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single b cell cloning

David Rizzo, Pierre-Julien Viailly, Sylvain Mareschal, Elodie Bohers, Jean-Michel Picquenot, Dominique Penther, Sydney Dubois, Vinciane Marchand, Philippe Bertrand, Catherine Maingonnat, Pascaline Etancelin, Jean Feuillard, Christian Bastard, Hervé Tilly, Fabrice Jardin, Philippe Ruminy
Little is known on the phylogenetic relationship between diagnostic and relapse clones of Diffuse Large B-Cell Lymphoma (DLBCL). We applied High Throughput Sequencing (HTS) of the VDJ locus of Immunoglobulin Heavy chain (IGHV) on fourteen DLBCL patients with serial samples, including tumor biopsies and/or peripheral blood mononuclear cells (PBMC). Phylogenetic data were consolidated with targeted sequencing and cytogenetics. Phylogeny clearly showed that DLBCL relapse could occur according either an early or a late divergent mode...
October 13, 2016: American Journal of Hematology
Xie Dejian, Shi Minglei, Zhang Yan, Wang Tianyi, Shen Wenlong, Ye Bingyu, Li Ping, He Chao, Zhang Xiangyuan, Zhao Zhihu
The CCCTC-binding factor (CTCF) is the main insulator protein described in vertebrates. It plays fundamental roles during diverse cellular processes. CTCF gene knockout mice led to death during embryonic development. To further explore the functions of CTCF, we employed a CRISPR/Cas9-based genome engineering strategy to in-frame insert the mitosis-special degradation domain (MD) of cyclin B into the upstream open reading frame of CTCF gene. Fusion protein is designed to degrade during mitosis leaded by MD. As a control group, mutation of a single arginine (R42A) within the destruction box inactivates the MD leading to constitutive expression of MD(*)-CTCF...
July 20, 2016: Yi Chuan, Hereditas
Louis Pérol, John M Lindner, Pamela Caudana, Nicolas Gonzalo Nunez, Audrey Baeyens, Andrea Valle, Christine Sedlik, Delphine Loirat, Olivier Boyer, Alain Créange, José Laurent Cohen, Ute Christine Rogner, Jun Yamanouchi, Martine Marchant, Xavier Charles Leber, Meike Scharenberg, Marie-Claude Gagnerault, Roberto Mallone, Manuela Battaglia, Pere Santamaria, Agnès Hartemann, Elisabetta Traggiai, Eliane Piaggio
Type 1 diabetes (T1D) is characterized by a chronic, progressive autoimmune attack against pancreas-specific antigens, effecting the destruction of insulin-producing β-cells. Here we show interleukin-2 (IL-2) is a non-pancreatic autoimmune target in T1D. Anti-IL-2 autoantibodies, as well as T cells specific for a single orthologous epitope of IL-2, are present in the peripheral blood of non-obese diabetic (NOD) mice and patients with T1D. In NOD mice, the generation of anti-IL-2 autoantibodies is genetically determined and their titre increases with age and disease onset...
October 6, 2016: Nature Communications
Peter Kuhn, Anders Carlsson, Madelyn S Luttgen, Kevin Keomanee Dizon, Patricia Troncoso, Paul Corn, Anand Kolatkar, James B Hicks, Christopher Logothetis, Amado J Zurita
PURPOSE: Recent studies demonstrate that prostate cancer clones from different metastatic sites are dynamically represented in the blood of patients over time, suggesting that the paired evaluation of tumor cells in circulation and bone marrow, the primary target for prostate cancer metastasis, may provide complementary information. EXPERIMENTAL DESIGN: We adapted our single-cell high-content liquid biopsy platform to bone marrow aspirates (BMA), to concurrently identify and characterize prostate cancer cells in patients' blood and bone and thus discern features associated to tumorigenicity and dynamics of metastatic progression...
October 4, 2016: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
L Brimberg, S Mader, V Jeganathan, R Berlin, T R Coleman, P K Gregersen, P T Huerta, B T Volpe, B Diamond
Autism spectrum disorder (ASD) occurs in 1 in 68 births, preferentially affecting males. It encompasses a group of neurodevelopmental abnormalities characterized by impaired social interaction and communication, stereotypic behaviors and motor dysfunction. Although recent advances implicate maternal brain-reactive antibodies in a causative role in ASD, a definitive assessment of their pathogenic potential requires cloning of such antibodies. Here, we describe the isolation and characterization of monoclonal brain-reactive antibodies from blood of women with brain-reactive serology and a child with ASD...
October 4, 2016: Molecular Psychiatry
Rachael J M Bashford-Rogers, Anne L Palser, Clare Hodkinson, Joanna Baxter, George A Follows, George S Vassiliou, Paul Kellam
Chronic lymphocytic leukaemia (CLL) is characterised by the accumulation of clonally-derived mature CD5(high) B-cells, however the cellular origin of CLL is still unknown. Patients with CLL also harbour variable numbers of CD5(low) B-cells, but the clonal relationship of these cells to the bulk disease is unknown and can have important implications for monitoring, treating and understanding the biology of CLL. Here we use B-cell receptors (BCRs) as molecular barcodes to first show that the great majority of CD5(low) B-cells in the blood of CLL patients are clonally related to CD5(high) CLL B-cells by single-cell BCR sequencing...
September 27, 2016: Experimental Hematology
M Krull, I Klare, B Ross, R Trenschel, D W Beelen, D Todt, E Steinmann, J Buer, P-M Rath, J Steinmann
BACKGROUND: Prevalence of vancomycin-resistant enterococci has increased in Germany. Here, we report the cluster of linezolid- and vancomycin-resistant Enterococcus faecium (LVRE) in a German department for hematologic stem cell transplantation (HSCT). METHODS: In this retrospective analysis we included all patients with LVRE in a university-based department for HSCT in 2014 and 2015. Patients chart reviews were used to investigate the epidemiology and clinical outcome...
2016: Antimicrobial Resistance and Infection Control
Jiun-Bo Chen, Louisa K James, Anna M Davies, Y-C Wu, Joanne Rimmer, Valerie J Lund, Jou-Han Chen, James M McDonnell, Yih-Chih Chan, George H Hutchins, Tse Wen Chang, Brian J Sutton, Harsha H Kariyawasam, Hannah J Gould
BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with local immunoglobulin hyperproduction and the presence of IgE antibodies against Staphylococcus aureus enterotoxins (SAEs). Aspirin-exacerbated respiratory disease (AERD) is a severe form of CRSwNP in which nearly all patients express anti-SAEs. OBJECTIVES: We aimed to understand antibodies reactive to SAEs and determine whether they recognize SAEs through their complementarity-determining regions (CDRs) or framework regions (FRs)...
September 19, 2016: Journal of Allergy and Clinical Immunology
Colm Keane, Clare Gould, Kimberley Jones, David Hamm, Dipti Talaulikar, Jonathan Ellis, Frank Vari, Simone Birch, Erica Han, Peter Wood, Kim-Anh Le Cao, Michael R Green, Pauline Crooks, Sanjiv Jain, Joshua Tobin, Raymond J Steptoe, Maher K Gandhi
PURPOSE: To investigate the relationship between the intra-tumoral T-cell receptor (TCR) repertoire and the tumor microenvironment (TME) in de novo Diffuse Large B-cell Lymphoma (DLBCL), and the TCR's impact on survival. EXPERIMENTAL DESIGN: We performed high-throughput unbiased TCRBeta sequencing on a population based cohort of 92 DLBCL patients treated with conventional (i.e. non-checkpoint blockade) frontline 'R-CHOP' therapy. Key immune checkpoint genes within the TME were digitally quantified by nanoString{trade mark, serif}...
September 20, 2016: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
Marianoel Pereira-Gómez, Juan-Vicente Bou, Iván Andreu, Rafael Sanjuán
The high levels of genetic diversity shown by hepatitis B virus (HBV) are commonly attributed to the low fidelity of its polymerase. However, the rate of spontaneous mutation of human HBV in vivo is currently unknown. Here, based on the evolutionary principle that the population frequency of lethal mutations equals the rate at which they are produced, we have estimated the mutation rate of HBV in vivo by scoring premature stop codons in 621 publicly available, full-length, molecular clone sequences derived from patients...
2016: PloS One
Jinny L Liu, Dan Zabetakis, Ellen R Goldman, George P Anderson
CD20 is a membrane protein with four integral membrane regions and a large extracellular loop between residues 142 and 187, which serves as a target binding region for rituximab (RTX) and most other anti-CD20 monoclonal antibodies. It is highly expressed in B-lymphoma cells and B lymphocytes and often serves as a target for immunotherapy. In this study, we developed single domain antibodies (sdAbs) for the sensitive detection of CD20. To achieve this, an immune sdAb library was prepared from llamas immunized with a fusion between the large loop from CD20 and Hoc, a highly antigenic protein from the T4 bacteriophage, (CD20-Hoc)...
October 2016: Molecular Immunology
Bahador Behrouz, Nour Amirmozafari, Nima Khoramabadi, Mahboobeh Bahroudi, Parisa Legaee, Mehdi Mahdavi
BACKGROUND: Pseudomonas aeruginosa is an important opportunistic human pathogen that causes serious infections in immunocompromised hosts. The single polar flagellum is an important factor in both virulence and colonization. OBJECTIVES: As flagellin is the major component of the flagellar filament, the main aims of the present study are to identify, clone, express, and purify the recombinant type B flagellin (r-B-flagellin) of P. aeruginosa, as well as to evaluate the functional activity of the rabbit polyclonal antibody raised against this r-B-flagellin...
June 2016: Iranian Red Crescent Medical Journal
Irvin Y Ho, Jeffrey J Bunker, Steven A Erickson, Karlynn E Neu, Min Huang, Mario Cortese, Bali Pulendran, Patrick C Wilson
Generating monoclonal antibodies from single B cells is a valuable tool for characterizing the specificity and functional properties of humoral responses. We and others developed protocols that have facilitated major advances in our understanding of B cell development, tolerance, and effector responses to HIV and influenza. Here, we demonstrate various refinements and dramatically reduce the time required to produce recombinant antibodies. Further, we present new methods for cloning and isolating antibodies from cells with lower immunoglobulin mRNA levels that may be resistant to traditional techniques...
November 2016: Journal of Immunological Methods
David B Page, Jianda Yuan, David Redmond, Y Hanna Wen, Jeremy C Durack, Ryan Emerson, Stephen Solomon, Zhiwan Dong, Phillip Wong, Christopher Comstock, Adi Diab, Janice Sung, Majid Maybody, Elizabeth Morris, Edi Brogi, Monica Morrow, Virgilio Sacchini, Olivier Elemento, Harlan Robins, Sujata Patil, James P Allison, Jedd D Wolchok, Clifford Hudis, Larry Norton, Heather L McArthur
In early-stage breast cancer, the degree of tumor-infiltrating lymphocytes (TIL) predicts response to chemotherapy and overall survival. Combination immunotherapy with immune checkpoint antibody plus tumor cryoablation can induce lymphocytic infiltrates and improve survival in mice. We used T-cell receptor (TCR) DNA sequencing to evaluate both the effect of cryoimmunotherapy in humans and the feasibility of TCR sequencing in early-stage breast cancer. In a pilot clinical trial, 18 women with early-stage breast cancer were treated preoperatively with cryoablation, single-dose anti-CTLA-4 (ipilimumab), or cryoablation + ipilimumab...
October 2016: Cancer Immunology Research
Asim Azhar, Ejaj Ahmad, Qamar Zia, Mohammad Owais, Ghulam Md Ashraf
Therapeutic proteins are engineered proteins produced in the laboratory for pharmaceutical use. With the advent of recombinant DNA technology, the proteins can be generated in specific host cells under defined conditions. In the process of production of genetically engineered animals, the gene of interest can be added at a single cell stage to produce a cloned animal from genetically engineered cells. Several recombinant cytokines, clotting factors etc have been licensed and are currently being utilized for the treatment of cancer, infectious diseases, hemophilia, anemia, multiple sclerosis, and hepatitis B/C...
September 1, 2016: Current Protein & Peptide Science
Tao Xu, Xiaoe Li, You Wu, Khawar Ali Shahzad, Wei Wang, Lei Zhang, Chuanlai Shen
The time-consuming and high-cost preparation of soluble pMHC complexes currently limits their wide uses in monitoring antigen-specific T cells. The single-chain trimer (SCT) of peptide-β2m-MHC class I heavy chain has then developed as an alternative strategy, but its gene fusion is hindered in many cases due to the incompatibility between the multiple restriction enzymes and the restriction endonuclease sites of plasmid vectors. In this study, overlap extension PCR and One-step cloning were adopted to overcome this restriction...
September 2, 2016: Journal of Microbiology and Biotechnology
Chaoyang Chen, Haodi Jia, Fei Zhang, Yanli Qin, Li Zong, Quan Yuan, Yongxiang Wang, Ningshao Xia, Jisu Li, Yumei Wen, Shuping Tong
Hepatitis B virus (HBV) has a 3.2-kb circular DNA genome. It employs four promoters in conjunction with a single polyadenylation signal to generate 3.5-, 2.4-, 2.1-, and 0.7-kb co-terminal RNAs. The 3.5-kb RNA is subdivided into the precore RNA for e antigen expression and pregenomic RNA for genome replication. When introduced to a genotype A clone, several core promoter mutations markedly enhanced HBV genome replication but suppressed e antigen expression through up regulation of pregenomic RNA at the expense of precore RNA...
August 23, 2016: Journal of General Virology
Jakob Kreye, Nina K Wenke, Mariya Chayka, Jonas Leubner, Rajagopal Murugan, Nikolaus Maier, Betty Jurek, Lam-Thanh Ly, Doreen Brandl, Benjamin R Rost, Alexander Stumpf, Paulina Schulz, Helena Radbruch, Anja E Hauser, Florence Pache, Andreas Meisel, Lutz Harms, Friedemann Paul, Ulrich Dirnagl, Craig Garner, Dietmar Schmitz, Hedda Wardemann, Harald Prüss
SEE ZEKERIDOU AND LENNON DOI101093/AWW213 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a recently discovered autoimmune syndrome associated with psychosis, dyskinesias, and seizures. Little is known about the cerebrospinal fluid autoantibody repertoire. Antibodies against the NR1 subunit of the NMDAR are thought to be pathogenic; however, direct proof is lacking as previous experiments could not distinguish the contribution of further anti-neuronal antibodies...
October 2016: Brain: a Journal of Neurology
Madori Osawa, Sohtaro Mine, Shinichiro Ota, Kengo Kato, Tsuyoshi Sekizuka, Makoto Kuroda, Michiyo Kataoka, Hitomi Fukumoto, Yuko Sato, Takayuki Kanno, Hideki Hasegawa, Keiji Ueda, Masashi Fukayama, Takuya Maeda, Soichiro Kanoh, Akihiko Kawana, Yuji Fujikura, Harutaka Katano
BACKGROUND: Primary effusion lymphoma is a rare distinct large B-cell neoplasm that is associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Over recent years, 9 KSHV-positive/Epstein-Barr virus (EBV)-negative PEL cell lines have been established. METHODS: Tumor cells were collected from the pleural effusion of a 49-year-old male with AIDS. Cells were grown in RPMI1640 culture medium supplemented with 10 % fetus bovine serum. Single cell cloning was performed successfully by a limiting dilution method in a 96-well plate...
2016: Infectious Agents and Cancer
Ditte L Hedegaard, Damien C Tully, Ian A Rowe, Gary M Reynolds, David J Bean, Ke Hu, Christopher Davis, Annika Wilhelm, Colin B Ogilvie, Karen A Power, Alexander W Tarr, Deirdre Kelly, Todd M Allen, Peter Balfe, Jane A McKeating
BACKGROUND & AIMS: The high replication and mutation rate of hepatitis C virus (HCV) results in a heterogeneous population of viral sequences in vivo. HCV replicates in the liver and infected hepatocytes occur as foci surrounded by uninfected cells that may promote compartmentalization of viral variants. Given recent reports showing interferon stimulated gene (ISG) expression in chronic hepatitis C, we hypothesized that local interferon responses may limit HCV replication and evolution...
August 13, 2016: Journal of Hepatology
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