Protein-engineered molecules carrying GAD65 epitopes and targeting CD35 selectively down-modulate disease-associated human B lymphocytes.

Type 1 diabetes mellitus is an autoimmune metabolic disorder characterized by chronic hyperglycemia, the presence of autoreactive T and B cells and autoantibodies against self-antigens. A membrane-bound enzyme on the pancreatic beta-cells, glutamic acid decarboxylase 65 (GAD65), is one of the main autoantigens in type 1 diabetes. Autoantibodies against GAD65 are potentially involved in beta-cell destruction and decline of pancreatic functions. The human complement receptor type 1 (CD35) on B and T lymphocytes has a suppressive activity on these cells. We hypothesized that it may be possible to eliminate GAD65-specific B cells from type 1 diabetes patients by using chimeric molecules, containing an anti-CD35 antibody, coupled to peptides resembling GAD65 B/T epitopes. These molecules are expected to selectively bind the anti-GAD65 specific B cells by the co-cross-linking of the immunoglobulin receptor and CD35 and to deliver a suppressive signal. Two synthetic peptides derived from GAD65 protein (GAD65 epitopes) and anti-CD35 monoclonal antibody were used for the construction of two chimeras. The immunomodulatory activity of the engineered antibodies was tested in vitro using peripheral blood mononuclear cells (PBMCs) from type 1 diabetes patients. A reduction in the number of anti-GAD65 IgG antibody-secreting plasma cells and increased percentage of apoptotic B lymphocytes was observed after treatment of these PBMCs with the engineered antibodies. The constructed chimeric molecules are able to selectively modulate the activity of GAD65-specific B lymphocytes and the production of anti-GAD65 IgG autoantibodies by co-cross-linking of the inhibitory CD35 and the B cell antigen receptor (BCR). This treatment presents a possible way to alter the autoimmune nature of these cells.

MHC Class II Binding Prediction by Molecular Docking.

Proteins of the Major Histocompatibility Complex (MHC) bind self and nonself peptide antigens or epitopes within the cell and present them at the cell surface for recognition by T cells. All T-cell epitopes are MHC binders but not all MCH binders are T-cell epitopes. The MHC class II proteins are extremely polymorphic. Polymorphic residues cluster in the peptide-binding region and largely determine the MHC's peptide selectivity. The peptide binding site on MHC class II proteins consist of five binding pockets. Using molecular docking, we have modelled the interactions between peptide and MHC class II proteins from locus DRB1. A combinatorial peptide library was generated by mutation of residues at peptide positions which correspond to binding pockets (so called anchor positions). The binding affinities were assessed using different scoring functions. The normalized scoring functions for each amino acid at each anchor position were used to construct quantitative matrices (QM) for MHC class II binding prediction. Models were validated by external test sets comprising 4540 known binders. Eighty percent of the known binders are identified in the best predicted 15 % of all overlapping peptides, originating from one protein.

Different approaches for determination of the attachment degree of polyethylene glycols to poly(anhydride) nanoparticles.

PURPOSE: The aim of the study was to find an appropriate method for determination of the attachment degree of polyethylene glycol (PEG) to poly(anhydride) nanoparticles. METHODS: The nanoparticles were modified with hydroxy-functionalized or with amino-functionalized PEGs. Three methods for their determination were applied and examined: in particular colorimetry, nuclear magnetic resonance ( (1) H-NMR), and elemental analysis (EA). RESULTS: The attachment degrees determined by (1) H-NMR and colorimetry were similar. The associated amounts of PEGs estimated on the basis of EA differed significantly than those determined by colorimetry and (1) H-NMR. CONCLUSION: The colorimetric determination was considered fast and simple technique, but (1) H-NMR contributed to more precise results.

Topical anal fissure treatment: placebo-controlled study of mononitrate and trinitrate therapies.

AIM: The present study aims to evaluate and compare the efficacy of two nitrate gels, containing isosorbide-5-mononitrate (ISMN) or glyceryl trinitrate (GTN), in the therapy of chronic anal fissure. MATERIALS AND METHODS: The patients were randomly assigned to three groups: 0.1% ISMN gel (21 patients), 0.1% GTN gel (21 patients) and a placebo group (ten patients). The ethic committee of our hospital approved the protocol and informed consent was obtained from all participants. All patients underwent clinical examination, visual inspection of the fissure and anal manometry prior to and after therapy. RESULTS: The chronic anal fissure was completely healed in 71% of the patients treated with ISMN, 67% with GTN and in 30% from the placebo group. One patient in the ISMN group reported mild headache. Three patients in the GTN group had anal burning. CONCLUSION: Both topical nitrate treatments (ISMN and GTN) were effective for chronic anal fissures. The reduction of the anal pressure was slightly higher after ISMN treatment (28%) than the treatment with GTN (23%). However, the statistical difference was not significant (p>0.05).

Towards the in silico identification of class II restricted T-cell epitopes: a partial least squares iterative self-consistent algorithm for affinity prediction.

MOTIVATION: The immunogenicity of peptides depends on their ability to bind to MHC molecules. MHC binding affinity prediction methods can save significant amounts of experimental work. The class II MHC binding site is open at both ends, making epitope prediction difficult because of the multiple binding ability of long peptides. RESULTS: An iterative self-consistent partial least squares (PLS)-based additive method was applied to a set of 66 peptides no longer than 16 amino acids, binding to DRB1*0401. A regression equation containing the quantitative contributions of the amino acids at each of the nine positions was generated. Its predictability was tested using two external test sets which gave r(pred) = 0.593 and r(pred) = 0.655, respectively. Furthermore, it was benchmarked using 25 known T-cell epitopes restricted by DRB1*0401 and we compared our results with four other online predictive methods. The additive method showed the best result finding 24 of the 25 T-cell epitopes. AVAILABILITY: Peptides used in the study are available from http://www.jenner.ac.uk/JenPep. The PLS method is available commercially in the SYBYL molecular modelling software package. The final model for affinity prediction of peptides binding to DRB1*0401 molecule is available at http://www.jenner.ac.uk/MHCPred. Models developed for DRB1*0101 and DRB1*0701 also are available in MHCPred.

Adenosine A2A receptor agonists: CoMFA-based selection of the most predictive conformation.

A step-wise comparative molecular field analysis (CoMFA)-based procedure was applied to a series of 51 2-oxyadenosines in order to select the most predictive conformation for binding to A2A adenosine receptor (AR). The highest correlation and predictive power were found for conformers with side chain at 2nd position oriented in the direction opposite to the exocyclic amino group on the adenine ring (torsion N1C2OR = 120 degrees) and fully extended. The interaction of ligand and receptor is under steric and electrostatic control. The steric contribution is of a greater importance for the predictivity than the electrostatic one. Hydrophobicity of the compounds investigated does not affect significantly either the affinity to A2A AR, nor the predictivity of the models.

Toward the quantitative prediction of T-cell epitopes: coMFA and coMSIA studies of peptides with affinity for the class I MHC molecule HLA-A*0201.

A set of 102 peptides with affinity for the class I MHC HLA-A0201 molecule was subjected to three-dimensional quantitative structure-affinity relationship (3D QSAR) studies using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). A test set of 50 peptides was used to determine the predictive value of the models. The CoMFA models gave q(2) and r(2)pred below 0.5. The best CoMSIA model has q(2) = 0.542 and r(2)pred = 0.679, and includes hydrophobic, steric, and H-bond donor fields. The hydrophobic interactions play a dominant role in peptide-MHC molecule binding. CoMSIA coefficient contour maps were used to analyze the structural features of the peptides accounting for the affinity in terms of the three positively contributing physicochemical properties: local hydrophobicity, steric bulk and hydrogen-bond-donor ability.

CoMFA-based comparison of two models of binding site on adenosine A1 receptor.

A set of 32 N6-substituted adenosines and 22 8-substituted xanthines with affinity for adenosine A1 receptors was subjected to three-dimensional quantitative structure-affinity relationship analysis using comparative molecular field analysis (CoMFA). The aim was to compare two modes of binding to the receptor--'N6-C8' and 'N6-N7'. Good models with high predictive power and stability were obtained. A comparison of these models gives the following results: (a) Inclusion of both steric and electrostatic fields in CoMFA generates better predictive models compared to models based on steric or electrostatic fields alone. (b) The 'N6-N7' CoMFA models are slightly better than the 'N6-C8' ones. (c) Steric restriction exists around the N6-H in the 'N6-N7' steric field map, which is absent in the 'N6-C8' steric field map. This report demonstrates that the 'N6-N7' mode of binding is a further development of the 'N6-C8' model with a slightly better predictive ability and more accurate steric and electrostatic overlaps between agonists and antagonists.

Reversed-phase high-performance liquid chromatography for evaluating the distribution of pharmaceutical substances in suppository base-phosphate buffer system.

The aim of this study was to assess the potency of the reversed-phase high-performance liquid chromatography (RP-HPLC) for in vitro evaluation of the distribution behavior of common drugs between one of the generally used suppository bases Witepsol H15 and the rectal liquid which is imitated by a phosphate buffer, pH 7.2. The distribution coefficients (log K) of nine compounds--paracetamol, caffeine, diclofenac, propyphenazone, indomethacin, codeine base, codeine phosphate, phenobarbital acid and phenobarbital sodium salt were determined by the classical shake-flask' method followed by RP-HPLC quantitative assay. The capacity factors log k' of the compounds were determined on reversed-phase C18 column at a number of methanol-5 mM phosphate buffer, pH 7.2 mobile phases containing different percentages of methanol (phiMeOH). The apparent capacity factors log k(w)app were derived by extrapolation of the methanol concentration to zero and using the correction for ionization, the real capacity factors log k'(w) were calculated. The lipophilicity of the compounds was assessed by the partition coefficients CLOGP and the distribution coefficients CLOGD7.2, calculated for the n-octanol-water system. Correlations between log k'(w) and CLOGP, log k(w)app and CLOGD7.2, log k(w)app and log K were found. The last correlation indicated that the parameter log k(w)app was suitable for evaluating the distribution behavior of the studied drugs in the examined Witepsol H15-rectal liquid system. The predictive power of this correlation was tested by a set of nine non-congeners. It was shown that the classical 'shake-flask' method for determination of the distribution behavior of the studied drugs between the suppository base Witepsol H15 and the phosphate buffer, pH 7.2 might be replaced by the RP-HPLC technique due to its priorities of rapid, stable and reproducible experiments.

2D and 3D QSAR analysis of some valproic acid metabolites and analogues as anticonvulsant agents.

PURPOSE: To investigate the structural features, responsible for the variations in anticonvulsant activity of a series of twenty six valproic acid (VPA) metabolites and analogues. METHODS: Different approaches for quantitative structure--activity relationship analysis (QSAR) as conventional 2D QSAR analysis and comparative molecular field analysis (3D QSAR) were used. The 2D QSAR was performed with more than twenty structure descriptors as the partition and distribution coefficients, topological, geometrical and electronic descriptors, and indicator variables. The electronic descriptors were calculated for the energetically most stable conformers. For the need of 3D QSAR steric and electrostatic potential maps were generated. Partial least squares (PLS) analysis has been carried out for the statistical evaluation of the models and weighted least squares (WLS) analysis was used for the visualization of the results. RESULTS: It was established that the two approaches--2D and 3D QSAR, prove the importance of the lipophilicity of the compounds for anticonvulsant activity. The results from both the approaches suggest that a substitution at alpha-position is essential for a higher activity. CONCLUSIONS: 3D QSAR is useful for describing the steric and electrostatic fields, important for the activity. For predicting the activity of new compounds 2D QSAR tools were proposed.

Biodegradable cross-linked prodrug of the bronchial dilator Vephylline. 2. Kinetics and quantum chemical studies on the release mechanism.

Experimental thermodynamics studies and quantum chemical reaction path calculations on the hydrolytic degradation of Poly-vephyllinemalate microspheres in acidic and basic media were performed. It was possible to make a conclusion on the release mechanism of free Vephylline as follows: a hydrolytic cleavage of the ester bonds between molecular fragments of R,S-malic acid takes place and leads to a soluble oligoester fraction. Then, further hydrolysis of the ester bonds between the xanthine fragment and R, S-malic acid leads to the release of Vephylline as free base. The hydrolytic process takes place in acidic solution with rapid degradation of the ester bonds between the malic acid monomers and by far slower degradation of the ester bonds between the malic acid and Vephylline. In basic solution both steps of the hydrolysis are fast processes leading to a complete release of free Vephylline within 1 h. The process of Vephylline release is under entropic control. The experimental results are well correlated to the results obtained after kinetics investigation and after AM1 quantum chemically calculated energy barriers in the reaction path leading to the tetrahedral intermediates of the hydrolytic reactions. This conclusion is in good accordance with an indirect study on the release mechanism of Vephylline from its polymeric prodrug, paying attention to the biological response, reported previously.