin.

The aggregation of teicoplanin was studied in four solutions: 0.06 M phosphate buffers pH 4.3 and 6.3, both with and without 10% (v/v) of acetonitrile. Conductometry, capillary electrophoresis, cyclic voltammetry, and static light scattering were employed to determine the critical micelle concentration (CMC) of teicoplanin. Dynamic light scattering was used to give an additional information on the size and the size distribution of the particles formed. While the CMC was found in solutions without acetonitrile, attempts to detect any CMC failed in solutions containing acetonitrile. The results point out to different solvating mechanism in solutions with and without acetonitrile, leading to two different schemes of association.

Enantioseparation of selected N-tert.-butyloxycarbonyl amino acids in high-performance liquid chromatography and capillary electrophoresis with a teicoplanin chiral selector.

Enantioseparation of N-tert.-butyloxycarbonyl amino acids (N-t-Boc-Aas) with teicoplanin chiral selector was performed in two different separation systems: A teicoplanin-based chiral stationary phase (CSP-TE) was used in reversed-phase HPLC, and the same chiral selector (CS) was added into a background electrolyte (BGE) in HPCE. The enantioselective interaction with the same CSP/CS can be influenced by several factors, such as mobile phase/background electrolyte composition: the buffer concentration, pH, the CS concentration, the presence of organic modifiers. In addition, the charge of the chiral selector related to the charge of the analyte and to EOF are important variables in CE. The effect of these parameters on enantioselectivity and enantioseparation of selected N-t-Boc-Aas was studied. The presence of a sufficient concentration (1% solution) of a triethylamine acetate buffer in the mobile phase was shown to be essential for enantioseparation of these blocked amino acids in HPLC. A certain concentration of teicoplanin aggregates (along with teicoplanin molecules) in the BGE is required to obtain enantioseparation of N-t-Boc-Aas in HPCE.

Capillary electrokinetic chromatography with charged linear polymers as a nonmicellar pseudostationary phase: determination of capacity factors and characterization by solvation parameters.

A permanent polycation, polydiallyldimethylammonium (PDADMA), is applied as a linear, polymeric, replaceable, and nonmicellar pseudostationary phase for the separation of neutral analytes by capillary electrokinetic chromatography. It is shown that this polymer used in the background electrolyte is able to separate the analytes even if it does not form micelles under the given conditions. The most favorable aspect for practical use lays in the simple replacement of the separation media after each run, thus generating highly reproducible conditions. To determine the capacity factors of the analytes, a new method, based on an isotachophoretic regime, has been introduced for the measurement of the electrophoretic mobility of the polymeric pseudo-stationary phase. The capacity factors in the separation system, derived from the mobilities of the polymer, the electroosmotic flow, and the mobilities of 15 individual aromatic analytes, range between 0.3 and 1.2 for the given separation media (aqueous solution of acetate buffer, pH 5.2, with 4% w/w PDADMA). The type of interaction in the pseudochromatographic system was clarified from solvation parameters based on the linear free energy relationship model. It was found that pi and n electron interactions and hydrogen-bond basicity of the polymer, as compared with the aqueous bulk phase, are the main cause of retention of the analytes.

Physical factors negatively affecting evaluation of long-term biodegradation experiments of polychlorinated biphenyls.

Behaviour of PCB added in the form of a technical mixture (Delor 106) and [14C]PCB 77 into different aqueous model cultivation media in long-term experiments was studied. The work was focused on monitoring PCB recovery from liquid media in the absence of micro-organisms. GC-ECD and radioanalysis were utilised for determination of PCB in the examined systems. Strong relations between duration of experiments, composition of media, degree of chlorination of biphenyls, and recovery of PCB were found. The lowest yields of PCB were observed for the extracts from model solutions based on N-limited cultivation media without veratryl alcohol and Tween 80. The addition of these components, mainly of Tween 80, caused a significant increase of PCB recoveries.

Enantiomer separation of dihydropyridine calcium antagonists with cyclodextrins as chiral selectors: structural correlation.

Native and substituted cyclodextrins (CDs) were used as chiral selectors both in high-performance liquid chromatography and capillary electromigration separations (HPCE and MEKC). Chromatographic data of five dihydropyridine calcium antagonists obtained on three beta-CD chiral stationary phases in reversed-phase mode were compared with those of capillary electrophoresis using beta-CDs in the presence and absence of sodium dodecyl sulfate (SDS). Competition of separated compounds with SDS molecules for penetration into the CD cavity can limit their necessary interaction with the chiral selector and consequently even preclude enantiomer separation. Some insight into this problem can be brought about by comparing the experimental data with computer-aided energy minimization of CD-solute and CD-SDS inclusion complexes.

Separation of type IX collagen from other cartilage collagens by hydrophobic interaction chromatography.

Collagen type IX was separated from other cartilage collagens (types II and XI) by hydrophobic interaction chromatography on a 25 cm X 8 mm I.D. stainless-steel column packed with Separon HEMA 1000 Bio. The mobile phase was 0.84 M ammonium sulphate with 0.1 M potassium dihydrogenphosphate (pH 6.5). Under these conditions only collagen type IX was eluted from the column; it could be monitored with UV detection (218 nm) or selectively with fluorescence detection (excitation 330 nm, emission filter 389 nm). The method can be used for the isolation and quantitation of collagen type IX. The assay was linear in the range 0-10 micrograms, the correlation coefficient was 0.99, precision 5.5% and accuracy 13%. The detection limit was about 0.6 microgram.