Selection Of Target Cells Research Articles

Correction of F508del in CFTR using CRISPR/Cas9 in cells derived from cystic fibrosis patients

BACKGROUND: Cystic fibrosis (CF) is the most common fatal and incurable genetic disease. The most frequent reason is the F508del mutation in the CFTR gene, which can theoretically be corrected by genome editing. Currently, pressing issue is the search for the most effective CRISPR/Cas9-based editing system for this mutation, as well as the selection of target cells that could support self-renewal and provide differentiated progeny of lung cells. Such promising targets can be presented by human induced pluripotent stem cells (hiPSCs) and human induced airway basal stem cells (hiBCs). AIM: To correct the F508del mutation in the CFTR gene in hiPSCs and hiBCs derived from cystic fibrosis patients using CRISPR/Cas9 technology. METHODS: We obtained three hiPSCs lines by reprogramming fibroblasts from patients with CF and three hiBCs lines by targeted differentiation of these hiPSCs. Based on three different variants of the Cas9 nuclease, three single guide RNAs (sgRNAs), and two single-stranded oligodeoxyribonucleotides (ssODNs) we created eight systems for editing the F508del mutation and transfected them into hiPSCs and hiBCs by electroporation. Then we analyzed the levels of non-homologous end joining (NHEJ), indels, and directed homologous repair (HDR) in the transfected cells by deep target sequencing. RESULTS: Eight editing systems were tested on hiPSCs. The highest efficiency of NHEJ, indels, and HDR was observed when using SpCas9(1.1). The mutation was significantly corrected using a combination of this nuclease with the sgRNA sgCFTR#sp1 (with an efficiency up to 6.6% of alleles in transfected cells). Only two editing systems), which seemed to be most effective on chiPSCs, were tested on hiBCs. The mutation was significantly corrected using both systems (with efficiency up to 20% of alleles in transfected cells). CONCLUSION: We demonstrated the possibility of highly effective correction of the F508del mutation in the CFTR gene in cells obtained from patients with CF using a designed system for editing this mutation based on the CRISPR/Cas9 system. Since hiBCs can be transfected and edited quite successfully, and can also be obtained in satisfactory quantities from hiPSCs, they are a promising platform for the development of gene therapy for cystic fibrosis.

Gibbs Haversine Reinforcement Learning Based Handover For 5g Enabled Seamless Mobility in Wireless Network

Seamless Mobility (SM) is crucial for bringing about better Quality of Service (QoS) like minimum handover latency with maximum throughput in 5G networks. In this work a method called, Jenkin Impulse Response Filtering and Reinforcement Learning-based Gibbs Haversine Distribution (JIRF-RLGHD) for optimal selection of target cells for the handover process to ensure seamless mobility is designed. The JIRF-RLGHD method is split into two sections. They are predicting the signal quality of both serving and adjacent wireless nodes using the Box Jenkin Impulse Response Filtering model. The second task remains in applying Reinforcement Learning-based Gibbs Haversine Distribution for optimal selection of target cells for handover to ensure seamless mobility in a wireless network. The overall proposed method was simulated on a Python programming interface language. The simulation results reveal that the JIRF-RLGHD method offers a greater delivery rate, and handover success with lesser handover latency at minimal packet loss rate. Numerical results show that the JIRF-RLGHD method performs better in terms of data delivery rate by 18%, and handover latency by 33% compared to existing methods.

(Invited) Discrimination and Selection of Target Cells from the Cell-Based Array Based on Dielectrophoresis

Introduction: We have developed manipulation techniques to form cell-based arrays by positive dielectrophoresis (p-DEP) and to retrieve target cells from cell−based arrays selectively by negative dielectrophoresis (n-DEP). The novel devices with microwell arrays on microband electrodes were employed to manipulate cells. Hybridomas with the secretion ability of antibodies were trapped to form cell-based arrays. Then, the ability was discriminated by trapping the antibodies to the antigen immobilized on the bottom of wells. Finally, individual cells trapped in microwells were selectively retrieved by regulating the generation of electric fields in individual microwells. The development of series of these techniques could be useful to recover hybridomas producing antibodies with high affinity in large populations of cells without repeated steps of a culture and a limiting serial-dilution. Experimental: The device comprises the upper substrate with indium-tin-oxide (ITO) microband electrodes and the lower substrate with microwell arrays on ITO microband electrodes. Hybridomas producing an anti-rabbit serum albumin (RSA) antibody suspended in the DEP medium were introduced in the channel. AC signal (3 MHz, 2 Vpp) was then applied to the upper and lower microband electrodes with opposite phase to form a cell-based array. Anti-RSA antibodies secreted from hybridomas trapped in the microwells were captured by RSA immobilized on the electrodes. Cell arrays were then treated with anti-mouse IgG antibody conjugated with Alexa 488 (a secondary antibody). Thereafter, a repulsive force of negative dielectrophoresis (n-DEP) was employed to retrieve the target hybridomas from the microwell array. To retrieve the target hybridoma, an AC signal in the n-DEP frequency region was applied to a pair of microband electrodes above and below the microwell with the target hybridoma. Results and discussion: Cell−based arrays were formed with the occupancy efficiency of over 90% in a few seconds by p-DEP. Antibodies secreted in microwell arrays were captured to discriminate target hybridomas in a few hours without repeated steps of a culture and a limiting serial-dilution. Furthermore, hybridomas trapped in microwells were retrieved from the cell−based array by applying an AC signal to band electrodes. The sequential system for forming cell−based arrays, discriminating hybridomas secreting specific antibodies, and retrieving target hybridomas was developed by using the novel microwell array device comprising 3-D microband array electrodes with an orthogonal arrangement.

High Capacity Immunomagnetic Isolation of Apheresis Samples in an Automated Closed System

Abstract Cell therapy development, including Chimeric antigen receptor (CAR)-T cells, involves many steps and requires processing on average 3 billion white blood cells or more. Automated large scale immunomagnetic cell isolation with high efficiency and throughput in a closed system is desired to enrich T cells from apheresis products. Current automated, high capacity methods are time consuming, high cost, low yield and can potentially influence the physiology of the target cells. We have developed an automated closed magnetic separation platform, MARS® Bar to address these problems. This platform uses three uniquely designed magnetic separation modules and each magnetic module can perform positive or negative selection of target cells in a fluidic chamber at high flow rate (1mL/min per module). On this platform we have developed cell isolation reagents and program with minimal cell preparation. In the pilot study, we isolated 116 million CD3+ T cells from 267million CD45+ cells in a single LSR chamber on a single MARS® module. The entire automated process took less than 15 minutes to complete and resulted in > 95% purity and > 96% recovery of CD3+ T cells. The isolated cells were then activated and expanded, with results showing high proliferation and the dominant population being central memory T cells. The unique MARS® Bar matrix-free design has unlimited capacity because of the automated release of positively captured cells and very low carry-over. This was validated by processing greater than 3 billion leukocytes at 20 million cells per mL within the three modules in parallel and intermittent release of captured cells for collection. The MARS® Bar can be easily integrated into standard T cell production workflow.

Single-cell sequencing reveals novel mechanisms of Aflatoxin B1-induced hepatotoxicity in S phase-arrested L02 cells.

Aflatoxin B1 (AFB1) is widely distributed in nature and is confirmed to be the most toxic of all the aflatoxins, whose predominant metabolism site is the liver. As a well-studied and vital mode of epigenetic modifications, aberrant methylation of the promoters in eukaryotic cells may cause the silence of essential genes, affecting their related transcriptional pathways and ultimately leading to the development of disease and cancers. This study investigated the mechanisms of AFB1-induced hepatotoxicity in S phase-arrested L02 cells using single-cell RNA-seq and single-cell reduced representation bisulfite sequencing (RRBS). AFB1 induced apoptosis and cell cycle S phase arrest, reduced mitochondrial membrane potential (ΔΨm), and increased reactive oxygen species (ROS) generation, as well as the DNA methylation level. Hepatotoxicity mechanism patterns induced by AFB1 in S phase-arrested L02 cells were revealed by combining single-cell RNA-seq with single-cell RRBS analysis, in which DNA methylation played a role via regulating the gonadotropin-releasing hormone receptor pathway, the Wnt signaling pathway, and the TGF-beta signaling pathway. Moreover, a novel strategy for precision toxicology exploration was obtained, including the selection of target cells, multi-group non-directional sequencing, and pathway analysis.

Advances in gene therapy for hemophilia

Hemophilia gene therapy is the process that vectors transfer normal FⅧ and FⅨ gene into patients with hemophilia, in order to correct genetic defects and produce normal FⅧ and FⅨ protein, which can meet the hemostatic requirements and bring new hope for treatment of hemophilia.The transformation of the target gene, as well as how to choose vectors and target cells are important parts of this process. The core technology of hemophilia gene therapy is gene transfer vector technology, and vectors for gene therapy can be divided into non-viral vectors and viral vectors.At present, many researches of gene therapy for hemophilia have made a breakthrough progress. However, hemophilia gene therapy still have some problems, such as the application of the vectors, the selection of target cells, the immune response to the vectors and the expression products, and the like, so further researchs are still needed. Therefore, this article summarizes the gene therapy process and clinical research progress of hemophilia. Key words: Hemophilia; Gene therapy; Genetic vectors; Targeted gene repair

Grass carp reovirus-GD108 fiber protein is involved in cell attachment.

Viral attachment to specific host receptors is the first step in viral infection and serves an essential function in the selection of target cells. In this study, structure analysis, neutralization assays, and cell attachment assays were carried out to evaluate the cell attachment functions of the outer capsid fiber protein of grass carp reovirus GD108 strain (GCRV-GD108). The GCRV-GD108 fiber protein contained 512 amino acids encoded by S7 segment and shared sequence similarities with mammalian reovirus cell attachment protein σ1 and adenovirus fiber. Structural analyses predicted the presence of a coiled-coil tail domain, three adenoviral shafts in the body domain, and a globular head domain, similar to other fiber proteins. Neutralization assays showed that polyclonal antibodies against the fiber protein could prevent viral infection in both fish and grass carp snout fibroblast cells (PSF), suggesting that the recombinant fiber protein could induce neutralized antibodies against GCRV-GD108. Cell attachment assays showed that recombinant fiber protein could bind to PSF cells, demonstrating that the fiber protein functioned as the cell attachment protein in GCRV-GD108. These results provided the basis for further studies of the pathogenesis of grass carp reovirus.

Real-time prediction of cell division timing in developing zebrafish embryo.

Combination of live-imaging and live-manipulation of developing embryos in vivo provides a useful tool to study developmental processes. Identification and selection of target cells for an in vivo live-manipulation are generally performed by experience- and knowledge-based decision-making of the observer. Computer-assisted live-prediction method would be an additional approach to facilitate the identification and selection of the appropriate target cells. Herein we report such a method using developing zebrafish embryos. We choose V2 neural progenitor cells in developing zebrafish embryo as their successive shape changes can be visualized in real-time in vivo. We developed a relatively simple mathematical method of describing cellular geometry of V2 cells to predict cell division-timing based on their successively changing shapes in vivo. Using quantitatively measured 4D live-imaging data, features of V2 cell-shape at each time point prior to division were extracted and a statistical model capturing the successive changes of the V2 cell-shape was developed. By applying sequential Bayesian inference method to the model, we successfully predicted division-timing of randomly selected individual V2 cells while the cell behavior was being live-imaged. This system could assist pre-selecting target cells desirable for real-time manipulation–thus, presenting a new opportunity for in vivo experimental systems.

Expression of SOX2 in oral squamous cell carcinoma and the association with lymph node metastasis.

Oral squamous cell carcinomas (OSCCs) are a growing problem in the world. The various existing treatments have not markedly improved the survival rate of patients with OSCC during the past three decades. Novel treatment strategies are required. Sex determining region Y-box 2 (SOX2) is a transcription factor that is involved in the maintenance of embryonic stem cell pluripotency and in multiple developmental processes. SOX2 expression was indicated to act as a prognostic factor in various types of tumors, including breast, colorectal, gastric and lung cancer and glioblastoma, and as a link between malignancy and stemness. Cancer stem cells (CSCs) may be responsible for the genesis, growth and metastatic spread of tumors. The poor survival outcomes for OSCC patients may be attributable to a poor selection of target cells for treatment, as current oral cancer therapies are generally aimed at the global mass of tumor. Therefore, the consideration that novel approaches to oral cancer may be targeted using SOX2 and CSCs appears reasonable. In order to better understand the oncogenic roles and the corresponding signal transduction pathways of the SOX2 protein, the present study emphasizes the role of SOX2 in OSCC, including the proteins associated with OSCC, and reviews the literature regarding the role of SOX2 in lymph node metastasis. The aim of the present study is to provide a reference for future studies that engage in research on the aforementioned subject.

Разработка нового метода детектирования экспрессии генов с помощью количественной ОТ-ПЦР единичных клеток без выделения РНК в сочетании с лазерной захватывающей микродиссекцией

Laser capture microdissection (LCM) combined with real time RT-PCR represents a powerful method for analyzing gene expression levels in selected cell types. To avoid degradation of RNA by endogenous and exogenous RNases and to ensure selection of target cells only, we designed a protocol in which the inactivation of exogenous and endogenous RNases by RNaseZap and RNA later is combined with immunofluorescent labeling, and labeled cells in sections used for subsequent LCM and real time RT-PCR. Immunolabelled neurons were captured onto the caps of RNase-free microcentrifuge tubes using LCM and lysed with 3% NP-40 without prior RNA purification. Subsequent reverse transcription for cDNA synthesis was performed in situ on the cap surface to avoid mRNA loss due to transfer between tubes. Applying this protocol, we determined immunoglobulin G (IgG), nerve growth factor (NGF) and GAPDH mRNA levels into small numbers of captured neurons with real time RT-PCR. Thus, this novel method combining LCM with real time RT-PCR without RNA purification appears to be an effective tool for the analysis of cell-specific target gene transcription in small numbers of cells isolated from RNA later-treated tissues.

Myxoid Liposarcoma-Associated EWSR1-DDIT3 Selectively Represses Osteoblastic and Chondrocytic Transcription in Multipotent Mesenchymal Cells

BackgroundLiposarcomas are the most common class of soft tissue sarcomas, and myxoid liposarcoma is the second most common liposarcoma. EWSR1-DDIT3 is a chimeric fusion protein generated by the myxoid liposarcoma-specific chromosomal translocation t(12;22)(q13;q12). Current studies indicate that multipotent mesenchymal cells are the origin of sarcomas. The mechanism whereby EWSR1-DDIT3 contributes to the phenotypic selection of target cells during oncogenic transformation remains to be elucidated.Methodology/Principal FindingsReporter assays showed that the EWSR1-DDIT3 myxoid liposarcoma fusion protein, but not its wild-type counterparts EWSR1 and DDIT3, selectively repressed the transcriptional activity of cell lineage-specific marker genes in multipotent mesenchymal C3H10T1/2 cells. Specifically, the osteoblastic marker Opn promoter and chondrocytic marker Col11a2 promoter were repressed, while the adipocytic marker Ppar-γ2 promoter was not affected. Mutation analyses, transient ChIP assays, and treatment of cells with trichostatin A (a potent inhibitor of histone deacetylases) or 5-Aza-2′-deoxycytidine (a methylation-resistant cytosine homolog) revealed the possible molecular mechanisms underlying the above-mentioned selective transcriptional repression. The first is a genetic action of the EWSR1-DDIT3 fusion protein, which results in binding to the functional C/EBP site within Opn and Col11a2 promoters through interaction of its DNA-binding domain and subsequent interference with endogenous C/EBPβ function. Another possible mechanism is an epigenetic action of EWSR1-DDIT3, which enhances histone deacetylation, DNA methylation, and histone H3K9 trimethylation at the transcriptional repression site. We hypothesize that EWSR1-DDIT3-mediated transcriptional regulation may modulate the target cell lineage through target gene-specific genetic and epigenetic conversions.Conclusions/SignificanceThis study elucidates the molecular mechanisms underlying EWSR1-DDIT3 fusion protein-mediated phenotypic selection of putative target multipotent mesenchymal cells during myxoid liposarcoma development. A better understanding of this process is fundamental to the elucidation of possible direct lineage reprogramming in oncogenic sarcoma transformation mediated by fusion proteins.

Role and fate of SP100 protein in response to Rep-dependent nonviral integration system

Previously, we studied an AAVS1 site-specific non-viral integration system with a Rep-donor plasmid and a plasmid containing adeno-associated virus integration element. Our earlier study focused on the plasmid vector itself, but the cellular response to the system was still unknown. SP100 is a member of the promyelocytic leukemia nuclear bodies. It is involved in many cellular processes such as transcriptional regulation and the cellular intrinsic immune response against viral infection. In this study, we revealed that SP100 inhibited the Rep-dependent nonviral integration. Conversely, transient expression of Rep78 increased the degradation of SP100. This degradation was inhibited by treatment with MG132, an inhibitor of the ubiquitin proteasome. SP100 and Rep78 are both located in the nucleolus, which provides the spatial possibility for their interaction. Rep78 was coimmunoprecipitated with the enhanced green fluorescent protein (EGFP)-SP100 fusion protein but not EGFP, which verified the interaction between Rep78 and SP100. These results have enriched our knowledge about the cellular protein SP100 and Rep-dependent nonviral integration. It may lead to an improvement in the application of Rep-related transgene integration method and in the selection of target cells.

Purification of cell subpopulations via immiscible filtration assisted by surface tension (IFAST)

The selective isolation of a sub-population of cells from a larger, mixed population is a critical preparatory process to many biomedical assays. Here, we present a new cell isolation platform with a unique set of advantages over existing devices. Our technology, termed Immiscible Filtration Assisted by Surface Tension, exploits physical phenomena associated with the microscale to establish fluidic barriers composed of immiscible liquids. By attaching magnetically-responsive particles to a target cell population via immunocapture, we can selectively transport this population across the immiscible barrier and into a separate aqueous solution. The high interfacial energy associated with the immiscible phase / aqueous phase boundaries prevents unwanted cells or other contaminants from inadvertently crossing the immiscible phase. We have demonstrated, using fluorescent particles, stromal cells, and whole blood as "background", that we can successfully isolate ~70% of a target breast cancer cell population with an average purity of >80%. Increased purity was obtained by coupling two immiscible barriers in series, a modification that only slightly increases operational complexity. Furthermore, several samples can be processed in parallel batches in a near-instantaneous manner without the requirement of any washing, which can cause dilution (negative selection) or significant uncontrolled loss (positive selection) of target cells. Finally, cells were observed to remain viable and proliferative following traverse through the immiscible phase, indicating that this process is suitable for a variety of downstream assays, including those requiring intact living cells.

Impact of sample acquisition and linear amplification on gene expression profiling of lung adenocarcinoma: laser capture micro-dissection cell-sampling versus bulk tissue-sampling

BackgroundThe methods used for sample selection and processing can have a strong influence on the expression values obtained through microarray profiling. Laser capture microdissection (LCM) provides higher specificity in the selection of target cells compared to traditional bulk tissue selection methods, but at an increased processing cost. The benefit gained from the higher tissue specificity realized through LCM sampling is evaluated in this study through a comparison of microarray expression profiles obtained from same-samples using bulk and LCM processing.MethodsExpression data from ten lung adenocarcinoma samples and six adjacent normal samples were acquired using LCM and bulk sampling methods. Expression values were evaluated for correlation between sample processing methods, as well as for bias introduced by the additional linear amplification required for LCM sample profiling.ResultsThe direct comparison of expression values obtained from the bulk and LCM sampled datasets reveals a large number of probesets with significantly varied expression. Many of these variations were shown to be related to bias arising from the process of linear amplification, which is required for LCM sample preparation. A comparison of differentially expressed genes (cancer vs. normal) selected in the bulk and LCM datasets also showed substantial differences. There were more than twice as many down-regulated probesets identified in the LCM data than identified in the bulk data. Controlling for the previously identified amplification bias did not have a substantial impact on the differences identified in the differentially expressed probesets found in the bulk and LCM samples.ConclusionLCM-coupled microarray expression profiling was shown to uniquely identify a large number of differentially expressed probesets not otherwise found using bulk tissue sampling. The information gain realized from the LCM sampling was limited to differential analysis, as the absolute expression values obtained for some probesets using this study's protocol were biased during the second round of amplification. Consequently, LCM may enable investigators to obtain additional information in microarray studies not easily found using bulk tissue samples, but it is of critical importance that potential amplification biases are controlled for.

Structure of Reovirus σ1 in Complex with Its Receptor Junctional Adhesion Molecule-A

Viral attachment to specific host receptors is the first step in viral infection and serves an essential function in the selection of target cells. Mammalian reoviruses are highly useful experimental models for studies of viral pathogenesis and show promise as vectors for oncolytics and vaccines. Reoviruses engage cells by binding to carbohydrates and the immunoglobulin superfamily member, junctional adhesion molecule-A (JAM-A). JAM-A exists at the cell surface as a homodimer formed by extensive contacts between its N-terminal immunoglobulin-like domains. We report the crystal structure of reovirus attachment protein σ1 in complex with a soluble form of JAM-A. The σ1 protein disrupts the JAM-A dimer, engaging a single JAM-A molecule via virtually the same interface that is used for JAM-A homodimerization. Thus, reovirus takes advantage of the adhesive nature of an immunoglobulin-superfamily receptor by usurping the ligand-binding site of this molecule to attach to the cell surface. The dissociation constant (KD) of the interaction between σ1 and JAM-A is 1,000-fold lower than that of the homophilic interaction between JAM-A molecules, indicating that JAM-A strongly prefers σ1 as a ligand. Analysis of reovirus mutants engineered by plasmid-based reverse genetics revealed residues in σ1 required for binding to JAM-A and infectivity of cultured cells. These studies define biophysical mechanisms of reovirus cell attachment and provide a platform for manipulating reovirus tropism to enhance vector targeting.

Rapid and Efficient Selection of Yeast Displaying a Target Protein Using Thermo‐responsive Magnetic Nanoparticles

Magnetic separation provides a relatively quick and easy-to-use method for cell isolation and protein purification. We have developed a rapid and efficient procedure to isolate yeast cells displaying a target polypeptide, namely, the Staphylococcus aureus ZZ domain, which serves as s model for protein interactions and can bind immunoglobulin G (IgG). We optimized selection of ZZ-displaying yeast cells using thermoresponsive magnetic nanoparticles. A model library was prepared by mixing various proportions of target yeast displaying the ZZ domain with control cells. Target cells in the model library that bound to the ZZ-specific binding partner, biotinylated IgG, were selected with biotinylated thermoresponsive magnetic nanoparticles using the biotin-avidin sandwich system. We determined ZZ expression levels and optimized the concentrations of both magnetic nanoparticles and avidin for efficient selection of target cells. After optimization, we successfully enriched the target cell population 4700-fold in a single round of selection. Moreover, only two rounds of selection were required to enrich the target cell population from 0.001% to nearly 100%. Our results suggest that magnetic separation will be useful for efficient exploration of novel protein-protein interactions and rapid isolation of biomolecules with novel functions.

Wnt4 Is a Local Repulsive Cue that Determines Synaptic Target Specificity

How synaptic specificity is molecularly coded in target cells is a long-standing question in neuroscience. Whereas essential roles of several target-derived attractive cues have been shown, less is known about the role of repulsion by nontarget cells. We conducted single-cell microarray analysis of two neighboring muscles (M12 and M13) in Drosophila, which are innervated by distinct motor neurons, by directly isolating them from dissected embryos. We identified a number of potential target cues that are differentially expressed between the two muscles, including M13-enriched Wnt4. When the functions of Wnt4, or putative receptors Frizzled 2 and Derailed-2 or Dishevelled were inhibited, motor neurons that normally innervate M12 (MN12s) formed smaller synapses on M12 but instead formed ectopic nerve endings on M13. Conversely, ectopic expression of Wnt4 in M12 inhibits synapse formation by MN12s. These results suggest that Wnt4, via Frizzled 2, Derailed-2, and Dishevelled, generates target specificity by preventing synapse formation on a nontarget muscle. Ectopic expression of five other M13-enriched genes, including beat-IIIc and Glutactin, also inhibits synapse formation by MN12s. These results demonstrate an important role for local repulsion in regulating cell-to-cell target specificity.

Vascular Endothelial Cells Have Impaired Capacity to Present Immunodominant, Antigenic Peptides: A Mechanism of Cell Type-Specific Immune Escape

Vascular endothelial cells (EC) are an exposed target tissue in the course of CTL-mediated alloimmune diseases such as graft-vs-host disease (GVHD) or solid organ transplant rejection. The outcome of an interaction between CTL and target cells is determined by the amount of Ag presented and the costimulatory signals delivered by the target cells. We compared human EC with leukocytes and epithelial cells as targets for peptide-specific, MHC class I-restricted CTL clones. EC were poor targets for immunodominant CTL. Both endogenously processed antigenic proteins and exogenously added antigenic peptides are presented at 50- to 5000-fold lower levels on EC compared with any other target cell analyzed. This quantitative difference fully explained the poor CTL-mediated killing of EC. There was no evidence that lack of costimulation would contribute significantly to this cell type-specific difference in CTL activation. An HLA-A2-specific CTL clone that killed a broad selection of HLA A2-positive target cells equally well, killed EC less efficiently. Our data suggest that EC present a different Ag repertoire compared with other cell types. By this mechanism, these cells may escape an attack by effector CTL, which have been educated by professional APCs and are specific for immunodominant antigenic peptides.

Evaluation of genetic patterns in different tumor areas of intermediate‐grade prostatic adenocarcinomas by high‐resolution genomic array analysis

Prostate cancer is known for its highly heterogeneous histological appearance. Data concerning the cytogenetic content of areas with different histology are sparse. We have genetically evaluated 10 prostatic adenocarcinomas with intermediate histopathological grades (Gleason score 7) that showed two distinctive growth patterns with different pathologies, that is, Gleason grades 3 and 4 (G3 and G4). The G3 and G4 tumor specimens were taken from spatially separated regions within the cancer mass. Array-based comparative genomic hybridization (aCGH) was performed to obtain genotypes from the 10 pairs of G3 and G4 cancer areas. The cancer DNAs were retrieved from formalin-fixed and paraffin-embedded tissues allowing optimal recognition and selection of target cells. A genome-wide 2,400-element BAC array that provided high-resolution detection of both deletions and amplifications was used. In the 20 G3 and G4 areas, 252 genomic aberrations (88 gains, 164 deletions) were noted, of which 86 were concurrent in G3 and G4 areas (34% overlap). Ninety-five of the 252 alterations were defined by a single BAC clone (54 gains, 41 deletions). Overlapping changes were more frequent for deletions (46%) than for gains (13%). Frequent coinciding deletions (> or = 20% of tumors) were seen on 8p (60%), 6q (30%), 1p (20%), 2q (20%), proximal 8q (20%), 10q (20%), 13q (20%), 16q (20%), and 18q (20%). A frequent overlapping gain (> or = 20% of tumors) was detected on distal 13q (20%). The patterns of imbalance could be found to coincide in the G3 and G4 areas of the majority of cancers. Array-based CGH can be used as a tool for the evaluation of genetic patterns in prostate cancer.

Affinity selection of target cells from cell surface displayed libraries: a novel procedure using thermo-responsive magnetic nanoparticles.

Biotinylated thermo-responsive magnetic nanoparticles for use in affinity selection from yeast cell surface display libraries were prepared by coating magnetite nanoparticles with a thermo-responsive polymer consisting of N-isopropyl acrylamide and a biotin derivative. These particles showed a reversible transition between flocculation and dispersion at around the lower critical solution temperature of 30 degrees C, above which the flocculated particles--which absorbed a large amount of avidin due to their large surface area--were quickly separable by magnet. The model library was constructed by mixing control yeast cells with target yeast cells co-displaying IgG binding protein (ZZ) and enhanced green fluorescence protein. Biotinylated IgG and avidin were subsequently added to the model library, and target cells were efficiently enriched with the biotinylated magnetic nanoparticles by avidin-biotin sandwich and ZZ-IgG interaction. The few target cells (0.001%) in the model library were enriched by up to 100% in only 5 days by an affinity selection procedure repeated four times. This novel method based on magnetic nanoparticles and a yeast cell surface display system could fulfill a wide range of applications in the analysis of protein-protein interactions and rapid isolation of novel biomolecules.