Ákos Végvári

Multiple sclerosis: Identification and clinical evaluation of novel CSF biomarkers.

Multiple sclerosis (MS) is a neuro-inflammatory and neurodegenerative disease that results in damage to myelin sheaths and axons in the central nervous system and which preferentially affects young adults. We performed a proteomics-based biomarker discovery study in which cerebrospinal fluid (CSF) from MS and control individuals was analyzed (n=112). Ten candidate biomarkers were selected for evaluation by quantitative immunoassay using an independent cohort of MS and control subjects (n=209). In relapsing-remitting MS (RRMS) patients there were significant increases in the CSF levels of alpha-1 antichymotrypsin (A1AC), alpha-1 macroglobulin (A2MG) and fibulin 1 as compared to control subjects. In secondary progressive MS (SPMS) four additional proteins (contactin 1, fetuin A, vitamin D binding protein and angiotensinogen (ANGT)) were increased as compared to control subjects. In particular, ANGT was increased 3-fold in SPMS, indicating a potential as biomarker of disease progression in MS. In PPMS, A1AC and A2MG exhibit significantly higher CSF levels than controls, with a trend of increase for ANGT. Classification models based on the biomarker panel could identify 70% of the RRMS and 80% of the SPMS patients correctly. Further evaluation was conducted in a pilot study of CSF from RRMS patients (n=36), before and after treatment with natalizumab.

Chiral separation of amino acids by ligand‐exchange capillary electrochromatography using continuous beds

A chiral ligand‐exchange phase for capillary electrochromatography based on continuous bed technology was developed. The chiral stationary phase is prepared by a one‐step in situ copolymerization procedure using methacrylamide, piperazine diacrylamide, vinylsulfonic acid and N‐(2‐hydroxy‐3‐allyloxypropyl)‐L‐4‐hydroxyproline. These chiral continuous beds are inexpensive and easy to prepare. They also have several advantages over silica‐based packed capillaries. Since the bed is covalently attached to the capillary wall, no frit is required. The applicability of this new approach to the chiral separation of underivatized amino acids is demonstrated.

Drug Localization in Different Lung Cancer Phenotypes by MALDI Mass Spectrometry Imaging

Lung cancer is a common cause of cancer mortality in the world, largely due to the risk factor of tobacco smoking. The drug therapy at the molecular level includes targeting the epidermal growth factor receptor (EGFR) tyrosine kinase activity by using inhibitors, such as erlotinib (Tarceva) and gefitinib (Iressa). The heterogeneity of disease phenotypes and the somatic mutations presented in patient populations have a great impact on the efficacy of treatments using targeted personalized medicine. In this study, we report on basic physical and chemical properties of erlotinib and gefitinib in three different lung cancer tumor phenotypes, using MALDI instrumentation in imaging mode, providing spatial localization of drugs without chemical labeling. Erlotinib and gefitinib were analyzed in i) planocellular lung carcinoma, ii) adenocarcinoma and iii) large cell lung carcinoma following their deposition on the tissue surfaces by piezo-dispensing, using a controlled procedure. The importance of high-resolution sampling was crucial in order to accurately localize the EGFR tyrosine kinase inhibitors deposited in heterogeneous cancer tissue compartments. This is the first report on personalized drug characterization with localizations at a lateral resolution of 30μm, which allowed us to map these compounds at attomolar concentrations within the lung tumor tissue microenvironments.

A new easy-to-prepare homogeneous continuous electrochromatographic bed for enantiomer recognition

Completely homogeneous polyacrylamide‐based gels were used for capillary electrochromatography (CEC) of drug enantiomers. Like continuous beds (also called continuous polymer rods, silica rods, monoliths) they do not require frits to support the bed because it is covalently linked to the capillary wall. A long lifetime is an important feature of the beds. The gel matrices can be prepared in any laboratory and for specific interactions they can be derivatized with appropriate ligands. The application range is, therefore, broad. For chiral electrochromatography, negatively and positively charged polyacrylamide gels copolymerized with 2‐hydroxy‐3‐allyloxy‐propyl‐β‐cyclodextrin (allyl‐β‐CD) were prepared. The latter monomer was synthesized from β‐CD and allylglycidyl ether by a very simple one‐step procedure. Eight acidic, neutral and basic drug compounds were resolved into their enantiomers, most of them with baseline separation. Interestingly, the resolution is independent of the electroendosmotic velocity, i.e., rapid analyses will not give low resolution. Upon increasing this velocity, the plate height for the fast enantiomer did not change (or decreased slightly), whereas that for the slow enantiomer increased. Only the last term in the van Deemter equation contributed significantly to the total plate height. The composition of the gel was chosen such that the „pores”︁ became large enough to guarantee a satisfactory electroendosmotic flow (EOF). This open gel structure explains why acetone diffused as in free solution, i.e., independently of the presence of the gel matrix. This finding also indicates that the separation of small molecules in polyacrylamide gels cannot be explained by „molecular‐sieving”︁, but rather by some type of adsorption (”︁aromatic adsorption”︁?).

Direct demonstration of tissue uptake of an inhaled drug: proof-of-principle study using matrix assisted laser desorption ionization mass spectrometry imaging

Drug therapy is often directed to specific organ and tissue compartments where the mode of action of the compound affects specifically targeted biological processes. However, the direct measurement of drug uptake in terms of a time kinetic and concentrations attained at the local sites has not been readily available as a clinical index for most drugs. A proof-of-principle study was conducted to test the utility of applying matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) to demonstrate the qualitative distribution pattern of a locally administered drug within tissue sites of targeted action. Here we have measured the occurrence of an inhaled bronchodilator, the muscarinic receptor antagonist ipratropium, within human bronchial biopsies obtained by fiber optic bronchoscopy shortly after dosing exposure. Cryo-preserved biopsy samples from five subjects being evaluated for airway obstruction or potential tumor development were prepared as thin frozen sections. Samples coated with a MALDI matrix were analyzed by a MALDI LTQ Orbitrap XL mass spectrometer at large (100 μm) and small (30 μm) raster sizes. Our results demonstrate that ipratropium is rapidly absorbed into the airway wall. Ipratropium parent ion (m/z 332.332) and daughter ions (m/z 166.2 and 290.2) were coincidently partitioned within submucosal spaces containing targeted airway smooth muscle in four out of five subjects. The signal intensity of ipratropium fragment ions provided estimates that local drug concentrations between 3 and 80 nM were achieved within the airway wall. To our knowledge, this is the first reported study in applying MALDI-MSI to demonstrate the localization of a drug administered at therapeutic levels. The study highlights the potential benefit of MALDI-MSI to provide important measurements of drug efficacy in clinical settings.

(Normal-Phase) Capillary Chromatography Using Acrylic Polymer-Based Continuous Beds

Microchromatographic separations of polar aromatic compounds (pyridine, 4-pyridylmethanol, 4-methoxyphenol, 2-naphthol, catechol, hydroquinone, resorcinol, 2,7-dihydroxynaphthalene) using continuous beds are described. The columns were prepared by a simple one-step in situ polymerization procedure: a solution of acrylic monomers, including the cross-linking agent piperazine diacrylamide, was polymerized in a fused-silica capillary pretreated with 3-(trimetoxysilyl) propyl methacrylate. The continuous bed formed contained a network of channels and was attached covalently to the wall of the silica capillary (100 mm I.D.) via its methacrylate groups. Therefore, the frit used in conventional, packed columns could be omitted. The separation mechanism is discussed, particularly with regard to whether the so-called aromatic adsorption to the matrix itself is involved, an interaction first described by Gelotte [1] (the ligands, isopropyl and sulfonate groups, are not required for separation). This discussion is relevant to the question of whether the separation technique described should be classified as normal-phase or adsorption chromatography. The mobile phase from the HPLC pump was split via an open capillary to get a flow rate through the continuous bed of about 100 nl /min. The beds were tested up to a pressure of 150 bar (8.8 bar /cm). A continuous bed synthesized at a relatively low molar fraction of the cross-linker in the monomer mixture (16.5%) and high total concentration of the monomers (31.9% (w/v)) afforded the highest efficiency for the separation of the polar 21 organic compounds. Plate numbers up to 150 000 m were obtained and the run-to-run reproducibility was high. The selectivity of the separations was adjusted by changing the composition of the mobile phase (hexane–ethanol–methanol). The sample was applied by a diffusion-based injection technique. (Less)

Enantioseparation of hydroxy acids on easy-to-prepare continuous beds for capillary electrochromatography.

This paper deals with the enantioseparation of hydroxy acids by ligand‐exchange capillary electrochromatography. A chiral continuous bed was easily prepared by in situ polymerization of monomers, including an L‐4‐hydroxyproline derivative. This phase showed chiral recognition for several hydroxy acids, in addition to amino acids.

Identification of novel candidate protein biomarkers for the post-polio syndrome - implications for diagnosis, neurodegeneration and neuroinflammation.

Survivors of poliomyelitis often develop increased or new symptoms decades after the acute infection, a condition known as post-polio syndrome (PPS). The condition affects 20-60% of previous polio patients, making it one of the most common causes of neurological deficits worldwide. The underlying pathogenesis is not fully understood and accurate diagnosis is not feasible. Herein we investigated whether it was possible to identify proteomic profile aberrations in the cerebrospinal fluid (CSF) of PPS patients. CSF from 15 patients with well-defined PPS were analyzed for protein expression profiles. The results were compared to data obtained from nine healthy controls and 34 patients with other non-inflammatory diseases which served as negative controls. In addition, 17 samples from persons with secondary progressive multiple sclerosis (SPMS) were added as relevant age-matched references for the PPS samples. The CSF of persons with PPS displayed a disease-specific and highly predictive (p=0.0017) differential expression of five distinct proteins: gelsolin, hemopexin, peptidylglycine alpha-amidating monooxygenase, glutathione synthetase and kallikrein 6, respectively, in comparison with the control groups. An independent ELISA confirmed the increase of kallikrein 6. We suggest that these five proteins should be further evaluated as candidate biomarkers for the diagnosis and development of new therapies for PPS patients.

Biobank resources for future patient care: developments, principles and concepts

The aim of the overview is to give a perspective of global biobank development is given in a view of positioning biobanking as a key resource for healthcare to identify new potential markers that can be used in patient diagnosis and complement the targeted personalized drug treatment. The fast progression of biobanks around the world is becoming an important resource for society where the patient benefit is in the focus, with a high degree of personal integrity and ethical standard. Biobanks are providing patient benefits by large scale screening studies, generating large database repositories. It is envisioned by all participating stakeholders that the biobank initiatives will become the future gateway to discover new frontiers within life science and patient care. There is a great importance of biobank establishment globally, as biobanks has been identified as a key area for development in order to speed up the discovery and development of new drugs and protein biomarker diagnostics. One of the major objectives in Europe is to establish concerted actions, where biobank networks are being developed in order to combine and have the opportunity to share and build new science and understanding from complex disease biology. These networks are currently building bridges to facilitate the establishments of best practice and standardizations.

Developments in biobanking workflow standardization providing sample integrity and stability

UNLABELLED Recommendations and outlines for standardization in biobanking processes are presented by a research team with long-term experience in clinical studies. These processes have important bearing on the use of samples in developing assays. These measurements are useful to document states of health and disease that are beneficial for academic research, commercial healthcare, drug development industry and government regulating agencies. There is a need for increasing awareness within proteomic and genomic communities regarding the basic concepts of collecting, storing and utilizing clinical samples. Quality control and sample suitability for analysis need to be documented and validated to ensure data integrity and establish contexts for interpretation of results. Standardized methods in proteomics and genomics are required to be practiced throughout the community allowing datasets to be comparable and shared for analysis. For example, sample processing of thousands of clinical samples, performed in 384 high-density sample tube systems in a fully automated workflow, preserves sample content and is presented showing validation criteria. Large studies will be accompanied by biological and molecular information with corresponding clinical records from patients and healthy donors. These developments position biobanks of human patient samples as an increasingly recognized major asset in disease research, future drug development and within patient care. BIOLOGICAL SIGNIFICANCE The current manuscript is of major relevance to the proteomic and genomic fields, as it outlines the standardization aspects of biobanking and the requirements that are needed to run future clinical studies that will benefit the patients where OMICS science will play a major role. A global view of the field is given where best practice and conventional acceptances are presented along with ongoing large-scale biobanking projects. The authors represent broadly stakeholders that cover the academic, pharma, biotech and healthcare fields with extensive experience and deliveries. This contribution will be a milestone paper to the proteomic and genomic scientists to present data in the future that will have impact to the life science area. This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics.