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single b cell cloning

Christine A Snidal, Qi Li, Brittany B Elliott, Henry K-H Mah, Robert H C Chen, Sabiha R Gardezi, Elise F Stanley
Neurotransmitter is released from presynaptic nerve terminals at fast-transmitting synapses by the action potential-gating of voltage dependent calcium channels (CaV), primarily of the CaV2.1 and CaV2.2 types. Entering Ca2+ diffuses to a nearby calcium sensor associated with a docked synaptic vesicle (SV) and initiates its fusion and discharge. Our previous findings that single CaVs can gate SV fusion argued for one or more tethers linking CaVs to docked SVs but the molecular nature of these tethers have not been established...
2018: Frontiers in Cellular Neuroscience
Cory A Perugino, Sultan B AlSalem, Hamid Mattoo, Emanuel Della-Torre, Vinay Mahajan, Gayathri Ganesh, Hugues Allard-Chamard, Zachary Wallace, Sydney B Montesi, Johannes Kreuzer, Wilhelm Haas, John H Stone, Shiv Pillai
BACKGROUND: The antigenic trigger that drives the expansion of circulating plasmablasts and CD4+ cytotoxic T cells (CD4+ CTLs) in patients with IgG4-Related Disease (IgG4-RD) is presently unknown. OBJECTIVE: We sought to sequence Ig genes from single cell clones of dominantly-expanded plasmablasts and generate recombinant human monoclonal antibodies to identify relevant antigens in IgG4-RD using mass spectrometry. METHODS: Paired heavy and light chain cDNAs from dominant plasmablast clones were expressed as monoclonal antibodies (mAbs) and used to purify antigens using immunoaffinity chromatography...
May 28, 2018: Journal of Allergy and Clinical Immunology
Zhaoping Liu, John O'Rourke
With advances in molecular engineering and humanization, monoclonal antibodies are one of the fastest-growing classes of biopharmaceuticals. During antibody discovery, antibody from hybridoma or primary B-cell supernatants is screened for the desired binding characteristics, and secondary screens measure antibody function and concentration, identify immunoglobulin G (IgG) isotype, and assess cell health. In order to expedite the antibody discovery process, we developed a high-throughput, multiplexed cell and bead-based competition assay that identifies and quantitates mouse IgG isotypes and assesses cell health...
May 1, 2018: SLAS Discovery
Joseph A Fraietta, Christopher L Nobles, Morgan A Sammons, Stefan Lundh, Shannon A Carty, Tyler J Reich, Alexandria P Cogdill, Jennifer J D Morrissette, Jamie E DeNizio, Shantan Reddy, Young Hwang, Mercy Gohil, Irina Kulikovskaya, Farzana Nazimuddin, Minnal Gupta, Fang Chen, John K Everett, Katherine A Alexander, Enrique Lin-Shiao, Marvin H Gee, Xiaojun Liu, Regina M Young, David Ambrose, Yan Wang, Jun Xu, Martha S Jordan, Katherine T Marcucci, Bruce L Levine, K Christopher Garcia, Yangbing Zhao, Michael Kalos, David L Porter, Rahul M Kohli, Simon F Lacey, Shelley L Berger, Frederic D Bushman, Carl H June, J Joseph Melenhorst
Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1-3 . In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5 . Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein...
May 30, 2018: Nature
Chang-Yu Chang, Fu-Ling Chang, Chen-Wei Chiang, Yan-Ni Lo, Tsai-Yu Lin, Wang-Chuan Chen, Keng-Chang Tsai, Yu-Ching Lee
To understand the mechanism for inhibition of hepatitis B virus (HBV) infection is important. In this study, single-chain variable fragment (scFv) antibodies were generated and directed to the pre-S2 epitope of HBV surface antigen (HBsAg). These human scFvs were isolated from a person with history of HBV infection by phage display technology. An evaluation of panning efficiency revealed that the eluted phage titer was increased, indicating that specific clones were enriched after panning. Selected scFvs were characterized with the recombinant HBsAg through Western blotting and enzyme-linked immunosorbent assay to confirm the binding ability...
May 30, 2018: Viral Immunology
Brian A Walker
Multiple myeloma is a malignancy of terminally differentiated plasma cells in the bone marrow. These plasma cells produce high levels of immunoglobulin which cause end-organ damage. Rearrangements within the immunoglobulin loci are a physiological part of B cell development, but these DNA level double-strand breaks may result in interchromosomal translocations. There are five main translocations involving the Ig loci: t(4;14) 12%, t(6;14) 1%, t(11;14) 15%, t(14;16) 3%, and t(14;20) 2%. These are primary events, found in all cells within the tumor clone and are associated with different prognosis...
2018: Methods in Molecular Biology
Jun S Wei, Igor B Kuznetsov, Shile Zhang, Young K Song, Shahab Asgharzadeh, Sivasish Sindiri, Xinyu Wen, Rajesh Patidar, Sushma Nagaraj, Ashley Walton, Jaime M Guidry Auvil, Daniela S Gerhard, Aysen Yuksel, Daniel R Catchpoole, Stephen M Hewitt, Paul M Sondel, Robert C Seeger, John M Maris, Javed Khan
PURPOSE: High-risk neuroblastoma is an aggressive disease. DNA sequencing studies have revealed a paucity of actionable genomic alterations and a low mutation burden, posing challenges to develop effective novel therapies. We used RNA sequencing (RNA-seq) to investigate the biology of this disease including a focus on tumor-infiltrating lymphocytes (TILs). EXPERIMENTAL DESIGN: We performed deep RNA-seq on pre-treatment diagnostic tumors from 129 high-risk and 21 low- or intermediate-risk patients with neuroblastomas...
May 21, 2018: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
Anna M Nicholson, Cora Olpe, Alice Hoyle, Ann-Sofie Thorsen, Teja Rus, Mathilde Colombé, Roxanne Brunton-Sim, Richard Kemp, Kate Marks, Phil Quirke, Shalini Malhotra, Rogier Ten Hoopen, Ashraf Ibrahim, Cecilia Lindskog, Meagan B Myers, Barbara Parsons, Simon Tavaré, Mark Wilkinson, Edward Morrissey, Douglas J Winton
We investigated the means and timing by which mutations become fixed in the human colonic epithelium by visualizing somatic clones and mathematical inference. Fixation requires two sequential steps. First, one of approximately seven active stem cells residing within each colonic crypt has to be mutated. Second, the mutated stem cell has to replace neighbors to populate the entire crypt in a process that takes several years. Subsequent clonal expansion due to crypt fission is infrequent for neutral mutations (around 0...
May 11, 2018: Cell Stem Cell
Katie Tungatt, Garry Dolton, Sophie B Morgan, Meriem Attaf, Anna Fuller, Thomas Whalley, Johanneke D Hemmink, Emily Porter, Barbara Szomolay, Maria Montoya, John A Hammond, John J Miles, David K Cole, Alain Townsend, Mick Bailey, Pierre J Rizkallah, Bryan Charleston, Elma Tchilian, Andrew K Sewell
There is increasing evidence that induction of local immune responses is a key component of effective vaccines. For respiratory pathogens, for example tuberculosis and influenza, aerosol delivery is being actively explored as a method to administer vaccine antigens. Current animal models used to study respiratory pathogens suffer from anatomical disparity with humans. The pig is a natural and important host of influenza viruses and is physiologically more comparable to humans than other animal models in terms of size, respiratory tract biology and volume...
May 2018: PLoS Pathogens
Manuel Buscarlet, Sylvie Provost, Yassamin Feroz Zada, Vincent Bourgoin, Luigina Mollica, Marie-Pierre Dubé, Lambert Busque
We analyzed DNA from PMN (granulocytes), CD14+ (monocytes), CD19+ (B-cells) and CD3+ (T-cells) of 107 individuals with clonal hematopoiesis of indeterminate potential (CHIP) to perform lineage restriction analysis of different gene mutations. Individuals were aged 55 to 96 (mean age: 70.0). Three lineage categories were defined: myeloid (PMN±monocytes), myelolympho-B (myeloid and B-cells), multipotent (myeloid, B and T-cells). Six individuals with aberrant patterns were excluded from analysis. Fifty-six had a single DNMT3A mutation, 24 had a single TET2 mutation, 7 had a single mutation in other genes (JAK2, ASXL1, CBL or TP53), and 14 had multiple mutations...
May 15, 2018: Blood
Alexsandro Dos Santos, Francine Campagnari, Ana Cristina Victorino Krepischi, Maria de Lourdes Ribeiro Câmara, Rita de Cássia E de Arruda Brasil, Ligia Vieira, Angela M Vianna-Morgante, Paulo A Otto, Peter L Pearson, Carla Rosenberg
A complex mosaicism of the short arm of chromosome 1 detected by SNP microarray analysis is described in a patient presenting a 4-Mb 1p36 terminal deletion and associated phenotypic features. The array pattern of chromosome 1p displayed an intriguing increase in divergence of the SNP heterozygote frequency from the expected 50% from the centromere towards the 1p36 breakpoint. This suggests that various overlapping segments of UPD were derived by somatic recombination between the 1p homologues. The most likely explanation was the occurrence of a series of events initiated in either a gamete or an early embryonic cell division involving a 1pter deletion rapidly followed by multiple telomere captures, resulting in additive, stepped increases in frequency of homozygosity towards the telomere...
May 12, 2018: Chromosome Research
Katsuhiko Ito, Kurako Kidokoro, Susumu Katsuma, Hideki Sezutsu, Keiro Uchino, Isao Kobayashi, Toshiki Tamura, Kimiko Yamamoto, Kazuei Mita, Toru Shimada, Keiko Kadono-Okuda
Bombyx mori densovirus type 1 (BmDV) is a pathogen that causes flacherie disease in the silkworm. The absolute nonsusceptibility to BmDV among certain silkworm strains is determined independently by two genes, nsd-1 and Nid-1. However, neither of these genes has been molecularly identified to date. Here, we isolated the nsd-1 gene by positional cloning and characterized the properties of its product, NSD-1. Sequence and biochemical analyses revealed that this gene encodes a Bombyx-specific mucin-like glycoprotein with a single transmembrane domain...
May 9, 2018: Scientific Reports
Nicole L Sullivan, Morgan A Reuter-Monslow, Janet Sei, Eberhard Durr, Carl W Davis, Cathy Chang, Megan McCausland, Andreas Wieland, David Krah, Nadine Rouphael, Aneesh K Mehta, Mark J Mulligan, Bali Pulendran, Rafi Ahmed, Kalpit A Vora
Herpes zoster (HZ/shingles) is the clinical manifestation of varicella zoster virus (VZV) reactivation. HZ typically develops as people age due to decreased cell-mediated immunity. However, the importance of antibodies for immunity against HZ prevention remains to be understood. The goal of this study was to examine the breadth and functionality of VZV-specific antibodies after vaccination with a live attenuated HZ vaccine (Zostavax). Direct enumeration of VZV-specific antibody secreting cells (ASC) via ELISPOT showed that Zostavax can induce both IgG and IgA ASCs seven days after vaccination, but not IgM ASCs...
May 9, 2018: Journal of Virology
Julius Lukes, Eliska Potuckova, Lucie Sramkova, Jan Stary, Julia Starkova, Jan Trka, Felix Votava, Jan Zuna, Marketa Zaliova
Fusion genes resulting from chromosomal rearrangements represent a hallmark of childhood acute lymphoblastic leukemia (ALL). Unlike more common fusion genes generated via simple reciprocal chromosomal translocations, formation of the ETV6-ABL1 fusion gene requires 3 DNA breaks and usually results from an interchromosomal insertion. We report a child with ALL in which a single interchromosomal insertion led to the formation of ETV6-ABL1 and two novel fusion genes: AIF1L-ETV6 and ABL1-AIF1L. We demonstrate the prenatal origin of this complex chromosomal rearrangement, which apparently initiated the leukemogenic process, by successful backtracking of the ETV6-ABL1 fusion into the patient's archived neonatal blood...
May 4, 2018: Genes, Chromosomes & Cancer
Laura Camacho-Jiménez, Alma B Peregrino-Uriarte, José A Martínez-Quintana, Gloria Yepiz-Plascencia
Some marine crustaceans like the white shrimp Litopenaeus vannamei are tolerant to environmental hypoxia. Under oxygen deprivation, shrimp tissues obtain energy by enhancing anaerobic glycolysis. In mammals, hypoxia increases the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which has been shown a "moonlighting" role in cells. However, the effect of hypoxia on the GAPDH expression has not been studied in crustaceans. In the present work, we obtained a 2744 bp gene sequence with a 999 bp ORF split by a single intron...
April 17, 2018: Marine Environmental Research
Agnès B Jousset, Rémy A Bonnin, Isabelle Rosinski-Chupin, Delphine Girlich, Gaëlle Cuzon, Nicolas Cabanel, Hélène Frech, Eric Farfour, Laurent Dortet, Philippe Glaser, Thierry Naas
Background: KPC-producing Klebsiella pneumoniae (KPC-Kp) have emerged globally over the last decade as a major nosocomial pathogen that threatens patient's care. These highly resistant bacteria are mostly associated with a single Kp clonal group (CG) CG258, but the reasons for its host and hospital adaptation remain largely unknown. Methods: We analyzed the in vivo evolution of a colistin-resistant KPC-Kp-CG258 strain that contaminated a patient following an endoscopy, and was responsible for a fatal bacteraemia 4...
April 21, 2018: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America
Lillian B Cohn, Israel T da Silva, Renan Valieris, Amy S Huang, Julio C C Lorenzi, Yehuda Z Cohen, Joy A Pai, Allison L Butler, Marina Caskey, Mila Jankovic, Michel C Nussenzweig
Despite suppressive combination antiretroviral therapy (ART), latent HIV-1 proviruses persist in patients. This latent reservoir is established within 48-72 h after infection, has a long half-life1,2 , enables viral rebound when ART is interrupted, and is the major barrier to a cure for HIV-1 3 . Latent cells are exceedingly rare in blood (∼1 per 1 × 106 CD4+ T cells) and are typically enumerated by indirect means, such as viral outgrowth assays4,5 . We report a new strategy to purify and characterize single reactivated latent cells from HIV-1-infected individuals on suppressive ART...
May 2018: Nature Medicine
Eric L Smith, Mette Staehr, Reed Masakayan, Ishan J Tatake, Terence J Purdon, Xiuyan Wang, Pei Wang, Hong Liu, Yiyang Xu, Sarah C Garrett-Thomson, Steven C Almo, Isabelle Riviere, Cheng Liu, Renier J Brentjens
B cell maturation antigen (BCMA) has recently been identified as an important multiple myeloma (MM)-specific target for chimeric antigen receptor (CAR) T cell therapy. In CAR T cell therapy targeting CD19 for lymphoma, host immune anti-murine CAR responses limited the efficacy of repeat dosing and possibly long-term persistence. This clinically relevant concern can be addressed by generating a CAR incorporating a human single-chain variable fragment (scFv). We screened a human B cell-derived scFv phage display library and identified a panel of BCMA-specific clones from which human CARs were engineered...
June 6, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
Joy E Tomlinson, Bettina Wagner, M Julia B Felippe, Gerlinde R Van de Walle
Immune phenotyping of equine peripheral blood mononuclear cells (PBMC) is commonly described by single or double marker labeling, which limits complex phenotypic descriptions and subpopulation identification. Our objective was to develop a new multispectral flow cytometry protocol to identify and sort equine lymphocyte subpopulations using commercially available, pre-conjugated monoclonal antibodies to cell surface markers. Two clones of anti-equine CD8 (CVS8 and CVS21) were compared in combination with CD3...
May 2018: Veterinary Immunology and Immunopathology
Shiny Nair, Joel Sng, Chandra Sekhar Boddupalli, Anja Seckinger, Marta Chesi, Mariateresa Fulciniti, Lin Zhang, Navin Rauniyar, Michael Lopez, Natalia Neparidze, Terri Parker, Nikhil C Munshi, Rachael Sexton, Bart Barlogie, Robert Orlowski, Leif Bergsagel, Dirk Hose, Richard A Flavell, Pramod K Mistry, Eric Meffre, Madhav V Dhodapkar
A role for antigen-driven stimulation has been proposed in the pathogenesis of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) based largely on the binding properties of monoclonal Ig. However, insights into antigen binding to clonal B cell receptors and in vivo responsiveness of the malignant clone to antigen-mediated stimulation are needed to understand the role of antigenic stimulation in tumor growth. Lysolipid-reactive clonal Ig were detected in Gaucher disease (GD) and some sporadic gammopathies...
April 19, 2018: JCI Insight
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