A portable optical waveguide resonance light-scattering scanner for microarray detection

A portable and low-cost planar waveguide based RLS microarray scanner was successfully fabricated to analyze 15 T2DM patient genotypes simultaneously.

Screening for Potential HLA-DRB1-Restricted T-Cell Epitopes in Factor VIII Using Peptide Microarrays

Therapeutic factor VIII (FVIII) infusions provoke CD4 T-cell proliferation and cytokine secretion leading to neutralizing anti-FVIII antibodies (inhibitors) in a substantial fraction of hemophilia A patients, leading to bleeding that is difficult to control. T-cell stimulation occurs when FVIII peptides bind to HLA-DRB1 proteins and are presented on the surface of antigen presenting cells, followed by T-cell recognition of these peptide-HLA-DRB1 complexes. Our laboratory is interested in identifying immunodominant, HLA-restricted T-cell epitopes in FVIII, in order to identify and then modify these immunological “hotspots” that cause inhibitors to develop. We have previously mapped several immunodominant HLA-restricted T-cell epitopes in FVIII recognized by the immune systems of inhibitor subjects, using HLA-DRB1 tetramers loaded with 20-mer FVIII peptides. However, because FVIII is such a large protein (>2,000 amino acid residues), methods more efficient than systematic mapping using overlapping peptides spanning its sequence would be useful. This study focuses on the binding of FVIII peptides to recombinant extracellular domains of 10 HLA-DRB1 proteins, which together represent HLA-DRB1 alleles found in >50% of the U.S population. PepStarTM (JPT Peptide Technologies) microarrays containing triplicate sets of FVIII peptides immobilized on glass slides via a flexible linker were incubated with HLA-DRB1 proteins at several dilutions, followed by incubation with a fluorescent-labeled antibody that recognizes the peptide-bound conformations of HLA-DRB1 proteins, and the slides were washed and read on a microarray scanner. The intensities of fluorescent signals from the triplicate sets of spots were quantified and also displayed as heat maps. Fluorescent signals ranged from background levels (indicating no binding) up to the detector saturation range. Between 20-40% of the FVIII peptides produced fluorescent signals indicating peptide binding to each of the HLA-DRB1 proteins tested, including positive control peptides containing FVIII and non-FVIII epitopes with known HLA-DRB1 restriction. The percentage of apparently high-affinity binding interactions was much lower. T-cell epitopes are generally 10-14 amino acid residues long. Therefore, strong signals from pairs of 20-mer peptides with a 12-residue overlapping sequence localized the FVIII sequence fitting into the HLA-DRB1 binding groove to the overlapping region, while disparate signals from other pairs of overlapping sequences localized the HLA-DRB1 binding sequence to the non-overlapping regions. For subsets of the FVIII peptides, the signal strengths were compared to (a) affinities (IC50 values) derived from quantitative, competition peptide-HLA-DRB1 binding assays, and (b) results of computer prediction algorithms MHCPanII and Propred. Peptides that bound strongly to one or more HLA-DRB1 protein were prioritized for further testing. This microarray-based screening procedure is an efficient method to generate manageable lists of potential T-cell epitopes in large antigenic proteins such as FVIII, where systematic epitope mapping using HLA-DRB1 tetramers loaded with peptides spanning the entire sequence is not feasible, due to both the high cost of such experiments and the blood volumes that can reasonably be requested from patient volunteers. Disclosures No relevant conflicts of interest to declare.

Do Leukemic Cells and Mesenchymal Stem Cells (MSCs) From AML Patients Share The Same Chromosomal Defect? A Cytogenetics, FISH and aCGH/Snpa Study

Recent evidence suggests that leukemia is not solely a cancer autonomous process, but rather a disease in which the bone marrow microenvironment, the niche, plays a crucial role too (Raaijmakers, 2011). MSCs are key component of the niche. Thus, several studies have tested whether these cells from haematological patients contain chromosomal defects identical or different from those present in leukemic cells. Based on these findings the principal aim of the present study was to evaluate whether leukemic and MSC from six AML patients shared the same cytogenetic defects after examination with three different technologies, conventional cytogenetics (CC), FISH and aCGH/SNPa. At the onset of the disease and after informed consent all the six patients were submitted to bone marrow (BM) aspiration. BM cells were submitted to CC and FISH analyses. In addition, MSC were isolated from BM cell suspension (10-15 ml) as previously described. Briefly, mononucleated cells were isolated from BM by density gradient centrifugation using Lympholyte®-H and seeded in 75 cm2 cell culture flasks at a cell density of 106 cells/cm2. Cells were cultured at 37°C, 5% CO2 in MEM-alpha medium containing 1% Penicillin/Streptomycin, 1% L-Glutamine and 10% fetal bovine serum. After 48-h adhesion, non-adherent cells were removed and culture medium replaced (Achille et al, 2011). Growth medium was changed every three days. MSCs were examined after the first passage and their phenotype was evaluated by flow cytometry. Cells were detached from culture using Tripsin-EDTA, washed twice with PBS and stained for ten minutes with the following fluorochrome-conjugated antibodies: anti-CD90-FITC, anti-CD105-PE, anti-CD14-FITC, anti-CD73-PE, anti-CD34-FITC, anti-CD80-PE, anti-CD133-APC, anti-CD31-PE and anti-CD45-APC-Alexa750. Stained cells were acquired with a Beckman Coulter Navios instrument and data analyzed with Kalooza software. The commercial FISH probes used were LSI D7S486/CEP7, LSI AMLETO from Abbot Molecular Inc. (Chicago, Il, USA) and ON c-Myc/SE8, SE10(D10Z1) from Kreatech (Amsterdam, NL). These probes were applied according to manufactures guidelines and cut-off values determined by applying a one-sided 95% confidence interval using a binomial distribution. aCGH/SNPa was carried out with the SureScan Microarray Scanner G4900DA (Agilent Technologies Inc. Santa Clara, CA). CC revealed a monosomy 7 in two patients, a del(7)(q31) in one, a trisomy 8 and a trisomy 10 in one patient each, a t(8;21)(q24,q22) translocation in the last patient. All these defects were confirmed by FISH. In order to establish whether leukemic cells and MSCs shared these same abnormalities, MSCs cultures were tested with FISH. MSC purity assessed by flow-cytometry was 50-87%. FISH revealed a normal pattern in all the cultures examined. In contrast, aCGH/SNPa revealed neither gains/losses nor LOH in four patients, a trisomy 5 in one and the LOH of a 3.8 Mb sized region located on 13q31.1 in one patient. This study, the first one that applied aCGH/SNPa to investigate the MSC chromosomal pattern, suggests that i) MSCs from chromosomally abnormal AML patients may show a normal FISH pattern, but may be either normal or contain chromosomal aberrations different from those present in leukemic cells on aCGH/SNPa analysis; ii) these defects are uncommonly seen in AML; iii) MSCs defects may flag that the leukemogenic event targets not only the hematopoietic tissue but also the stromal cell compartment, i.e. the niche; iii) aCGH/SNPa provides an in-depth view of MSC chromosomal pattern allowing the identification of potential clonal markers. Disclosures: No relevant conflicts of interest to declare.

Multiplexed Bead-Based Mesofluidic System for Detection of Food-Borne Pathogenic Bacteria

ABSTRACT In the present study, a simple and rapid multiplexed bead-based mesofluidic system (BMS) was developed for simultaneous detection of food-borne pathogenic bacteria, including Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, Salmonella, Enterobacter sakazakii, Shigella, Escherichia coli O157:H7, and Campylobacter jejuni. This system is based on utilization of isothiocyanate-modified microbeads that are 250 μm in diameter, which were immobilized with specific amino-modified oligonucleotide probes and placed in polydimethylsiloxane microchannels. PCR products from the pathogens studied were pumped into microchannels to hybridize with the oligonucleotide-modified beads, and hybridization signals were detected using a conventional microarray scanner. The short sequences of nucleic acids (21 bases) and PCR products characteristic of bacterial pathogens could be detected at concentrations of 1 pM and 10 nM, respectively. The detection procedure could be performed in less than 30 min with high sensitivity and specificity. The assay was simple and fast, and the limits of quantification were in the range from 500 to 6,000 CFU/ml for the bacterial species studied. The feasibility of identification of food-borne bacteria was investigated with samples contaminated with bacteria, including milk, egg, and meat samples. The results demonstrated that the BMS method can be used for effective detection of multiple pathogens in different foodstuffs.