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https://www.readbyqxmd.com/read/28425565/immunolocalization-of-jian-carp-cyprinus-carpio-var-jian-cathepsin-b-cloning-expression-characterization-and-antibody-preparation
#1
Shu-Hong Li, Ran Li, Hai-Xia Zhong, Xiu-Hua Chen, Ai-Ping Liu, Juan Yang, Qiang Hu, Qin-Qin Ke, Mei-Liang Li
Cathepsin B (CatB) cDNA of 759 bp from Jian carp (Cyprinus carpio var. Jian) with amino acid similarity of 99.6% to common carp was cloned. The mature CatB was expressed in Escherichia coli BL21 transferred with vector CatB-pET-30a. It was purified and identified as a single band (29 kDa) on SDS-PAGE. Optimum CatB activity was observed at 40 °C and pH 5.5. Mouse anti-CatB polyclonal antibody with a high titer of 1:256000 was prepared successfully and shown to specifically recognize the antigen both in prokaryotic cells and in the tissues of Jian carp according to western blotting and immunohistochemistry results...
April 20, 2017: Journal of Food Science
https://www.readbyqxmd.com/read/28423700/hla-class-i-loss-in-metachronous-metastases-prevents-continuous-t-cell-recognition-of-mutated-neoantigens-in-a-human-melanoma-model
#2
Barbara Schrörs, Silke Lübcke, Volker Lennerz, Martina Fatho, Anne Bicker, Catherine Wölfel, Patrick Derigs, Thomas Hankeln, Dirk Schadendorf, Annette Paschen, Thomas Wölfel
T lymphocytes against tumor-specific mutated neoantigens can induce tumor regression. Also, the size of the immunogenic cancer mutanome is supposed to correlate with the clinical efficacy of checkpoint inhibition. Herein, we studied the susceptibility of tumor cell lines from lymph node metastases occurring in a melanoma patient over several years towards blood-derived, neoantigen-specific CD8+ T cells. In contrast to a cell line established during early stage III disease, all cell lines generated at later time points from stage IV metastases exhibited partial or complete loss of HLA class I expression...
March 9, 2017: Oncotarget
https://www.readbyqxmd.com/read/28377179/rapid-selection-of-brca1-proficient-tumor-cells-during-neoadjuvant-therapy-for-ovarian-cancer-in-brca1-mutation-carriers
#3
Anna P Sokolenko, Elena L Savonevich, Alexandr O Ivantsov, Grigory A Raskin, Ekatherina S Kuligina, Tatiana V Gorodnova, Elena V Preobrazhenskaya, Maxim A Kleshchov, Vladislav I Tiurin, Marina S Mukhina, Khristina B Kotiv, Andrey V Shulga, Sergey G Kuznetsov, Igor V Berlev, Evgeny N Imyanitov
Ovarian carcinomas (OC) often demonstrate rapid tumor shrinkage upon neoadjuvant chemotherapy (NACT). However, complete pathologic responses are very rare and the mechanisms underlying the emergence of residual tumor disease remain elusive. We hypothesized that the change of somatic BRCA1 status may contribute to this process. The loss-of-heterozygosity (LOH) at the BRCA1 locus was determined for 23 paired tumor samples obtained from BRCA1 germ-line mutation carriers before and after NACT. We observed a somatic loss of the wild-type BRCA1 allele in 74% (17/23) of OCs before NACT...
April 1, 2017: Cancer Letters
https://www.readbyqxmd.com/read/28373404/precancer-atlas-to-drive-precision-prevention-trials
#4
Avrum Spira, Matthew B Yurgelun, Ludmil Alexandrov, Anjana Rao, Rafael Bejar, Kornelia Polyak, Marios Giannakis, Ali Shilatifard, Olivera J Finn, Madhav Dhodapkar, Neil E Kay, Esteban Braggio, Eduardo Vilar, Sarah A Mazzilli, Timothy R Rebbeck, Judy E Garber, Victor E Velculescu, Mary L Disis, Douglas C Wallace, Scott M Lippman
Cancer development is a complex process driven by inherited and acquired molecular and cellular alterations. Prevention is the holy grail of cancer elimination, but making this a reality will take a fundamental rethinking and deep understanding of premalignant biology. In this Perspective, we propose a national concerted effort to create a Precancer Atlas (PCA), integrating multi-omics and immunity - basic tenets of the neoplastic process. The biology of neoplasia caused by germline mutations has led to paradigm-changing precision prevention efforts, including: tumor testing for mismatch repair (MMR) deficiency in Lynch syndrome establishing a new paradigm, combinatorial chemoprevention efficacy in familial adenomatous polyposis (FAP), signal of benefit from imaging-based early detection research in high-germline risk for pancreatic neoplasia, elucidating early ontogeny in BRCA1-mutation carriers leading to an international breast cancer prevention trial, and insights into the intricate germline-somatic-immunity interaction landscape...
April 1, 2017: Cancer Research
https://www.readbyqxmd.com/read/28368006/mechanisms-of-central-tolerance-for-b-cells
#5
REVIEW
David Nemazee
Immune tolerance hinders the potentially destructive responses of lymphocytes to host tissues. Tolerance is regulated at the stage of immature B cell development (central tolerance) by clonal deletion, involving apoptosis, and by receptor editing, which reprogrammes the specificity of B cells through secondary recombination of antibody genes. Recent mechanistic studies have begun to elucidate how these divergent mechanisms are controlled. Single-cell antibody cloning has revealed defects of B cell central tolerance in human autoimmune diseases and in several human immunodeficiency diseases caused by single gene mutations, which indicates the relevance of B cell tolerance to disease and suggests possible genetic pathways that regulate tolerance...
April 3, 2017: Nature Reviews. Immunology
https://www.readbyqxmd.com/read/28364361/a-novel-method-to-generate-single-cell-derived-cancer-associated-fibroblast-clones
#6
Hiroko Hashimoto, Yoshitaka Suda, Tomoyuki Miyashita, Atsushi Ochiai, Masahiro Tsuboi, Kenkichi Masutomi, Tohru Kiyono, Genichiro Ishii
BACKGROUND: Cancer-associated fibroblasts (CAFs) communicate with cancer cells to play important roles in tumor progression. However, CAFs have heterogeneous phenotypes and functions. To understand how much of this heterogeneity relates to different biological responses, a more efficient method of generating single-cell-derived CAF clones is required. METHOD: We transduced two primary CAF cultures (CAFs-608 and CAFs-621) from lung adenocarcinoma with human telomerase reverse transcriptase (hTERT), mutant forms of cyclin dependent kinase 4 (CDK4R24C) independently and in combination (hTERT/CDK4R24C)...
March 31, 2017: Journal of Cancer Research and Clinical Oncology
https://www.readbyqxmd.com/read/28356257/exploring-human-antimicrobial-antibody-responses-on-a-single-b-cell-level
#7
Daniel Hofmann, Jonathan R Lai
Analysis of monoclonal antibodies (mAbs) derived from single B cell cloning has been highly beneficial for antimicrobial immunotherapy, vaccine design, and advancing our understanding of pathogen-triggered effects on the human immunoglobulin repertoire. Sequencing of single B cells variable domains, and characterization of binding and functional activities of mAbs derived from those sequences, provides in-depth insight into not only sites of susceptibility for antibody-mediated neutralization or opsonization of the pathogen, but also on the dynamics of protective antibody evolution during infection...
March 29, 2017: Clinical and Vaccine Immunology: CVI
https://www.readbyqxmd.com/read/28341999/immunological-memory-to-hyperphosphorylated-tau-in-asymptomatic-individuals
#8
Gabriel Pascual, Jehangir S Wadia, Xueyong Zhu, Elissa Keogh, Başak Kükrer, Jeroen van Ameijde, Hanna Inganäs, Berdien Siregar, Gerrard Perdok, Otto Diefenbach, Tariq Nahar, Imke Sprengers, Martin H Koldijk, Els C Brinkman-van der Linden, Laura A Peferoen, Heng Zhang, Wenli Yu, Xinyi Li, Michelle Wagner, Veronica Moreno, Julie Kim, Martha Costa, Kiana West, Zara Fulton, Lucy Chammas, Nancy Luckashenak, Lauren Fletcher, Trevin Holland, Carrie Arnold, R Anthony Williamson, Jeroen J Hoozemans, Adrian Apetri, Frederique Bard, Ian A Wilson, Wouter Koudstaal, Jaap Goudsmit
Several reports have described the presence of antibodies against Alzheimer's disease-associated hyperphosphorylated forms of tau in serum of healthy individuals. To characterize the specificities that can be found, we interrogated peripheral IgG(+) memory B cells from asymptomatic blood donors for reactivity to a panel of phosphorylated tau peptides using a single-cell screening assay. Antibody sequences were recovered, cloned, and expressed as full-length IgGs. In total, 52 somatically mutated tau-binding antibodies were identified, corresponding to 35 unique clonal families...
May 2017: Acta Neuropathologica
https://www.readbyqxmd.com/read/28337768/tet2-mutations-in-b-cells-of-patients-affected-by-angioimmunoblastic-t-cell-lymphoma
#9
Friederike H Schwartz, Qian Cai, Eva Fellmann, Sylvia Hartmann, Mikko I Mäyränpää, Marja-Liisa Karjalainen-Lindsberg, Christer Sundström, René Scholtysik, Martin-Leo Hansmann, Ralf Küppers
Angioimmunoblastic T cell lymphomas (AITL) frequently carry mutations in the TET2 and IDH2 genes. TET2 mutations represent early genetic lesions as they were already detected in hematopoietic precursor cells of AITL patients. We show by analysis of whole tissue sections and microdissected PD1(+) cells that the frequency of TET2-mutated AITL is presumably even higher than reported (12/13 cases in our collection, 92%). In two thirds of informative AITL (6/9) also a fraction of B cells was TET2-mutated. Investigation of four AITL by TET2 and IGHV gene sequencing of single microdissected B cells showed that between 10-60% of polyclonal B cells in AITL lymph nodes harboured the identical TET2 mutations of the respective T cell lymphoma clone...
March 24, 2017: Journal of Pathology
https://www.readbyqxmd.com/read/28332627/cross-neutralizing-anti-hiv-1-human-single-chain-variable-fragments-scfvs-against-cd4-binding-site-and-n332-glycan-identified-from-a-recombinant-phage-library
#10
Lubina Khan, Rajesh Kumar, Ramachandran Thiruvengadam, Hilal Ahmad Parray, Muzamil Ashraf Makhdoomi, Sanjeev Kumar, Heena Aggarwal, Madhav Mohata, Abdul Wahid Hussain, Raksha Das, Raghavan Varadarajan, Jayanta Bhattacharya, Madhu Vajpayee, K G Murugavel, Suniti Solomon, Subrata Sinha, Kalpana Luthra
More than 50% of HIV-1 infection globally is caused by subtype_C viruses. Majority of the broadly neutralizing antibodies (bnAbs) targeting HIV-1 have been isolated from non-subtype_C infected donors. Mapping the epitope specificities of bnAbs provides useful information for vaccine design. Recombinant antibody technology enables generation of a large repertoire of monoclonals with diverse specificities. We constructed a phage recombinant single chain variable fragment (scFv) library with a diversity of 7.8 × 10(8) clones, using a novel strategy of pooling peripheral blood mononuclear cells (PBMCs) of six select HIV-1 chronically infected Indian donors whose plasma antibodies exhibited potent cross neutralization efficiency...
March 23, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28321219/computational-model-reveals-limited-correlation-between-germinal-center-b-cell-subclone-abundancy-and-affinity-implications-for-repertoire-sequencing
#11
Polina Reshetova, Barbera D C van Schaik, Paul L Klarenbeek, Marieke E Doorenspleet, Rebecca E E Esveldt, Paul-Peter Tak, Jeroen E J Guikema, Niek de Vries, Antoine H C van Kampen
Immunoglobulin repertoire sequencing has successfully been applied to identify expanded antigen-activated B-cell clones that play a role in the pathogenesis of immune disorders. One challenge is the selection of the Ag-specific B cells from the measured repertoire for downstream analyses. A general feature of an immune response is the expansion of specific clones resulting in a set of subclones with common ancestry varying in abundance and in the number of acquired somatic mutations. The expanded subclones are expected to have BCR affinities for the Ag higher than the affinities of the naive B cells in the background population...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/28301575/generation-of-murine-tumor-cell-lines-deficient-in-mhc-molecule-surface-expression-using-the-crispr-cas9-system
#12
Krishna Das, David Eisel, Clarissa Lenkl, Ashish Goyal, Sven Diederichs, Elke Dickes, Wolfram Osen, Stefan B Eichmüller
In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY), were transfected with an expression plasmid encoding a β2m-specific single guide (sg)RNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay...
2017: PloS One
https://www.readbyqxmd.com/read/28269762/cloning-and-molecular-characterization-of-the-cdnas-encoding-the-variable-regions-of-an-anti-cd20-monoclonal-antibody
#13
Dariush Shanehbandi, Jafar Majidi, Tohid Kazemi, Behzad Baradaran, Leili Aghebati-Maleki
BACKGROUND: CD20-based targeting of B-cells in hematologic malignancies and autoimmune disorders is associated with outstanding clinical outcomes. Isolation and characterization of VH and VL cDNAs encoding the variable regions of the heavy and light chains of monoclonal antibodies (MAb) is necessary to produce next generation MAbs and their derivatives such as and bispecific antibodies (bsAb) and single-chain variable fragments (scFv). OBJECTIVE: This study was aimed at cloning and characterization of the VH and VL cDNAs from a hybridoma cell against the CD20 antigen...
February 24, 2017: Human Antibodies
https://www.readbyqxmd.com/read/28249223/pronounced-effect-of-hapten-binding-on-thermal-stability-of-an-anti-4-hydroxy-3-nitrophenyl-acetyl-antibody-possessing-a-glycine-residue-at-position-95-of-the-heavy-chain
#14
Yusui Sato, Satomi Inaba, Harumi Fukada, Takachika Azuma, Masayuki Oda
Immune response to T-cell-dependent antigens is highly dynamic; several B-cell clones responsible for antibody production appear alternately during immunization. It was previously shown that at least two-types of antibodies are secreted after immunization with (4-hydroxy-3-nitrophenyl)acetyl (NP); one has Tyr and another has Gly at position 95 of the heavy chain (referred to as Tyr95- and Gly95-type). The former appeared at an early stage, while the latter appeared at a late stage, i.e., after secondary immunization, although Fv domains of these antibodies were encoded by same genes of variable heavy and light chains...
February 26, 2017: Molecular Immunology
https://www.readbyqxmd.com/read/28245848/a-streamlined-method-for-the-design-and-cloning-of-shrnas-into-an-optimized-dox-inducible-lentiviral-vector
#15
Sander B Frank, Veronique V Schulz, Cindy K Miranti
BACKGROUND: Short hairpin RNA (shRNA) is an established and effective tool for stable knock down of gene expression. Lentiviral vectors can be used to deliver shRNAs, thereby providing the ability to infect most mammalian cell types with high efficiency, regardless of proliferation state. Furthermore, the use of inducible promoters to drive shRNA expression allows for more thorough investigations into the specific timing of gene function in a variety of cellular processes. Moreover, inducible knockdown allows the investigation of genes that would be lethal or otherwise poorly tolerated if constitutively knocked down...
February 28, 2017: BMC Biotechnology
https://www.readbyqxmd.com/read/28212528/the-crispr-cas9%C3%A2-system-efficiently-reverts-the-tumorigenic-ability-of-bcr-abl-in-vitro-and-in-a-xenograft-model-of-chronic-myeloid-leukemia
#16
Ignacio García-Tuñón, María Hernández-Sánchez, José Luis Ordoñez, Veronica Alonso-Pérez, Miguel Álamo-Quijada, Rocio Benito, Carmen Guerrero, Jesús María Hernández-Rivas, Manuel Sánchez-Martín
CRISPR/Cas9 technology was used to abrogate p210 oncoprotein expression in the Boff-p210 cell line, a pro-B line derived from interlukin-3-dependent Baf/3, that shows IL-3-independence arising from the constitutive expression of BCR-ABL p210. Using this approach, pools of Boff-p210-edited cells and single edited cell-derived clones were obtained and functionally studied in vitro. The loss of p210 expression in Boff-p210 cells resulted in the loss of ability to grow in the absence of IL-3, as the Baf/3 parental line, showing significantly increased apoptosis levels...
February 9, 2017: Oncotarget
https://www.readbyqxmd.com/read/28202626/cancer-cells-exhibit-clonal-diversity-in-phenotypic-plasticity
#17
Robert Austin Mathis, Ethan S Sokol, Piyush B Gupta
Phenotypic heterogeneity in cancers is associated with invasive progression and drug resistance. This heterogeneity arises in part from the ability of cancer cells to switch between phenotypic states, but the dynamics of this cellular plasticity remain poorly understood. Here we apply DNA barcodes to quantify and track phenotypic plasticity across hundreds of clones in a population of cancer cells exhibiting epithelial or mesenchymal differentiation phenotypes. We find that the epithelial-to-mesenchymal cell ratio is highly variable across the different clones in cancer cell populations, but remains stable for many generations within the progeny of any single clone-with a heritability of 0...
February 2017: Open Biology
https://www.readbyqxmd.com/read/28179494/hierarchical-clustering-can-identify-b-cell-clones-with-high-confidence-in-ig-repertoire-sequencing-data
#18
Namita T Gupta, Kristofor D Adams, Adrian W Briggs, Sonia C Timberlake, Francois Vigneault, Steven H Kleinstein
Adaptive immunity is driven by the expansion, somatic hypermutation, and selection of B cell clones. Each clone is the progeny of a single B cell responding to Ag, with diversified Ig receptors. These receptors can now be profiled on a large scale by next-generation sequencing. Such data provide a window into the microevolutionary dynamics that drive successful immune responses and the dysregulation that occurs with aging or disease. Clonal relationships are not directly measured, but they must be computationally inferred from these sequencing data...
March 15, 2017: Journal of Immunology: Official Journal of the American Association of Immunologists
https://www.readbyqxmd.com/read/28125682/the-number-of-overlapping-aid-hotspots-in-germline-ighv-genes-is-inversely-correlated-with-mutation-frequency-in-chronic-lymphocytic-leukemia
#19
Chaohui Yuan, Charles C Chu, Xiao-Jie Yan, Davide Bagnara, Nicholas Chiorazzi, Thomas MacCarthy
The targeting of mutations by Activation-Induced Deaminase (AID) is a key step in generating antibody diversity at the Immunoglobulin (Ig) loci but is also implicated in B-cell malignancies such as chronic lymphocytic leukemia (CLL). AID has previously been shown to preferentially deaminate WRC (W = A/T, R = A/G) hotspots. WGCW sites, which contain an overlapping WRC hotspot on both DNA strands, mutate at much higher frequency than single hotspots. Human Ig heavy chain (IGHV) genes differ in terms of WGCW numbers, ranging from 4 for IGHV3-48*03 to as many as 12 in IGHV1-69*01...
2017: PloS One
https://www.readbyqxmd.com/read/28114943/joining-the-in-vitro-immunization-of-alpaca-lymphocytes-and-phage-display-rapid-and-cost-effective-pipeline-for-sdab-synthesis
#20
Lubos Comor, Saskia Dolinska, Katarina Bhide, Lucia Pulzova, Irene Jiménez-Munguía, Elena Bencurova, Zuzana Flachbartova, Lenka Potocnakova, Evelina Kanova, Mangesh Bhide
BACKGROUND: Camelids possess unique functional heavy chain antibodies, which can be produced and modified in vitro as a single domain antibody (sdAb or nanobody) with full antigen binding ability. Production of sdAb in conventional manner requires active immunization of Camelidae animal, which is laborious, time consuming, costly and in many cases not feasible (e.g. in case of highly toxic or infectious antigens). RESULTS: In this study, we describe an alternative pipeline that includes in vitro stimulation of naïve alpaca B-lymphocytes by antigen of interest (in this case endothelial cell binding domain of OspA of Borrelia) in the presence of recombinant alpaca interleukins 2 and 4, construction of sdAb phage library, selection of antigen specific sdAb expressed on phages (biopanning) and confirmation of binding ability of sdAb to the antigen...
January 23, 2017: Microbial Cell Factories
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