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Sample records for monoclonal plasma cell

  1. Efficient Methods To Isolate Human Monoclonal Antibodies from Memory B Cells and Plasma Cells.

    Science.gov (United States)

    Corti, Davide; Lanzavecchia, Antonio

    2014-10-01

    In this article, we highlight the advantages of isolating human monoclonal antibodies from the human memory B cells and plasma cell repertoires by using high-throughput cellular screens. Memory B cells are immortalized with high efficiency using Epstein-Barr virus (EBV) in the presence of a toll-like receptor (TLR) agonist, while plasma cells are maintained in single-cell cultures by using interleukin 6 (IL-6) or stromal cells. In both cases, multiple parallel assays, including functional assays, can be used to identify rare cells that produce antibodies with unique properties. Using these methods, we have isolated potent and broadly neutralizing antibodies against a variety of viruses, in particular, a pan-influenza-A-neutralizing antibody and an antibody that neutralizes four different paramyxoviruses. Given the high throughput and the possibility of directly screening for function (rather than just binding), these methods are instrumental to implement a target-agnostic approach to identify the most effective antibodies and, consequently, the most promising targets for vaccine design. This approach is exemplified by the identification of unusually potent cytomegalovirus-neutralizing antibodies that led to the identification of the target, a pentameric complex that we are developing as a candidate vaccine.

  2. Cytogenetic profiles in multiple myeloma and monoclonal gammopathy of undetermined significance: a study in highly purified aberrant plasma cells.

    Science.gov (United States)

    Schmidt-Hieber, Martin; Gutiérrez, María Laura; Pérez-Andrés, Martin; Paiva, Bruno; Rasillo, Ana; Tabernero, Maria Dolores; Sayagués, José Maria; Lopez, Antonio; Bárcena, Paloma; Sanchez, María Luz; Gutiérrez, Norma C; San Miguel, Jesus F; Orfao, Alberto

    2013-02-01

    Cytogenetic studies in clonal plasma cell disorders have mainly been done in whole bone marrow or CD138(+) microbead-enriched plasma cells and suggest that recurrent immunoglobulin heavy chain translocations - e.g. t(4;14) -are primary oncogenetic events. The aim of this study was to determine cytogenetic patterns of highly purified aberrant plasma cells (median purity ≥ 98%) in different clonal plasma cell disorders. We analyzed aberrant plasma cells from 208 patients with multiple myeloma (n=148) and monoclonal gammopathy of undetermined significance (n=60) for the presence of del(13q14), del(17p13) and t(14q32) using multicolor interphase fluorescence in situ hybridization. Additionally, immunoglobulin heavy chain gene arrangements were analyzed and complementarity determining region 3 was sequenced in a subset of patients and combined multicolor interphase fluorescence in situ hybridization/immunofluorescent protein staining analyses were performed in selected cases to confirm clonality and cytogenetic findings. At diagnosis, 96% of cases with multiple myeloma versus 77% of monoclonal gammopathy of undetermined significance cases showed at least one cytogenetic alteration and/or hyperdiploidy. The cytogenetic heterogeneity of individual cases reflected coexistence of cytogenetically-defined aberrant plasma cell clones, and led to the assumption that karyotypic alterations were acquired stepwise. Cases of multiple myeloma and monoclonal gammopathy of undetermined significance frequently showed different but related cytogenetic profiles when other cytogenetic alterations such as deletions/gains of the immunoglobulin heavy chain or the fibroblast growth factor receptor 3 were additionally considered. Interestingly, in 24% of multiple myeloma versus 62% of monoclonal gammopathy of undetermined significance patients with an immunoglobulin heavy chain translocation, aberrant plasma cells with and without t(14q32) coexisted in the same patient. Our data suggest that

  3. Target-selective homologous recombination cloning for high-throughput generation of monoclonal antibodies from single plasma cells

    Directory of Open Access Journals (Sweden)

    Isobe Masaharu

    2011-04-01

    Full Text Available Abstract Background Molecular cloning of functional immunoglobulin genes from single plasma cells is one of the most promising technologies for the rapid development of monoclonal antibody drugs. However, the proper insertion of PCR-amplified immunoglobulin genes into expression vectors remains an obstacle to the high-throughput production of recombinant monoclonal antibodies. Results We developed a single-step cloning method, target-selective homologous recombination (TS-HR, in which PCR-amplified immunoglobulin variable genes were selectively inserted into vectors, even in the presence of nonspecifically amplified DNA. TS-HR utilizes Red/ET-mediated homologous recombination with a target-selective vector (TS-vector with unique homology arms on its termini. Using TS-HR, immunoglobulin variable genes were cloned directly into expression vectors by co-transforming unpurified PCR products and the TS-vector into E. coli. Furthermore, the high cloning specificity of TS-HR allowed plasmids to be extracted from pools of transformed bacteria without screening single colonies for correct clones. We present a one-week protocol for the production of recombinant mouse monoclonal antibodies from large numbers of single plasma cells. Conclusion The time requirements and limitations of traditional cloning procedures for the production of recombinant immunoglobulins have been significantly reduced with the development of the TS-HR cloning technique.

  4. Infiltration patterns in monoclonal plasma cell disorders: correlation of magnetic resonance imaging with matched bone marrow histology

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    Andrulis, Mindaugas [Institute of Pathology, University of Heidelberg, Heidelberg (Germany); Bäuerle, Tobias [Department of Diagnostic and Interventional Radiology, University of Hamburg, Hamburg (Germany); Goldschmidt, Hartmut [Department of Hematology and Oncology, University of Heidelberg, Heidelberg (Germany); Delorme, Stefan [Department of Radiology, German Cancer Research Center (DKFZ), Heidelberg (Germany); Landgren, Ola [Multiple Myeloma Section, Metabolism Branch, National Cancer Institute, Bethesda (United States); Schirmacher, Peter [Institute of Pathology, University of Heidelberg, Heidelberg (Germany); Hillengass, Jens, E-mail: jens.hillengass@med.uni-heidelberg.de [Department of Hematology and Oncology, University of Heidelberg, Heidelberg (Germany); Department of Radiology, German Cancer Research Center (DKFZ), Heidelberg (Germany)

    2014-06-15

    Objectives: To investigate how plasma cell infiltration patterns detected by MRI match the plasma cell distribution in bone marrow biopsy. Methods: We assessed 50 patients with monoclonal plasma cell disorders of all clinical stages. MRI infiltration pattern was compared with matched BM histology from the same anatomic region. Results: MRI revealed a minimal (n = 11, 22%), focal (n = 5, 10%), diffuse (n = 14, 28%) and mixed (n = 20, 40%) infiltration pattern. Diffuse MRI pattern was predominant in smoldering myeloma patients whereas the MRI patterns with “focal component” (i.e. focal and mixed) were most common in symptomatic myeloma (p < 0.01). In histology an interstitial (n = 13, 26%), nodular (n = 23, 46%) and packed marrow (n = 14, 28%) was found respectively. All three histological types of infiltration were observed in patients with diffuse and mixed MRI patterns. Minimal MRI pattern was found in all MGUS patients and was associated with an interstitial BM infiltration. In two patients with minimal MRI pattern an extensive micro-nodular BM infiltration was found in histology. Conclusions: Infiltration patterns in MRI represent different histological growth patterns of plasma cells, but the MRI resolution is not sufficient to visualize micro-nodular aggregates of plasma cells.

  5. Advances in the treatment of monoclonal gammopaties: The emerging role of targeted therapy in plasma cell dyscrasias

    Directory of Open Access Journals (Sweden)

    Aldo M Roccaro

    2008-09-01

    Full Text Available Aldo M Roccaro1, Irene M Ghobrial1, Simona Blotta1, Steven P Treon1, Michele Malagola2, Kenneth C Anderson1, Paul G Richardson1, Domenico Russo21Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA; 2Unit of Blood Diseases and Cell Therapies, University of Brescia Medical School, Brescia, ItalyAbstract: The paradigm for the treatment of monoclonal gammopaties has dramatically changed: therapeutic options in multiple myeloma (MM have evolved from the introduction of melphalan and prednisone in the 1960s, high-dose chemotherapy and stem cell transplantation in the late 1980s and 1990s, to the rapid introduction of small novel molecules within the last seven years. Based on the understanding of the complex interaction of the MM cells with the bone marrow microenvironment and the signaling pathways that are dysregulated in this process, a number of novel therapeutic agents are now available. Specifically, three novel agents with a specific-targeted anti-MM activity, have been FDA-approved for the treatment of this disease, namely Bortezomib, thalidomide, and lenalidomide which are now all playing a key role in the treatment of MM. The success of targeted therapy in MM has since led to the development and investigation of more than 30 new compounds in this disease and in other plasma cell dyscrasias such as Waldenström’s macroglobulinemia and primary amyloidosis, both in the preclinical settings and as part of clinical trials.Keywords: monoclonal gammopaties, targeted therapies

  6. Imaging of multiple myeloma and related monoclonal plasma cell diseases. An update; Bildgebung des multiplen Myeloms und verwandter monoklonaler Plasmazellerkrankungen. Ein Update

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    Weber, Marc-Andre [Universitaetsklinikum, Heidelberg (Germany). Abt. Diagnostische und Interventionelle Radiologie; Delorme, Stefan [Deutsches Krebsforschungszentrum, Heidelberg (Germany). Abt. Radiologie; Hillengass, Jens [Universitaetsklinikum, Heidelberg (Germany). Sektion Multiples Myelom

    2014-09-15

    Multiple myeloma is a hematologic disorder characterized by the infiltration and proliferation of monoclonal plasma cells mainly in the bone marrow. The main symptoms are hypercalcemia, renal impairment, cytopenia/anemia and bone disease - summarized as CRAB-criteria. Symptomatic multiple myeloma is consistently preceded by asymptomatic premalignant stages called monoclonal gammopathy of undetermined significance and smoldering multiple myeloma. Staging of multiple myeloma is based on the measurement of the monoclonal protein in serum and urine as well as the assessment of impairment of hematopoiesis, renal function and mineralized bone. In the last decade the development of novel therapeutic agents has led to an increase in response rates and survival time of patients with multiple myeloma, which further stresses the value of response assessment by imaging. Cross sectional imaging like MRI and CT is currently replacing conventional radiological surveys in the initial work-up and follow-up of patients with monoclonal plasma cell diseases. The added value of MRI is to improve initial staging by unraveling a diffuse infiltration of bone marrow by plasma cells, a focal pattern or a combination of both. Furthermore, a complete remission of myeloma confirmed by MRI and CT goes along with a better prognosis compared to a complete response based only on serological parameters.

  7. Chromosomal aberrations are shared by malignant plasma cells and a small fraction of circulating CD19+ cells in patients with myeloma and monoclonal gammopathy of undetermined significance

    National Research Council Canada - National Science Library

    Zojer, Niklas; Schuster-Kolbe, Judith; Assmann, Irene; Ackermann, Jutta; Strasser, Kathrin; Hubl, Wolfgang; Drach, Johannes; Ludwig, Heinz

    2002-01-01

    In the present study, we aimed to identify distinct structural and numerical chromosomal aberrations in peripheral blood B cells of patients with myeloma and monoclonal gammopathy of undetermined significance (MGUS...

  8. Interphase fluorescence in situ hybridization in multiple myeloma and monoclonal gammopathy of undetermined significance without and with positive plasma cell identification

    DEFF Research Database (Denmark)

    Christensen, Jacob H; Abildgaard, Niels; Plesner, Torben

    2007-01-01

    Interphase fluorescence in-situ hybridization (i-FISH) was used to investigate 192 patients with multiple myeloma (MM; n = 182) and benign monoclonal gammopathy of undetermined significance (MGUS; n = 10). Of the 182 MM cases, 132 were investigated without and 50 with positive plasma cell......32. Of these, translocations t(4;14) constituted 9% and t(11;14), 20%. Finally, based on the small number of cytogenetically abnormal cases, it is recommended to include cytogenetics (and, for example, the DNA index) in the prognostic armamentarium....

  9. Sensitivity of whole-body CT and MRI versus projection radiography in the detection of osteolyses in patients with monoclonal plasma cell disease

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    Wolf, Maya B., E-mail: m.mueller-wolf@dkfz.de [Department of Diagnostic and Interventional Radiology, University Hospital Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg Germany (Germany); Department of Radiology, German Cancer Research Center (Dkfz), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Murray, Fritz, E-mail: fritz.murray@hotmail.de [Department of Diagnostic and Interventional Radiology, University Hospital Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg Germany (Germany); Kilk, Kerstin, E-mail: k_fechtner@hotmail.com [Department of Diagnostic and Interventional Radiology, University Hospital Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg Germany (Germany); Hillengass, Jens, E-mail: jens.hillengass@med.uni-heidelberg.de [Department of Haematology, Oncology, Rheumatology, University Hospital Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg (Germany); Delorme, Stefan, E-mail: s.delorme@dkfz-heidelberg.de [Department of Radiology, German Cancer Research Center (Dkfz), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Heiss, Christiane, E-mail: c.heiss@dkfz-heidelberg.de [Department of Biostatistics, German Cancer Research Center (Dkfz), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Neben, Kai, E-mail: k.neben@klinikum-mittelbaden.de [Department of Haematology, Oncology, Rheumatology, University Hospital Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg (Germany); Goldschmidt, Hartmut, E-mail: hartmut.goldschmidt@med.uni-heidelberg.de [Department of Haematology, Oncology, Rheumatology, University Hospital Heidelberg and National Center for Tumour Diseases, Im Neuenheimer Feld 410, 69120 Heidelberg (Germany); Kauczor, Hans-Ulrich, E-mail: hu.kauczor@med.uni-heidelberg.de [Department of Diagnostic and Interventional Radiology, University Hospital Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg Germany (Germany); and others

    2014-07-15

    Purpose: To compare sensitivity of whole-body Computed Tomography (wb-CT) and whole-body Magnetic Resonance Imaging (wb-MRI) with Projection Radiography (PR) regarding each method's ability to detect osteolyses in patients with monoclonal plasma cell disease. Patients and methods: The bone status of 171 patients was evaluated. All patients presented with multiple myeloma (MM) of all stages, monoclonal gammopathy of unknown significance (MGUS) or solitary plasmacytoma. Two groups were formed. Group A consisted of 52 patients (26 females, 26 males) with an average age of 62 years (range, 45–89 years) who received, both, PR and wb-CT as part of their diagnostic work-up. Group B comprised 119 patients (58 females, 61 males) averaging 57 years of age (range, 20–80 years) who received, both, PR and wb-MRI. Two experienced radiologists were blinded regarding the disease status and assessed the number and location of osteolyses in consensus. A distinction was made between axial and extra-axial lesions. Results: In group A, wb-CT revealed osteolyses in 12 patients (23%) that were not detected in PR. CT was superior in detecting lesions in patients with osteopenia and osteoporosis. Compared with PR, wb-CT was significantly more sensitive in detecting osteolyses than PR (p < 0.001). This was particularly true for axial lesions. Additionally, CT revealed clinically relevant incidental findings in 33 patients (63%). In group B, wb-MRI revealed lesions in 19 patients (16%) that were not detected in PR. All lesions detected by PR were also detected by wb-MRI and wb-CT. Wb-MRI and wb-CT are each superior to PR in detecting axial lesions. Conclusion: Wb-CT can detect 23% more focal lesions than PR, especially in the axial skeleton. Therefore, this imaging method should be preferred over PR in the diagnostic work-up and staging of patients with monoclonal plasma cell disease.

  10. Sperm-immobilizing monoclonal antibody to human seminal plasma antigens.

    Science.gov (United States)

    Shigeta, M; Watanabe, T; Maruyama, S; Koyama, K; Isojima, S

    1980-01-01

    Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6783353

  11. Monoclonal antibodies directed against protoplasts of soybean cells: analysis of the lateral mobility of plasma membrane-bound antibody MVS-1.

    Science.gov (United States)

    Metcalf, T N; Villanueva, M A; Schindler, M; Wang, J L

    1986-04-01

    A monoclonal antibody (MVS-1) was used to monitor the lateral mobility of a defined component (Mr approximately 400,000) of the plasma membrane of soybean protoplasts prepared from suspension cultures of Glycine max (SB-1 cell line). The diffusion coefficient (D) of antibody MVS-1 bound to its target was determined (D = 3.2 X 10(-10) cm2/s) by fluorescence redistribution after photobleaching. Pretreatment of the protoplasts with soybean agglutinin (SBA) resulted in a 10-fold reduction of the lateral mobility of antibody MVS-1 (D = 4.1 X 10(-11) cm2/s). This lectin-induced modulation could be partially reversed by prior treatment of the protoplasts with either colchicine or cytochalasin B. When used together, these drugs completely reversed the modulation effect induced by SBA. These results have refined our previous analysis of the effect of SBA on receptor mobility to the level of a defined receptor and suggest that the binding of SBA to the plasma membrane results in alterations in the plasma membrane such that the lateral diffusion of other receptors is restricted. These effects are most likely mediated by the cytoskeletal components of the plant cell.

  12. Plasma Cell Disorders.

    Science.gov (United States)

    Castillo, Jorge J

    2016-12-01

    Plasma cell disorders are benign, premalignant, and malignant conditions characterized by the presence of a monoclonal paraprotein detected in serum or urine. These conditions are biologically, pathologically, and clinically heterogeneous. There have been major advances in the understanding of the biology of these diseases, which are promoting the development of therapies with novel mechanisms of action. Novel agents such as proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies have gained approval in the United States and Europe for the treatment of plasma cell disorders. Such therapies are translating into higher rates of response and survival and better toxicity profiles. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Secondary monoclonal gammopathy of undetermined significance is frequently associated with high response rate and superior survival in patients with plasma cell dyscrasias.

    Science.gov (United States)

    Zou, Dehui; An, Gang; Zhu, Guoqing; Wang, Jinhong; Shi, Lihui; Meng, Hengxing; Xu, Yan; Sui, Weiwei; Deng, Shuhui; Zhan, Fenghuang; Qiu, Lugui

    2014-03-01

    Secondary monoclonal gammopathy of undetermined significance (MGUS) is a special phenomenon that occurs during the treatment of multiple myeloma (MM). The incidence, biological characteristics, and prognostic value of secondary MGUS in patients with MM remain undefined. We proceed with a retrospective systematic review of serum immunofixation electrophoresis studies performed in 438 cases of patients with plasma cell dyscrasias, including 409 cases of newly diagnosed MM and 29 cases of primary plasma cell leukemia. Secondary MGUS was more common in patients with myeloma who had undergone stem cell transplantation than in those who had not (17 [29.8%] of 57 versus 5 [1.4%] of 352, P < .001). The clinical parameters and cytogenetic characteristics in patients with or without secondary MGUS were comparable. The complete response rates in patients with or without secondary MGUS were 81.8% and 21.8% respectively (P < .01). For the cohort as a whole, secondary MGUS was associated with significantly prolonged progression-free survival (median, 52.0 months versus 22.5 months; P = .002) and overall survival (median, not reached versus 35.0 months; P < .001). The presence of secondary MGUS retained independent prognostic value with a moderate impact on overall survival (hazard ratio .128 [95% confidence interval .018 to .922]; P = .041) in the multivariate Cox regression model. However, when analysis was restricted to patients undergoing stem cell transplantation, no statistical differences in progression-free survival and overall survival were found. In conclusion, we observe that secondary MGUS was frequently observed in MM patients after transplantation and conferred a survival prolongation. The favorable survival in patients with secondary MGUS may be explained by beneficial effect from myeloablative therapy. Copyright © 2014 American Society for Blood and Marrow Transplantation. All rights reserved.

  14. Monoclonal antibodies reactive with hairy cell leukemia

    NARCIS (Netherlands)

    Visser, L; Shaw, A; Slupsky, J; Vos, H; Poppema, S

    Monoclonal antibodies reactive with hairy cell leukemia were developed to aid in the diagnosis of this subtype of B cell chronic lymphocytic leukemia and to gain better insight into the origin of hairy cells. Three antibodies were found to be of value in the diagnosis of hairy cell leukemia.

  15. Monoclonal antibodies reactive with hairy cell leukemia

    NARCIS (Netherlands)

    Visser, L; Shaw, A; Slupsky, J; Vos, H; Poppema, S

    1989-01-01

    Monoclonal antibodies reactive with hairy cell leukemia were developed to aid in the diagnosis of this subtype of B cell chronic lymphocytic leukemia and to gain better insight into the origin of hairy cells. Three antibodies were found to be of value in the diagnosis of hairy cell leukemia. Antibod

  16. The application of Gadopentate-Dimeneglumin has no impact on progression free and overall survival as well as renal function in patients with monoclonal plasma cell disorders if general precautions are taken

    Energy Technology Data Exchange (ETDEWEB)

    Hillengass, J. [University of Heidelberg, Department of Hematology, Oncology and Rheumatology, Heidelberg (Germany); German Cancer Research Center Heidelberg, Department of Radiology, Heidelberg (Germany); Stoll, J.; Wagner, B.; Goldschmidt, H. [University of Heidelberg, Department of Hematology, Oncology and Rheumatology, Heidelberg (Germany); Zechmann, C.M. [Rinecker Proton Therapy Center, Munich (Germany); Kunz, C.; Heiss, C. [German Cancer Research Center Heidelberg, Department of Biostatistics, Heidelberg (Germany); Sumkauskaite, M. [University of Heidelberg, Department of Radiology, Heidelberg (Germany); Moehler, T.M. [InVentiv Health Clinical, Wiesbaden (Germany); Schlemmer, H.P.; Delorme, S. [German Cancer Research Center Heidelberg, Department of Radiology, Heidelberg (Germany)

    2014-10-31

    The current analysis investigated the prognostic significance of gadopentetate dimeglumine on survival and renal function in patients with monoclonal plasma cell disorders. In this study 263 patients who had received gadopentetate dimeglumine within a prospective trial investigating dynamic contrast-enhanced magnetic resonance imaging (MRI) were compared with 335 patients who had undergone routine, unenhanced MRI. We found no significant prognostic impact of the application of contrast agent on progression-free survival in patients with either monoclonal gammopathy of undetermined significance, smouldering or symptomatic myeloma and no significant prognostic impact on overall survival in patients with symptomatic myeloma. Since renal impairment is a frequent complication of myeloma, and decreased renal function is associated with a higher risk of complications in patients receiving contrast agents, we evaluated the impact of contrast agent on renal function after 1 year. In the present analysis the only significant adverse impact on kidney function occurred in symptomatic myeloma patients who already had impaired renal parameters at baseline. Here, the renal function did not recover during therapy, whereas it did so in patients with normal or only slightly impaired renal function. If general recommendations are adhered to, gadopentetate dimeglumine can be safely applied in patients with monoclonal plasma cell disease. (orig.)

  17. Monoclonal paraprotein influences baseline B-cell repertoire diversity and perturbates influenza vaccination-induced B-cell response

    NARCIS (Netherlands)

    Tete, Sarah M.; Kipling, David; Westra, Johanna; de Haan, Aalzen; Bijl, Marc; Dunn-Walters, Deborah K.; Sahota, Surinder S.; Bos, Nicolaas A.

    2015-01-01

    Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasma cells in the bone marrow, secreting monoclonal (M) paraprotein. It is associated with increased susceptibility to infections, which may reflect altered B-cell repertoire. To investigate this, we examin

  18. Monoclonal paraprotein influences baseline B-cell repertoire diversity and perturbates influenza vaccination-induced B-cell response

    NARCIS (Netherlands)

    Tete, Sarah M.; Kipling, David; Westra, Johanna; de Haan, Aalzen; Bijl, Marc; Dunn-Walters, Deborah K.; Sahota, Surinder S.; Bos, Nicolaas A.

    2015-01-01

    Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasma cells in the bone marrow, secreting monoclonal (M) paraprotein. It is associated with increased susceptibility to infections, which may reflect altered B-cell repertoire. To investigate this, we examin

  19. Aspectos morfológicos da infiltração da medula óssea por condições exibindo diferenciação plasmocitária e gamopatia monoclonal Morphological aspects of bone marrow infiltration by diseases characterized by plasma cell proliferation and monoclonal gamopathy

    Directory of Open Access Journals (Sweden)

    Victor P. Andrade

    2009-08-01

    Full Text Available As doenças linfoproliferativas com diferenciação plasmocitária e pico monoclonal são causas de dificuldade diagnóstica no estudo de espécimes de medula óssea. Conhecer os aspectos clínicos, morfológicos, imunofenotípicos e citogenéticos é fundamental para o diagnóstico correto. Descrevemos os aspectos práticos mais relevantes para a interpretação de biópsias de medula óssea frente às situações mais frequentes.Some lymphoproliferative diseases with plasma cell differentiation and monoclonal gammopathy are challenging to diagnose when dealing with bone marrow biopsies. Knowledge of clinical, morphological, phenotypic and cytogenetic aspects is crucial to establish the correct diagnosis. We describe practical relevant aspects that help in the interpretation of bone marrow biopsies in these situations.

  20. Circulating clonotypic B cells in multiple myeloma and monoclonal gammopathy of undetermined significance.

    Science.gov (United States)

    Thiago, Leandro S; Perez-Andres, Martin; Balanzategui, Ana; Sarasquete, Maria E; Paiva, Bruno; Jara-Acevedo, Maria; Barcena, Paloma; Sanchez, Maria Luz; Almeida, Julia; González, Marcos; San Miguel, Jesus F; Garcia-Sanz, Ramón; Orfao, Alberto

    2014-01-01

    The B-cell compartment in which multiple myeloma stem cells reside remains unclear. We investigated the potential presence of mature, surface-membrane immunoglobulin-positive B lymphocytes clonally related to the tumor bone marrow plasma cells among different subsets of peripheral blood B cells from ten patients (7 with multiple myeloma and 3 with monoclonal gammopathies of undetermined significance). The presence of clonotypic immunoglobulin heavy chain gene rearrangements was determined in multiple highly-purified fractions of peripheral blood B-lymphocytes including surface-membrane IgM(+) CD27(-) naïve B-lymphocytes, plus surface-membrane IgG(+), IgA(+) and IgM(+) memory CD27(+) B cells, and normal circulating plasma cells, in addition to (mono)clonal plasma cells, by a highly-specific and sensitive allele-specific oligonucleotide polymerase chain reaction directed to the CDR3 sequence of the rearranged IGH gene of tumor plasma cells from individual patients. Our results showed systematic absence of clonotypic rearrangements in all the different B-cell subsets analyzed, including M-component isotype-matched memory B-lymphocytes, at frequencies undetermined significance are usually devoid of clonotypic B cells while the presence of immunophenotypically aberrant myeloma plasma cells in peripheral blood of myeloma patients is a relatively frequent finding.

  1. [Single B cell monoclonal antibody technologies and applications].

    Science.gov (United States)

    Chi, Xiangyang; Yu, Changming; Chen, Wei

    2012-06-01

    Monoclonal antibodies (mAbs) contribute a lot to the development of numerous fields in life science as a pivotal tool in modern biological research. Development of the PCR methods and maturation of antibody production have made it possible to generate mAbs from single human B cells by single cell RT-PCR with successional cloning and expression in vitro. Compared to traditional monoclonal antibody technologies, single B cell technologies require relatively fewer cells, which are highly efficient in obtaining specific mAbs in a rapid way with preservation of the natural heavy and light chain pairing. With so many advantages, single B cell technologies have been proved to be an attractive approach for retrieval of naive and antigen-experienced antibody repertoires generated in vivo, design of rationale structure-based vaccine, evaluation and development of basic B cell biology concepts in health and autoimmunity, and prevention of infectious diseases by passive immunization and therapy for disorders. Accordingly, this review introduced recent progresses in the single B cell technologies for generating monoclonal antibodies and applications.

  2. Immune defects in the risk of infection and response to vaccination in monoclonal gammopathy of undetermined significance and multiple myeloma

    NARCIS (Netherlands)

    Tete, Sarah M.; Bijl, Marc; Sahota, Surinder S.; Bos, Nicolaas

    2014-01-01

    The plasma cell proliferative disorders monoclonal gammopathy of undetermined significance (MGUS) and malignant multiple myeloma (MM) are characterized by an accumulation of transformed clonal plasma cells in the bone marrow and production of monoclonal immunoglobulin. They typically affect an older

  3. Cell culture processes for monoclonal antibody production

    OpenAIRE

    LI Feng; Vijayasankaran, Natarajan; Shen, Amy (Yijuan); Kiss, Robert; Amanullah, Ashraf

    2010-01-01

    Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including (1) cell lines capable of synthesizin...

  4. Cell culture processes for monoclonal antibody production.

    Science.gov (United States)

    Li, Feng; Vijayasankaran, Natarajan; Shen, Amy Yijuan; Kiss, Robert; Amanullah, Ashraf

    2010-01-01

    Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including 1) cell lines capable of synthesizing the required molecules at high productivities that ensure low operating cost; 2) culture media and bioreactor culture conditions that achieve both the requisite productivity and meet product quality specifications; 3) appropriate on-line and off-line sensors capable of providing information that enhances process knowledge; and 4) good understanding of culture performance at different scales to ensure smooth scale-up. Successful implementation also requires appropriate strategies for process development, scale-up and process characterization and validation that enable robust operation that is compliant with current regulations. This review provides an overview of the state-of-the art technology in key aspects of cell culture, e.g., engineering of highly productive cell lines and optimization of cell culture process conditions. We also summarize the current thinking on appropriate process development strategies and process advances that might affect process development.

  5. Cell culture processes for monoclonal antibody production

    Science.gov (United States)

    Li, Feng; Vijayasankaran, Natarajan; Shen, Amy (Yijuan); Kiss, Robert

    2010-01-01

    Animal cell culture technology has advanced significantly over the last few decades and is now generally considered a reliable, robust and relatively mature technology. A range of biotherapeutics are currently synthesized using cell culture methods in large scale manufacturing facilities that produce products for both commercial use and clinical studies. The robust implementation of this technology requires optimization of a number of variables, including (1) cell lines capable of synthesizing the required molecules at high productivities that ensure low operating cost; (2) culture media and bioreactor culture conditions that achieve both the requisite productivity and meet product quality specifications; (3) appropriate on-line and off-line sensors capable of providing information that enhances process control; and (4) good understanding of culture performance at different scales to ensure smooth scale-up. Successful implementation also requires appropriate strategies for process development, scale-up and process characterization and validation that enable robust operation and ensure compliance with current regulations. This review provides an overview of the state-of-the art technology in key aspects of cell culture, e.g., generation of highly productive cell lines and optimization of cell culture process conditions. We also summarize the current thinking on appropriate process development strategies and process advances that might affect process development. PMID:20622510

  6. Hospital population screening reveals overrepresentation of CD5(-) monoclonal B-cell lymphocytosis and monoclonal gammopathy of undetermined significance of IgM type.

    Science.gov (United States)

    Voigtlaender, Minna; Vogler, Birthe; Trepel, Martin; Panse, Jens; Jung, Roman; Bokemeyer, Carsten; Bacher, Ulrike; Binder, Mascha

    2015-09-01

    Monoclonal B-cell lymphocytosis (MBL) and monoclonal gammopathy of undetermined significance (MGUS) result from clonal expansions of mature B or plasma cells. Here, we set out to determine the immunophenotypic/monoclonal immunoglobulin (M protein) features and co-prevalence of MBL and MGUS in a hospital-based cohort of 1909 non-hematooncological patients. Of the evaluable cases, 3.8 % showed evidence for MBL by immunophenotyping, while 9.8 % were screened positive for M protein by immunofixation. With six concomitant cases (0.4 %), MBL and MGUS were not statistically associated. At least in two of these coincident cases, MBL and MGUS were of different clonal origin since both clones had divergent light chain restriction. CD5(-) MBL (57.1 %) and IgM+ MGUS (24.7 %) were strikingly overrepresented compared to population-based screenings and did not progress to overt lymphoma or myeloma during the observation period (mean follow-up of 117 weeks or 110 weeks, respectively). Prevalence and phenotypes suggest that a substantial proportion of incidental MBL and MGUS in hospitalized patients may be attributed to transiently expanded B-cell clones in the context of disease-related immune stimulation rather than reflecting veritable precursors of clonal B-cell malignancies.

  7. Plasma Cell Dyscrasia; LCDD vs Immunotactoid glomerulopathy

    Directory of Open Access Journals (Sweden)

    Jabur Wael

    2008-01-01

    Full Text Available Light chain deposit disease is a plasma cell disorder characterized by production of a large amount of monoclonal immunoglobulin light chain or part of it, which is usually deposited as an amorphous substance in the kidneys. Immunotactoid glomerulopathy is an uncommon disease, which might be related to plasma cell dyscrasia, and characteristically manifest as organized glomerular ultra structural fibrils or microtubules. In this article, we report a case of a combined presentation of light chain disease and immunotactoid glomerulopathy in a patient with multiple myeloma and reversible advanced renal failure.

  8. Gingival plasma cell granuloma

    Directory of Open Access Journals (Sweden)

    Phadnaik Mangesh

    2010-01-01

    Full Text Available Plasma cell granuloma is a rare reactive lesion composed of polyclonal plasma cells. It manifests primarily in the lungs, but may occur in various other anatomic locations like the oral cavity. Intraoral plasma cell granulomas involving the tongue, lip, oral mucosa and gingiva have been reported in the past. This case presents a 54-year-old female with chronic periodontitis and mandibular anterior gingival overgrowth treated by Phase I therapy (scaling and root planing and excisional biopsy. Histological examination revealed inflammatory cell infiltrate containing sheets of plasma cells. Immunohistochemistry for kappa and lambda light chains showed a polyclonal staining pattern confirming a diagnosis of plasma cell granuloma. This case highlights the need to biopsy for unusual lesions to rule out potential neoplasms.

  9. [Acute plasma cell leukemia].

    Science.gov (United States)

    Monsalbe, V; Domíngues, C; Roa, I; Busel, D; González, S

    1989-01-01

    Plasma Cell Leukemia is a very rare form of plasmocytic dyscrasia, whose clinical and pathological characteristics warrant its recognition as a distinct subentity. We report the case of a 60 years old man who presented a rapidly fatal acute plasma cell leukemia, with multiple osteolytic lesions, hipercalcemia, renal and cardiac failure.

  10. "Angular" plasma cell cheilitis.

    Science.gov (United States)

    da Cunha Filho, Roberto Rheingantz; Tochetto, Lucas Baldissera; Tochetto, Bruno Baldissera; de Almeida, Hiram Larangeira; Lorencette, Nádia Aparecida; Netto, José Fillus

    2014-03-17

    Plasma cell cheilitis is an extremely rare disease, characterized by erythematous-violaceous, ulcerated and asymptomatic plaques, which evolve slowly. The histological characteristics include dermal infiltrate composed of mature plasmocytes. We report a case of Plasma cell angular cheilitis in a 58-year-old male, localized in the lateral oral commissure.

  11. "Angular" plasma cell cheilitis

    OpenAIRE

    da Cunha Filho, Roberto Rheingantz; Tochetto, Lucas Baldissera; Tochetto, Bruno Baldissera; de Almeida Jr, Hiram Larangeira; Lorencette, Nadia Aparecida; Netto, Jose Fillus

    2014-01-01

    Plasma cell cheilitis is an extremely rare disease, characterized by erythematous-violaceous, ulcerated and asymptomatic plaques, which evolve slowly. The histological characteristics include dermal infiltrate composed of mature plasmocytes. We report a case of Plasma cell angular cheilitis in a 58-year-old male, localized in the lateral oral commissure.

  12. Monoclonal antibodies directed against protoplasts of soybean cells : Generation of hybridomas and characterization of a monoclonal antibody reactive with the cell surface.

    Science.gov (United States)

    Villanueva, M A; Metcalf, T N; Wang, J L

    1986-09-01

    Splenocytes, derived from mice that had been immunized with protoplasts prepared from suspension cultures of root cells of Glycine max (L.) Merr. (SB-1 cell line), were fused with a murine myeloma cell line. The resulting hybridoma cultures were screened for the production of antibodies directed against the soybean protoplasts and were then cloned. One monoclonal antibody, designated MVS-1, was found to bind to the outer surface of the plasma membrane on the basis of several criteria: (a) agglutination of the protoplasts; (b) binding of fluorescence-labeled immunoglobulin on protoplasts yielding a ring staining pattern with prominent intensity at the edges; and (c) saturable binding by protoplasts of (125)I-labeled Antibody MVS-1. The antigenic target of Antibody MVS-1, identified by immunoblotting techniques, contained a polypeptide of relative molecular mass (Mr) approx. 400000 under both reducing and non-reducing conditions. When the antigenic target of Antibody MVS-1 was chromatographed in potassium phosphate buffer, the position of elution corresponded to that of a high-molecular-weight species (Mr 400000). These results provide the protein characterization required for the analysis of the mobility of Antibody MVS-1 bound to the plasma membrane of SB-1 cells.

  13. Gingival plasma cell granuloma

    Directory of Open Access Journals (Sweden)

    Amitkumar B Pandav

    2012-01-01

    Full Text Available Plasma cell granuloma, also known as inflammatory pseudotumor is a tumor-like lesion that manifests primarily in the lungs. But it may occur in various other anatomic locations like orbit, head and neck, liver and rarely in the oral cavity. We here report an exceedingly rare case of gingival plasma cell granuloma in a 58 year old woman who presented with upper gingival polypoidal growth. The histopathological examination revealed a mass composed of proliferation of benign spindle mesenchymal cells in a loose myxoid and fibrocollagenous stroma along with dense infiltrate of chronic inflammatory cells predominantly containing plasma cells. Immunohistochemistry for kappa and lambda light chains showed a polyclonal staining pattern confirming a diagnosis of plasma cell granuloma.

  14. Plasma Cell Cheilitis

    Directory of Open Access Journals (Sweden)

    Thami Gurvinder P

    1999-01-01

    Full Text Available A case of plasma cell cheilitis with good response to glucocorticoids, is described for its rarity and probable aetiological correlation with habit of use of nasal snuff is discussed.

  15. Monoclonal gammopathy of undetermined significance and smoldering multiple myeloma.

    Science.gov (United States)

    Kyle, Robert A; San-Miguel, Jesus F; Mateos, Maria-Victoria; Rajkumar, S Vincent

    2014-10-01

    Monoclonal gammopathy of undetermined significance (MGUS) is characterized by an M spike less than 3 g/dL and a bone marrow containing fewer than 10% plasma cells without evidence of CRAB (hypercalcemia, renal insufficiency, anemia, or bone lesions). Light chain MGUS has an abnormal free light chain (FLC) ratio, increased level of the involved FLC, no monoclonal heavy chain, and fewer than 10% monoclonal plasma cells in the bone marrow. Smoldering multiple myeloma has an M protein of at least 3 g/dL and/or at least 10% monoclonal plasma cells in the bone marrow without CRAB features. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Plasma cell leukemia

    DEFF Research Database (Denmark)

    Fernández de Larrea, C; Kyle, R A; Durie, B G M

    2013-01-01

    Plasma cell leukemia (PCL) is a rare and aggressive variant of myeloma characterized by the presence of circulating plasma cells. It is classified as either primary PCL occurring at diagnosis or as secondary PCL in patients with relapsed/refractory myeloma. Primary PCL is a distinct clinic......-pathological entity with different cytogenetic and molecular findings. The clinical course is aggressive with short remissions and survival duration. The diagnosis is based upon the percentage (≥ 20%) and absolute number (≥ 2 × 10(9)/l) of plasma cells in the peripheral blood. It is proposed that the thresholds...... regimens and bortezomib-based regimens are recommended followed by high-dose therapy with autologous stem cell transplantation if feasible. Allogeneic transplantation can be considered in younger patients. Prospective multicenter studies are required to provide revised definitions and better understanding...

  17. Defining process design space for monoclonal antibody cell culture.

    Science.gov (United States)

    Abu-Absi, Susan Fugett; Yang, LiYing; Thompson, Patrick; Jiang, Canping; Kandula, Sunitha; Schilling, Bernhard; Shukla, Abhinav A

    2010-08-15

    The concept of design space has been taking root as a foundation of in-process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non-key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product-related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified.

  18. Monoclonal antibody to human endothelial cell surface internalization and liposome delivery in cell culture.

    Science.gov (United States)

    Trubetskaya, O V; Trubetskoy, V S; Domogatsky, S P; Rudin, A V; Popov, N V; Danilov, S M; Nikolayeva, M N; Klibanov, A L; Torchilin, V P

    1988-02-01

    A monoclonal antibody (mAb), E25, is described that binds to the surface of cultured human endothelial cells. Upon binding E25 is rapidly internalized and digested intracellularly. Selective liposome targeting to the surface of the cells is performed using a biotinylated E25 antibody and an avidin-biotin system. Up to 30% of the cell-adherent liposomal lipid is internalized.

  19. Purification of human seminal plasma no. 7 antigen by immunoaffinity chromatography on bound monoclonal antibody.

    Science.gov (United States)

    Isojima, S; Koyama, K; Fujiwara, N

    1982-01-01

    Human seminal plasma (HSP) No. 7 antigen was purified by immunoaffinity chromatography on bound 1C4 monoclonal antibody (Moab) (Shigeta et al., 1980b). The pooled HSP protein was applied to a CNBr-activated Sepharose 4B column of bound 1C4 Moab gamma globulin and the antibody bound fraction (fr) eluted was further purified by rechromatography in the same way. The purified antigen in the antibody bound fr obtained by rechromatography gave a single band on SDS-PAGE in a position corresponding to a molecular weight of 15,000 daltons. This preparation was 196.2 times more effective than the original HSP protein in neutralizing the sperm immobilizing activity of 1C4 Moab. The purified HSP No. 7 antigen contained iron, but was different from lactoferrin and transferrin. It did not show any enzymatic activities, such as those of acid phosphatase, LDH or trypsin inhibitor, and shared antigenicity with human milk protein. It was present in seminal plasma as a molecule with a higher molecular weight but seemed to be cleaved to a monomer of 15,000 daltons during purification procedures. This antigen is present on spermatozoa as sperm-coating antigen and the corresponding antibody can immobilize spermatozoa with complement. Images Fig. 3 PMID:7127911

  20. PLASMA CELL LEUKEMIA

    Science.gov (United States)

    de Larrea, Carlos Fernandez; Kyle, Robert A.; Durie, Brian GM; Ludwig, Heinz; Usmani, Saad; Vesole, David H.; Hajek, Roman; Miguel, Jésus San; Sezer, Orhan; Sonneveld, Pieter; Kumar, Shaji K.; Mahindra, Anuj; Comenzo, Ray; Palumbo, Antonio; Mazumber, Amitabha; Anderson, Kenneth C.; Richardson, Paul G.; Badros, Ashraf Z.; Caers, Jo; Cavo, Michele; LeLeu, Xavier; Dimopoulos, Meletios A.; Chim, CS; Schots, Rik; Noeul, Amara; Fantl, Dorotea; Mellqvist, Ulf-Henrik; Landgren, Ola; Chanan-Khan, Asher; Moreau, Philippe; Fonseca, Rafael; Merlini, Giampaolo; Lahuerta, JJ; Bladé, Joan; Orlowski, Robert Z.; Shah, Jatin J.

    2014-01-01

    Plasma cell leukemia (PCL) is a rare and aggressive variant of myeloma characterized by the presence of circulating plasma cells. It is classified as either primary PCL occurring at diagnosis or as secondary PCL in patients with relapsed/refractory myeloma. Primary PCL is a distinct clinic-pathologic entity with different cytogenetic and molecular findings. The clinical course is aggressive with short remissions and survival duration. The diagnosis is based upon the percentage (≥ 20%) and absolute number (≥ 2 × 10 9/L) of plasma cells in the peripheral blood. It is proposed that the thresholds for diagnosis be reexamined and consensus recommendations are made for diagnosis, as well as, response and progression criteria. Induction therapy needs to begin promptly and have high clinical activity leading to rapid disease control in an effort to minimize the risk of early death. Intensive chemotherapy regimens and bortezomib-based regimens are recommended followed by high-dose therapy with autologous stem-cell transplantation (HDT/ASCT) if feasible. Allogeneic transplantation can be considered in younger patients. Prospective multicenter studies are required to provide revised definitions and better understanding of the pathogenesis of PCL. PMID:23288300

  1. Secondary monoclonal gammopathy of undetermined significance after allogeneic stem cell transplantation in multiple myeloma.

    Science.gov (United States)

    Schmitz, Marian F; Otten, Henny G; Franssen, Laurens E; van Dorp, Suzanne; Strooisma, Theo; Lokhorst, Henk M; van de Donk, Niels W C J

    2014-12-01

    In the course of multiple myeloma, patients may develop a M-protein band different from the original: secondary monoclonal gammopathy of undetermined significance. In this retrospective single center analysis, we describe the occurrence and clinical relevance of secondary monoclonal gammopathy of undetermined significance after allogeneic stem cell transplantation (post-transplant monoclonal gammopathy of undetermined significance). A total of 138 patients who had undergone 139 allogeneic stem cell transplantations (39.6% in the upfront setting and 60.4% for relapsed multiple myeloma) were included in the study. Sixty-seven (48.2%) patients developed secondary monoclonal gammopathy of undetermined significance, after a median latency of 6.9 months. Secondary monoclonal gammopathy of undetermined significance occurred more often in patients who achieved at least very good partial response after allogeneic stem cell transplantation, compared to partial response or less (54.8% vs. 26.5%; P=0.005). The incidence was also higher in the upfront setting as compared to relapsed disease, or with a sibling donor compared to matched unrelated donor, but less often after T-cell depletion. Importantly, development of post-transplant monoclonal gammopathy of undetermined significance as a time-dependent variable independently predicted for superior progression-free and overall survival (median progression-free survival 37.5 vs. 6.3 months, Pundetermined significance should not be confused with relapse or progression of disease. (Trial registered with trialregister.nl; HOVON 108: NTR 2958.). Copyright© Ferrata Storti Foundation.

  2. Effects of the Monoclonal Antibody against Porcine 40 kDa Adipocyte-specific Plasma Membrane Protein on Adipocytes and Carcass Composition

    Institute of Scientific and Technical Information of China (English)

    Shizheng GAO; Changrong GE; Xi ZHANG; Yonggang LIU

    2007-01-01

    The effects of the mouse monoclonal antibody against 40 kDa adipocyte-specific plasma membrane protein on porcine adipocytes and carcass composition were investigated in vitro and in vivo.Results revealed that the in vitro complement-mediated cytotoxicity of this monoclonal antibody can lead to adipocyte lysis, remarkable reduction of adipocyte lipid accumulation (P<0.01), and significant decrease of well-differentiated fat cells (P<0.01). Treatment of adipocytes with this antibody alone in vitro did not induce cell lysis, but could lead to noticeable reduction of well-differentiated cells and lipid accumulation (P<0.05) at the pre-adipocyte stage. In vivo, pigs injected with 0.5 mg/kg or 1.0 mg/kg of antibody showed smaller adipocyte sizes (P<0.01) and reduced lipid accumulation of adipocytes (P<0.01). Our results also indicated that pigs intraperitoneally or subcutaneously immunized with 0.5 mg/kg of monoclonal antibody at 15 kg or 1.0 mg/kg antibody at 60 kg had a higher lean meat percentage (P<0.05), larger loin eye area (P<0.05), lower fat meat percentage (P<0.05), less backfat thickness (P<0.05) and smaller leaf fat weight (P<0.05) than the control pigs, but other carcass traits such as caul fat weight, heart weight, liver weight, spleen weight,kidney weight, lung weight, and dressing percentage were not significantly affected. These results suggested that this monoclonal antibody could be applied to restrain excessive fat deposition in porcine production.

  3. Staining of Langerhans Cells with Monoclonal Antibodies to Macrophages and Lymphoid Cells

    Science.gov (United States)

    Haines, Kathleen A.; Flotte, Thomas J.; Springer, Timothy A.; Gigli, Irma; Thorbecke, G. Jeanette

    1983-06-01

    Langerhans cells are Ia-bearing antigen-presenting cells in the epidermis that share many functions with macrophages. We have used monoclonal antibodies to the macrophage antigens, Mac-2 and -3, Ia antigen, Fc fragment receptor, and the common leukocyte antigen CLA to compare the cell surface antigens of these cells with those of interdigitating and follicular dendritic cells and of macrophages in lymphoid tissues. Immunoperoxidase staining was carried out with epidermal sheets from BALB/c mice and epidermal cell suspensions enriched for Langerhans cells by Fc rosetting. Langerhans cells stained for all of these antigens. Comparison with the staining properties of other dendritic cells and macrophages, in combination with previous observations, indicates a close relationship of Langerhans cells to the interdigitating cells of lymphoid tissues.

  4. A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells

    Science.gov (United States)

    Colcher, D.; Horan Hand, P.; Nuti, M.; Schlom, J.

    1981-05-01

    Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a ``pancarcinoma'' reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

  5. Antigen-specific monoclonal antibodies isolated from B cells expressing constitutively active STAT5.

    Directory of Open Access Journals (Sweden)

    Ferenc A Scheeren

    Full Text Available BACKGROUND: Fully human monoclonal antibodies directed against specific pathogens have a high therapeutic potential, but are difficult to generate. METHODOLOGY/PRINCIPAL FINDINGS: Memory B cells were immortalized by expressing an inducible active mutant of the transcription factor Signal Transducer and Activator of Transcription 5 (STAT5. Active STAT5 inhibits the differentiation of B cells while increasing their replicative life span. We obtained cloned B cell lines, which produced antibodies in the presence of interleukin 21 after turning off STAT5. We used this method to obtain monoclonal antibodies against the model antigen tetanus toxin. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel and relatively simple method of immortalizing antigen-specific human B cells for isolation of human monoclonal antibodies. These results show that STAT5 overexpression can be employed to isolate antigen specific antibodies from human memory B cells.

  6. Plasma cell granuloma of lip

    Directory of Open Access Journals (Sweden)

    B Sabarinath

    2012-01-01

    Full Text Available Plasma cells are medium-sized round-to-oval cells with eccentrically placed nuclei, usually found in the red pulp of the spleen, tonsils, medulla of the lymph nodes, nasal mucosa, upper airway, lamina propria of the gastrointestinal tract, and sites of inflammation. Plasma cell granuloma is a rare reactive tumor-like proliferation composed chiefly of plasmacytic infiltrate. Here, we present a case of plasma cell granuloma of lip in a female patient.

  7. Statistical analysis of data from limiting dilution cloning to assess monoclonality in generating manufacturing cell lines.

    Science.gov (United States)

    Quiroz, Jorge; Tsao, Yung-Shyeng

    2016-07-08

    Assurance of monoclonality of recombinant cell lines is a critical issue to gain regulatory approval in biological license application (BLA). Some of the requirements of regulatory agencies are the use of proper documentations and appropriate statistical analysis to demonstrate monoclonality. In some cases, one round may be sufficient to demonstrate monoclonality. In this article, we propose the use of confidence intervals for assessing monoclonality for limiting dilution cloning in the generation of recombinant manufacturing cell lines based on a single round. The use of confidence intervals instead of point estimates allow practitioners to account for the uncertainty present in the data when assessing whether an estimated level of monoclonality is consistent with regulatory requirements. In other cases, one round may not be sufficient and two consecutive rounds are required to assess monoclonality. When two consecutive subclonings are required, we improved the present methodology by reducing the infinite series proposed by Coller and Coller (Hybridoma 1983;2:91-96) to a simpler series. The proposed simpler series provides more accurate and reliable results. It also reduces the level of computation and can be easily implemented in any spreadsheet program like Microsoft Excel. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1061-1068, 2016.

  8. Culture and Identification of Monoclonal Neural Stem Cells Derived from Cerebral Cortex

    Institute of Scientific and Technical Information of China (English)

    TAO Kaixiong; CHEN Jingbo; WANG Guobin; SHU Xiaogang

    2006-01-01

    To isolate and culture the purified monoclonal neural stem cells from the cerebral cortex of new born mice, new-born mice cerebral cortex was isolated and dissociated to single-cell suspension by mechanical trituration. The dissociated single cells were cultured in serum-free medium. After the formation of neurospheres, single-cell clone culture was performed by limiting dilution and the proliferated single-cell clones were harvested for subculture. Immunocytochemistry was used to detect the specific marker of neuroepithelial stem cells (Nestin) of the primary and monoclonal neurospheres. In the differentiated cells we detected the specific antigen of NF-200 and GFAP. Our results showed that the primary neurospheres expressed Nestin antigen positively. By limiting dilution, we cultured the cell lines from single-cell clone and the monoclonal neurospheres expressed Nestin and had capabilities of self-renewal, proliferation and the potentiality of differentiation into neurons and glial cells. It is concluded that monoclonal neural stem cells which have the ability of proliferation and multi-directional differentiation can be isolated and cultured from the cerebral cortex of new-born mice by limiting dilution.

  9. Reactivity of alveolar epithelial cells in primary culture with type I cell monoclonal antibodies.

    Science.gov (United States)

    Danto, S I; Zabski, S M; Crandall, E D

    1992-03-01

    An understanding of the process of alveolar epithelial cell growth and differentiation requires the ability to trace and analyze the phenotypic transitions that the cells undergo. This analysis demands specific phenotypic probes to type II and, especially, type I pneumocytes. To this end, monoclonal antibodies have been generated to type I alveolar epithelial cells using an approach designed to enhance production of lung-specific clones from a crude lung membrane preparation. The monoclonal antibodies were screened by a combination of enzyme-linked immunosorbent assay and immunohistochemical techniques, with the determination of type I cell specificity resting primarily on immunoelectron microscopic localization. Two of these new markers of the type I pneumocyte phenotype (II F1 and VIII B2) were used to analyze primary cultures of type II cells growing on standard tissue culture plastic and on a variety of substrata reported to affect the morphology of these cells in culture. On tissue culture plastic, the antibodies fail to react with early (days 1 to 3) type II cell cultures. The cells become progressively more reactive with time in culture to a plateau of approximately 6 times background by day 8, with a maximum rate of increase between days 3 and 5. This finding is consistent with the hypothesis that type II cells in primary culture undergo at least partial differentiation into type I cells. Type II cells grown on laminin, which reportedly delays the loss of type II cell appearance, and on fibronectin, which has been reported to facilitate cell spreading and loss of type II cell features, develop the type I cell markers during cultivation in vitro with kinetics similar to those on uncoated tissue culture plastic. Cells on type I collagen and on tissue culture-treated Nuclepore filters, which have been reported to support monolayers with type I cell-like morphology, also increase their expression of the II F1 and VIII B2 epitopes around days 3 to 5. Taken

  10. A phase II trial of chimeric monoclonal antibody G250 for advanced renal cell carcinoma patients.

    NARCIS (Netherlands)

    Bleumer, I.; Knuth, A.; Oosterwijk, E.; Hofmann, R.; Varga, Z.; Lamers, C.B.H.W.; Kruit, W.; Melchior, S.; Mala, C.; Ullrich, S.; Mulder, P.; Mulders, P.F.A.; Beck, J.L.M.

    2004-01-01

    Chimeric monoclonal antibody G250 (WX-G250) binds to a cell surface antigen found on >90% of renal cell carcinoma (RCC). A multicentre phase II study was performed to evaluate the safety and efficacy of WX-G250 in metastatic RCC (mRCC) patients. In all, 36 patients with mRCC were included. WX-G250 w

  11. Plasma cell granuloma of gingiva

    Directory of Open Access Journals (Sweden)

    Balaji Manohar

    2011-01-01

    Full Text Available Plasma cell granuloma is a rare benign lesion characterized by the infiltration of plasma cells; primarily occurring in the lungs. It is also seen to occur in the brain, kidney stomach, heart, and so on. In the intraoral region it is seen to involve the tongue, oral mucosa, and gingiva. This case presents a 42-year-old female, with an enlargement in the maxillary anterior region, treated by excisional biopsy. Histological evaluation revealed plasma cell infiltrates in the connective tissue. The immunohistochemistry revealed kappa and lambda light chains with a polyclonal staining pattern, which confirmed the diagnosis of plasma cell granuloma.

  12. CHO cell line specific prediction and control of recombinant monoclonal antibody N-glycosylation.

    Science.gov (United States)

    Grainger, Rhian K; James, David C

    2013-11-01

    Here we demonstrate that it is possible to predict and control N-glycan processing of a secreted recombinant monoclonal antibody during manufacturing process development using a combination of statistical modelling and comparative measurement of cell surface glycans using fluorescent lectins. Using design of experiments--response surface modelling (DoE-RSM) methodology to adjust the relative media concentrations of known metabolic effectors of galactosylation (manganese, galactose, and uridine) we have shown that β1,4-galactosylation of the same recombinant IgG4 monoclonal antibody produced by different CHO cell lines can be precisely controlled in a cell line specific manner. For two cell lines, monoclonal antibody galactosylation could be increased by over 100% compared to control, non-supplemented cultures without a reduction in product titre and with minimal effect on cell growth. Analysis of galactosylation effector interactions by DoE-RSM indicated that Mn²⁺ alone was necessary but not sufficient to improve galactosylation, and that synergistic combinations of Gal and Urd were necessary to maximize galactosylation, whilst minimizing the deleterious effect of Urd on cell growth. To facilitate rapid cell culture process development we also tested the hypothesis that substrate-level control of cellular galactosylation would similarly affect both cell surface and secreted monoclonal antibody glycans, enabling facile indirect prediction of product glycan processing. To support this hypothesis, comparative quantitation of CHO cell surface β1,4-galactosylation by flow cytometry using fluorescent derivatives of RCA and ConA lectins revealed that substrate-controlled variation in monoclonal antibody galactosylation and cell surface galactosylation were significantly correlated. Taken together, these data show that precision control of a complex, dynamic cellular process essential for the definition of protein product molecular heterogeneity and bioactivity is

  13. Plasma exchange and chlorambucil in polyneuropathy associated with monoclonal IgM gammopathy. IgM-associated Polyneuropathy Study Group.

    Science.gov (United States)

    Oksenhendler, E; Chevret, S; Léger, J M; Louboutin, J P; Bussel, A; Brouet, J C

    1995-01-01

    The study compared chlorambucil alone with chlorambucil in combination with plasma exchange in patients with polyneuropathy associated with monoclonal IgM. Forty four patients were prospectively randomly assigned, in a comparative open trial, to receive either 0.1 mg/kg/day chlorambucil orally, for 12 months or chlorambucil associated with 15 courses of plasma exchange, during the first four months of treatment. They were evaluated by a neuropathy disability score and nerve conduction studies. No difference was found between the two treatment groups. The average neuropathy disability score improved by 2.1 points from baseline (21.0 to 18.9) in the chlorambucil group and by 1.8 points (20.4 to 18.6) in the chlorambucil + plasma exchange group (P = 0.70). The mean motor nerve conduction velocity decreased from 20.0 to 18.2 m/s in the chlorambucil group and increased from 20.5 to 22.5 m/s in the chlorambucil + plasma exchange group (P = 0.51). A slight improvement of the sensory component of the neuropathy disability score (from 10.5 to 8.3) was noted in both groups (P = 0.01). At the end of the study and according to self evaluation, 15 patients--eight from the chlorambucil group and seven from the chlorambucil + plasma exchange group--reported clinical improvement, whereas 15--eight from the chlorambucil group and seven from the chlorambucil + plasma exchange group--reported clinical worsening. Neuropathy remained stable in the others. Thus plasma exchange seemed to confer no additional benefit in the treatment of polyneuropathy associated with monoclonal IgM. PMID:7673949

  14. Inhibition of iodothyronine transport into rat liver cells by a monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Mol, J.A.; Krenning, E.P.; Docter, R.; Rozing, J.; Hennemann, G.

    1986-06-15

    The role of the rat liver plasma membrane in the regulation of uptake and subsequent deiodination of thyroxine (T4) or the biologically active thyroid hormone 3,3',5-triiodothyronine (T3) was investigated. Here we report on the production of monoclonal antibodies raised against rat hepatocytes. Two antibodies were selected. Antibody ER-22 did bind to a Mr 52,000 membrane protein and inhibited the 1- and 5-min uptake of both T4 and T3 by primary cultured rat hepatocytes in a dose-dependent fashion. As the uptake of T4 and T3 depends on the presence of a sodium gradient over the plasma membrane, the inhibitory potency of ER-22 on the Na+,K+-ATPase activity was investigated. No inhibition of the uptake of 86Rb+ could be determined, indicating that antibody ER-22 is not directed against the Na+,K+-ATPase but probably the carrier protein itself. Clearance of T3 from the medium and concomitant iodide production by cultured rat hepatocytes during a 20-h incubation in the presence of ER-22 were both inhibited by 50% with respect to a control incubation in the absence of monoclonal antibody, pointing to the importance of carrier-mediated transport in cellular uptake and metabolism of T3. A second monoclonal antibody did bind to two other plasma membrane proteins but did not inhibit transport of thyroid hormone.

  15. Closed inductively coupled plasma cell

    Science.gov (United States)

    Manning, Thomas J.; Palmer, Byron A.; Hof, Douglas E.

    1990-01-01

    A closed inductively coupled plasma cell generates a relatively high power, low noise plasma for use in spectroscopic studies. A variety of gases can be selected to form the plasma to minimize spectroscopic interference and to provide a electron density and temperature range for the sample to be analyzed. Grounded conductors are placed at the tube ends and axially displaced from the inductive coil, whereby the resulting electromagnetic field acts to elongate the plasma in the tube. Sample materials can be injected in the plasma to be excited for spectroscopy.

  16. Purification of infectious canine parvovirus from cell culture by affinity chromatography with monoclonal antibodies.

    NARCIS (Netherlands)

    J. Groen (Jan); N. Juntti; J.S. Teppema; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert); G.F. Rimmelzwaan (Guus)

    1987-01-01

    textabstractImmuno affinity chromatography with virus neutralizing monoclonal antibodies, directed to the haemagglutinating protein of canine parvovirus (CPV) was used to purify and concentrate CPV from infected cell culture. The procedure was monitored by testing the respective fractions in an infe

  17. Immunohistochemical identification of mast cells in formaldehyde-fixed tissue using monoclonal antibodies specific for tryptase.

    Science.gov (United States)

    Walls, A F; Jones, D B; Williams, J H; Church, M K; Holgate, S T

    1990-10-01

    An avidin-biotin enhanced immunoperoxidase procedure using monoclonal antibodies (AA1, AA3, and AA5) prepared against human mast cell tryptase resulted in intense staining of mast cells in paraffin-embedded tissue. The distribution of mast cells observed was similar to that seen when adjacent serial sections were stained using a standard procedure with toluidine blue, though the immunoperoxidase technique permitted the identification of significantly more mast cells. With monoclonal antibody AA1, immunostaining was entirely specific for mast cell granules, and there was negligible background staining in a range of tissues including lung, tonsil, colon, gastric mucosa, skin, and pituitary. There was no staining of antibody on basophils or on any other normal blood leukocyte. The technique was effective with tissue fixed in either Carnoy's or neutral buffered formalin, though the internal mast cell structure was better preserved with formaldehyde fixation. The immunoperoxidase staining procedure with monoclonal antibody AA1 is a highly specific and sensitive means for the detection of mast cells in routinely processed tissues.

  18. Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins.

    Science.gov (United States)

    Luo, Shen; Zhang, Baolin

    2015-01-01

    Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5-6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5-6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.

  19. Dialysis cultures with immobilized hybridoma cells for effective production of monoclonal antibodies

    OpenAIRE

    Pörtner, Ralf; Lüdemann, Ines; Märkl, Herbert

    1997-01-01

    An industrial scale reactor concept for continuous cultivation of immobilized animal cells (e.g. hybridoma cells) in a radial-flow fixed bed is presented, where low molecular weight metabolites are removed via dialysis membrane and high molecular products (e.g. monoclonal antibodies) are enriched. In a new “nutrient-split” feeding strategy concentrated medium is fed directly to the fixed bed unit, whereas a buffer solution is used as dialysis fluid. This feeding strategy was investigated in a...

  20. Plasma Etching Improves Solar Cells

    Science.gov (United States)

    Bunyan, S. M.

    1982-01-01

    Etching front surfaces of screen-printed silicon photovoltaic cells with sulfur hexafluoride plasma found to increase cell performance while maintaining integrity of screen-printed silver contacts. Replacement of evaporated-metal contacts with screen-printed metal contacts proposed as one way to reduce cost of solar cells for terrestrial applications.

  1. Monoclonal T-cell receptors: new reagents for cancer therapy.

    Science.gov (United States)

    Stauss, Hans J; Cesco-Gaspere, Michela; Thomas, Sharyn; Hart, Daniel P; Xue, Shao-An; Holler, Angelika; Wright, Graham; Perro, Mario; Little, Ann-Margaret; Pospori, Constantina; King, Judy; Morris, Emma C

    2007-10-01

    Adoptive transfer of antigen-specific T lymphocytes is an effective form of immunotherapy for persistent virus infections and cancer. A major limitation of adoptive therapy is the inability to isolate antigen-specific T lymphocytes reproducibly. The demonstration that cloned T-cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T-cell therapy. TCR gene-modified lymphocytes display antigen-specific function in vitro, and were shown to protect against virus infection and tumor growth in animal models. A recent trial in humans demonstrated that TCR gene-modified T cells persisted in all and reduced melanoma burden in 2/15 patients. In future trials, it may be possible to use TCR gene transfer to equip helper and cytotoxic T cells with new antigen-specificity, allowing both T-cell subsets to cooperate in achieving improved clinical responses. Sequence modifications of TCR genes are being explored to enhance TCR surface expression, while minimizing the risk of pairing between introduced and endogenous TCR chains. Current T-cell transduction protocols that trigger T-cell differentiation need to be modified to generate "undifferentiated" T cells, which, upon adoptive transfer, display improved in vivo expansion and survival. Both, expression of only the introduced TCR chains and the production of naïve T cells may be possible in the future by TCR gene transfer into stem cells.

  2. Application of dielectric spectroscopy for monitoring high cell density in monoclonal antibody producing CHO cell cultivations.

    Science.gov (United States)

    Párta, László; Zalai, Dénes; Borbély, Sándor; Putics, Akos

    2014-02-01

    The application of dielectric spectroscopy was frequently investigated as an on-line cell culture monitoring tool; however, it still requires supportive data and experience in order to become a robust technique. In this study, dielectric spectroscopy was used to predict viable cell density (VCD) at industrially relevant high levels in concentrated fed-batch culture of Chinese hamster ovary cells producing a monoclonal antibody for pharmaceutical purposes. For on-line dielectric spectroscopy measurements, capacitance was scanned within a wide range of frequency values (100-19,490 kHz) in six parallel cell cultivation batches. Prior to detailed mathematical analysis of the collected data, principal component analysis (PCA) was applied to compare dielectric behavior of the cultivations. PCA analysis resulted in detecting measurement disturbances. By using the measured spectroscopic data, partial least squares regression (PLS), Cole-Cole, and linear modeling were applied and compared in order to predict VCD. The Cole-Cole and the PLS model provided reliable prediction over the entire cultivation including both the early and decline phases of cell growth, while the linear model failed to estimate VCD in the later, declining cultivation phase. In regards to the measurement error sensitivity, remarkable differences were shown among PLS, Cole-Cole, and linear modeling. VCD prediction accuracy could be improved in the runs with measurement disturbances by first derivative pre-treatment in PLS and by parameter optimization of the Cole-Cole modeling.

  3. Unique association of Waldenström macroglobulinemia with optic neuritis and monoclonal T cell expansion.

    Science.gov (United States)

    Morita, Ken; Yoshimi, Akihide; Masuda, Akiko; Ichikawa, Motoshi; Yatomi, Yutaka; Kurokawa, Mineo

    2013-08-01

    Waldenström macroglobulinemia is a lymphoplasmacytic lymphoma characterized by production of the immunoglobulin M (IgM) monoclonal protein. Commonly involved sites are the bone marrow, lymph nodes, and spleen. Lymphoplasmacytic infiltration of the central nervous system (CNS), in contrast, is referred to as Bing-Neel syndrome, and is an extremely rare phenomenon. Here, we present a unique case of Waldenström macroglobulinemia with optic neuritis accompanied by monoclonal expansion of T cells, which recovered after administration of CNS-targeting chemotherapy. Although the underlying causal relationships in this case remain obscure, aberrantly expanded T cells may have contributed to the development of optic neuritis, and we should be reminded that some types of cranial neuropathy in Waldenström macroglobulinemia may be reversible.

  4. Production of Human Monoclonal Rheumatoid Factor Secreting Hybridomas Derived from Rheumatoid Synovial Cells

    Science.gov (United States)

    1989-01-01

    antisera to human [gM and human IgG heavy chains , kappa and lambda lightTable 2: Rheumatoid synowal cell tRF subelass speclicity profiles chains , and...antisera to whole mouse Ig including light chains .(ELISA)frompantie MKin Table 1& AD7 RF was a human 1gM k monoclonal antibody without Well IgGI gG2

  5. Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

    Directory of Open Access Journals (Sweden)

    Cavazzoni Andrea

    2012-12-01

    Full Text Available Abstract Background The epidermal growth factor receptor (EGFR is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.

  6. ANTIBODIES DEFINING RAT ENDOTHELIAL-CELLS - RECA-1, A PAN-ENDOTHELIAL CELL-SPECIFIC MONOCLONAL-ANTIBODY

    NARCIS (Netherlands)

    DUIJVESTIJN, AM; VANGOOR, H; KLATTER, F; MAJOOR, GD; VANBUSSEL, E; VRIESMAN, PJCV

    1992-01-01

    We have been searching for antibodies reactive with rat endothelial cells. Two monoclonal antibodies (mAb), named RECA-1 and RECA-2 were produced and tested in immunoperoxidase staining on frozen sections of various rat tissues. Staining patterns were compared to those obtained with the mAbs OX-2, O

  7. ANTIBODIES DEFINING RAT ENDOTHELIAL-CELLS - RECA-1, A PAN-ENDOTHELIAL CELL-SPECIFIC MONOCLONAL-ANTIBODY

    NARCIS (Netherlands)

    DUIJVESTIJN, AM; VANGOOR, H; KLATTER, F; MAJOOR, GD; VANBUSSEL, E; VRIESMAN, PJCV

    1992-01-01

    We have been searching for antibodies reactive with rat endothelial cells. Two monoclonal antibodies (mAb), named RECA-1 and RECA-2 were produced and tested in immunoperoxidase staining on frozen sections of various rat tissues. Staining patterns were compared to those obtained with the mAbs OX-2, O

  8. The Effects of Anti-Hcg Monoclonal Antibodies on Human Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Mirshahi M

    2011-12-01

    Full Text Available Background: Human cancer cell lines express human choriogonadotropin (hCG, its subunits and derivatives, regardless of their origin and type. It appears that hCG is a common phenotype in human cancer cell lines. In this research, the effects of hCG targeting monoclonal antibodies (7D9, T18H7 and T8B12 on human cancer cell lines were evaluated. Methods: Monoclonal antibody secreting hybridomas were proliferated and injected intraperitoneally to Balb/C mice after treatment with pristine. Two weeks later, ascites fluid was collected. Purification of aforementioned antibodies from ascites fluid was performed using G-protein affinity followed by ion exchange chromatography. SDS-PAGE and ELISA confirmed the structure and functional integrity of the purified antibodies, respectively. Two human cancer cell lines "Hela" and "MDA" were treated by the purified antibodies. Three days later, different wells were imaged and the cells counted. Results: SDS-PAGE gel (None-reducing indicated consistency of band migration patterns with control antibodies. ELISA test using hCG antigens indicated that the produced antibodies could detect hCG antigens. Cell lines were cultured and treated with different concentrations of each antibody. Counting and imaging different wells of treated plates, indicated that 7D9 antibody had a more significant (P<0.01 cytotoxic effect on cancer cell lines than the control cells. Conclusion: HCG targeting monoclonal antibodies can be used for targeted cancer therapy, as human cancer cells express hCG gene. 7D9 antibody that exhibits protease activity is a proper candidate for this purpose, as it possesses both antagonistic and enzymatic properties.

  9. Monoclonal Antibodies as Probes for Unique Antigens in Secretory Cells of Mixed Exocrine Organs

    Science.gov (United States)

    Basbaum, C. B.; Mann, J. K.; Chow, A. W.; Finkbeiner, W. E.

    1984-07-01

    In the past, it has been difficult to identify the secretory product and control mechanisms associated with individual cell types making up mixed exocrine organs. This report establishes the feasibility of using immunological methods to characterize both the biochemical constituents and regulatory mechanisms associated with secretory cells in the trachea. Monoclonal antibodies directed against components of tracheal mucus were produced by immunizing mice with dialyzed, desiccated secretions harvested from tracheal organ culture. An immunofluorescence assay revealed that of the total 337 hybridomas screened, 100 produced antibodies recognizing goblet cell granules; 64, gland cell granules; and 3, antigen confined to the ciliated apical surface of the epithelium. The tracheal goblet cell antibody described in this report was strongly cross-reactive with intestinal goblet cells, as well as with a subpopulation of submandibular gland cells, but not with cells of Brunner's glands or the ciliated cell apical membrane. The serous cell antibody was not cross-reactive with goblet, Brunner's gland, or submandibular cells, or the ciliated cell apical membrane. The antibody directed against the apical membrane of ciliated cells did not cross-react with gland or goblet cells or the apical membrane of epithelial cells in the duodenum. Monoclonal antibodies, therefore, represent probes by which products unique to specific cells or parts of cells in the trachea can be distinguished. The antibodies, when used in enzyme immunoassays, can be used to quantitatively monitor secretion by individual cell types under a variety of physiological and pathological conditions. They also provide the means for purification and characterization of cell-specific products by immunoaffinity chromatography.

  10. Towards Stratified Medicine in Plasma Cell Myeloma

    Science.gov (United States)

    Egan, Philip; Drain, Stephen; Conway, Caroline; Bjourson, Anthony J.; Alexander, H. Denis

    2016-01-01

    Plasma cell myeloma is a clinically heterogeneous malignancy accounting for approximately one to 2% of newly diagnosed cases of cancer worldwide. Treatment options, in addition to long-established cytotoxic drugs, include autologous stem cell transplant, immune modulators, proteasome inhibitors and monoclonal antibodies, plus further targeted therapies currently in clinical trials. Whilst treatment decisions are mostly based on a patient’s age, fitness, including the presence of co-morbidities, and tumour burden, significant scope exists for better risk stratification, sub-classification of disease, and predictors of response to specific therapies. Clinical staging, recurring acquired cytogenetic aberrations, and serum biomarkers such as β-2 microglobulin, and free light chains are in widespread use but often fail to predict the disease progression or inform treatment decision making. Recent scientific advances have provided considerable insight into the biology of myeloma. For example, gene expression profiling is already making a contribution to enhanced understanding of the biology of the disease whilst Next Generation Sequencing has revealed great genomic complexity and heterogeneity. Pathways involved in the oncogenesis, proliferation of the tumour and its resistance to apoptosis are being unravelled. Furthermore, knowledge of the tumour cell surface and its interactions with bystander cells and the bone marrow stroma enhance this understanding and provide novel targets for cell and antibody-based therapies. This review will discuss the development in understanding of the biology of the tumour cell and its environment in the bone marrow, the implementation of new therapeutic options contributing to significantly improved outcomes, and the progression towards more personalised medicine in this disorder. PMID:27775669

  11. Towards Stratified Medicine in Plasma Cell Myeloma

    Directory of Open Access Journals (Sweden)

    Philip Egan

    2016-10-01

    Full Text Available Plasma cell myeloma is a clinically heterogeneous malignancy accounting for approximately one to 2% of newly diagnosed cases of cancer worldwide. Treatment options, in addition to long-established cytotoxic drugs, include autologous stem cell transplant, immune modulators, proteasome inhibitors and monoclonal antibodies, plus further targeted therapies currently in clinical trials. Whilst treatment decisions are mostly based on a patient’s age, fitness, including the presence of co-morbidities, and tumour burden, significant scope exists for better risk stratification, sub-classification of disease, and predictors of response to specific therapies. Clinical staging, recurring acquired cytogenetic aberrations, and serum biomarkers such as β-2 microglobulin, and free light chains are in widespread use but often fail to predict the disease progression or inform treatment decision making. Recent scientific advances have provided considerable insight into the biology of myeloma. For example, gene expression profiling is already making a contribution to enhanced understanding of the biology of the disease whilst Next Generation Sequencing has revealed great genomic complexity and heterogeneity. Pathways involved in the oncogenesis, proliferation of the tumour and its resistance to apoptosis are being unravelled. Furthermore, knowledge of the tumour cell surface and its interactions with bystander cells and the bone marrow stroma enhance this understanding and provide novel targets for cell and antibody-based therapies. This review will discuss the development in understanding of the biology of the tumour cell and its environment in the bone marrow, the implementation of new therapeutic options contributing to significantly improved outcomes, and the progression towards more personalised medicine in this disorder.

  12. Flow cytometric immunophenotypic characteristics of plasma cell leukemia

    Directory of Open Access Journals (Sweden)

    Barbara Kruk

    2011-04-01

    Full Text Available The aim of this prospective study was to define the flow cytometric characteristics of simultaneously investigated bone marrow and peripheral blood plasma cells antigens expression in 36 plasma cell leukemia (PCL patients. The immunophenotypic profile of plasma cells was determined with a panel of monoclonal antibodies. The antigen expression intensity was calculated as relative fluorescence intensity (RFI. Bone marrow plasma cells showed expression of particular antigens in the following proportion of cases: CD49d 100%, CD29 94%, CD54 93%, CD44 83%, CD56 60%, CD18 26%, CD11b 29%, CD11a 19%, CD117 27%, CD71 30%, CD126 100% and CD19 0%, while the expression of those antigens on peripheral blood plasma cells was present in the following percentage of patients: CD49d 100%, CD29 96%, CD54 93%, CD44 95%, CD56 56%, CD18 50%, CD11b 53%, CD11a 29%, CD117 26%, CD71 28%, CD126 100% and CD19 0%. The expression of CD54 was significantly higher than that of adhesion molecules belonging to the integrin b2 family: CD11a, CD18 and CD11b, on both bone marrow and peripheral blood cells (p < 0.01. Expression of CD18, CD11a and CD11b was differential between two cell compartments: lower on bone marrow and higher on peripheral blood cells. We found that plasma cells in the bone marrow of patients with plasma cell leukaemia showed significantly greater granularity and size than those in the peripheral blood (p = 0.0001 and p = 0.04, respectively. However, no differences in cell size or granularity were revealed between bone marrow plasma cells from patients with PCL and multiple myeloma. In conclusion, impaired expression of adhesion molecules such as CD11a/CD18 (LFA-1 or CD56 may explain hematogenic dissemination characterizing PCL. The following pattern of adhesion molecule expression according to the proportion of plasma cells expressing a given antigen in peripheral blood and bone marrow and arranged in diminishing order may be established: CD49d > CD44 > CD54

  13. A monoclonal antibody reactive with normal and leukemic human myeloid progenitor cells.

    Science.gov (United States)

    Griffin, J D; Linch, D; Sabbath, K; Larcom, P; Schlossman, S F

    1984-01-01

    Anti-MY9 is an IgG2b murine monoclonal antibody selected for reactivity with immature normal human myeloid cells. The MY9 antigen is expressed by blasts, promyelocytes and myelocytes in the bone marrow, and by monocytes in the peripheral blood. Erythrocytes, lymphocytes and platelets are MY9 negative. All myeloid colony-forming cells (CFU-GM), a fraction of erythroid burst-forming cells (BFU-E) and multipotent progenitors (CFU-GEMM) are MY9 positive. This antigen is further expressed by the leukemic cells of a majority of patients with AML and myeloid CML-BC. Leukemic stem cells (leukemic colony-forming cells, L-CFC) from most patients tested were also MY9 positive. In contrast, MY9 was not detected on lymphocytic leukemias. Anti-MY9 may be a valuable reagent for the purification of hematopoietic colony-forming cells and for the diagnosis of myeloid-lineage leukemias.

  14. Non-CLL-like monoclonal B-Cell lymphocytosis in the general population: Prevalence and phenotypic/genetic characteristics

    NARCIS (Netherlands)

    W.G. Nieto (Wendy); C. Teodosio (Cristina); A. López (Antonio); A. Rodríguez-Caballero (Arancha); A. Romero (Alfonso); P. Bárcena (Paloma); M.L. Gutierrez; A.W. Langerak (Anton); P. Fernandez-Navarro (Paulino); A. Orfao; J. Almeida (Julia); A.O.M.C.C.S. Santa Marta de Tormes; B.H.P.C.S. Garrido Sur; C.L.M.T.C.S. Ledesma; C.R.J.M.C.S. Alba de Tormes; C.L.R.C.S.F. Villalobos; D.V.P.J.C.S. Peñaranda; F.E.E.C.S. Pizarrales-Vidal; G.R.B.L.C.S. La Alberca; G.S.F.C.S. Periurbana Norte; G.M.J.C.S. Guijuelo; G.M.J.M.C.S. Vitigudino; J.R.M.J.C.S. Garrido Norte; J.C.T.B.C.S. Elena Ginel Diez; M.P.M.C.S. Fuentes de Oñoro; M.L.J.C.S. San Juan; M.D.M.P.C.S. Miguel Armijo; S.A.B.C.S. Aldeadavila de La Ribera; S.P.R.C.S. San Jose

    2010-01-01

    textabstractBackground: Monoclonal B-cell lymphocytosis (MBL) indicates <5 × 109peripheral blood (PB) clonal B-cells/L in healthy individuals. In most cases, MBL cells show similar phenotypic/genetic features to chronic lymphocytic leukemia cells - CLL-like MBL - but little is known about

  15. Non-CLL-like monoclonal B-Cell lymphocytosis in the general population: Prevalence and phenotypic/genetic characteristics

    NARCIS (Netherlands)

    W.G. Nieto (Wendy); C. Teodosio (Cristina); A. López (Antonio); A. Rodríguez-Caballero (Arancha); A. Romero (Alfonso); P. Bárcena (Paloma); M.L. Gutierrez; A.W. Langerak (Ton); P. Fernandez-Navarro (Paulino); A. Orfao; J. Almeida (Julia); A.O.M.C.C.S. Santa Marta de Tormes; B.H.P.C.S. Garrido Sur; C.L.M.T.C.S. Ledesma; C.R.J.M.C.S. Alba de Tormes; C.L.R.C.S.F. Villalobos; D.V.P.J.C.S. Peñaranda; F.E.E.C.S. Pizarrales-Vidal; G.R.B.L.C.S. La Alberca; G.S.F.C.S. Periurbana Norte; G.M.J.C.S. Guijuelo; G.M.J.M.C.S. Vitigudino; J.R.M.J.C.S. Garrido Norte; J.C.T.B.C.S. Elena Ginel Diez; M.P.M.C.S. Fuentes de Oñoro; M.L.J.C.S. San Juan; M.D.M.P.C.S. Miguel Armijo; S.A.B.C.S. Aldeadavila de La Ribera; S.P.R.C.S. San Jose

    2010-01-01

    textabstractBackground: Monoclonal B-cell lymphocytosis (MBL) indicates <5 × 109peripheral blood (PB) clonal B-cells/L in healthy individuals. In most cases, MBL cells show similar phenotypic/genetic features to chronic lymphocytic leukemia cells - CLL-like MBL - but little is known about non-CLL-li

  16. [MALT B cell lymphoma with kidney damage and monoclonal gammopathy: a case study and literature review].

    Science.gov (United States)

    Peces, R; Vega-Cabrera, C; Peces, C; Pobes, A; Fresno, M F

    2010-01-01

    We report a case of low-grade B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) involving the left kidney and simultaneous onset of a monoclonal gammopathy IgM kappa. No predisposing local inflammatory condition was identified. Following left nephrectomy, the renal specimen showed the centrocyte like cells and lymphoid cells in the lymphoepithelial lesions were positive for CD20 and CD79α. The neoplastic cells expressed monotypic cytoplasmic IgM kappa. The demonstration of bone marrow cells of B-lineage expressing the same monoclonal protein as the tumor suggested bone marrow involvement, even in the absence of identical morphology. Despite chemotherapy and rituximab treatment, clinical follow-up showed right kidney extension with high-grade transformation, and finally systemic dissemination. This case illustrates that the kidney is among the sites that may be involved by MALT B-cell lymphomas in a primary or secondary fashion, and the need for expanded investigation of the possible dissemination. We review the literature on this unusual extranodal lymphoma.

  17. Monoclonal antibody-based analysis of cell wall remodeling during xylogenesis.

    Science.gov (United States)

    Shinohara, Naoki; Kakegawa, Koichi; Fukuda, Hiroo

    2015-11-01

    Xylogenesis, a process by which woody tissues are formed, entails qualitative and quantitative changes in the cell wall. However, the molecular events that underlie these changes are not completely understood. Previously, we have isolated two monoclonal antibodies, referred to as XD3 and XD27, by subtractive screening of a phage-display library of antibodies raised against a wall fraction of Zinnia elegans xylogenic culture cells. Here we report the biochemical and immunohistochemical characterization of those antibodies. The antibody XD3 recognized (1→4)-β-D-galactan in pectin fraction. During xylogenesis, the XD3 epitope was localized to the primary wall of tracheary-element precursor cells, which undergo substantial cell elongation, and was absent from mature tracheary elements. XD27 recognized an arabinogalactan protein that was bound strongly to a germin-like protein. The XD27 epitope was localized to pre-lignified secondary walls of tracheary elements. Thus these cell-wall-directed monoclonal antibodies revealed two molecular events during xylogenesis. The biological significance of these events is discussed in relation to current views of the plant cell wall.

  18. Dialysis cultures with immobilized hybridoma cells for effective production of monoclonal antibodies.

    Science.gov (United States)

    Pörtner, R; Lüdemann, I; Märkl, H

    1997-01-01

    An industrial scale reactor concept for continuous cultivation of immobilized animal cells (e.g. hybridoma cells) in a radial-flow fixed bed is presented, where low molecular weight metabolites are removed via dialysis membrane and high molecular products (e.g. monoclonal antibodies) are enriched. In a new "nutrient-split" feeding strategy concentrated medium is fed directly to the fixed bed unit, whereas a buffer solution is used as dialysis fluid. This feeding strategy was investigated in a laboratory scale reactor with hybridoma cells for production of monoclonal antibodies. A steady state monoclonal antibody concentration of 478 mg l(-1) was reached, appr. 15 times more compared to the concentration reached in chemostat cultures with suspended cells. Glucose and glutamine were used up to 98%. The experiments were described successfully with a kinetic model for immobilized growing cells. Conclusions were drawn for scale-up and design of the large scale system. c(Glc) - glucose concentration, mmol l(-1); c(Gln) - glutamine concentration, mmol l(-1); c(Amm) - ammonia concentration, mmol l(-1); c(Lac) - lactate concentration, mmol l(-1); c(MAb) - MAb concentration, mg l(-1); D - dilution rate, d(-1); D(i) - dilution rate in the inner chamber of the membrane dialysis reactor, d(-1); D(0) - dilution rate in the outer chamber of the membrane dialysis reactor, d(-1); q*(FB,Glc) - volume specific glucose uptake rate related to the fixed bed volume, mmol l(FB) (-1) h(-1); q*(FB,Gln) - volume specific glutamine uptake rate related to the fixed bed volume, mmol l(FB) (-1) h(-1).

  19. Plasma Thrombin Generation and Sensitivity to Activated Protein C Among Patients With Myeloma and Monoclonal Gammopathy of Undetermined Significance.

    Science.gov (United States)

    Crowley, Maeve P; Kevane, Barry; O'Shea, Susan I; Quinn, Shane; Egan, Karl; Gilligan, Oonagh M; Ní Áinle, Fionnuala

    2016-09-01

    The etiology of the prothrombotic state in myeloma has yet to be definitively characterized. Similarly, while recent evidence suggests that patients with monoclonal gammopathy of undetermined significance (MGUS) may also be at increased risk of thrombosis, the magnitude and the etiology of this risk have also yet to be defined. The present study aims to characterize patterns of plasma thrombin generation and sensitivity to the anticoagulant activity of activated protein C (APC) at the time of initial diagnosis of myeloma and in response to therapy in comparison to that observed among patients with MGUS and matched, healthy volunteers. Patients presenting with newly diagnosed/newly relapsed myeloma (n = 8), MGUS (n = 8), and matched healthy volunteers (n = 8) were recruited. Plasma thrombin generation was determined by calibrated automated thrombography. Peak thrombin generation was significantly higher in patients with myeloma (383.4 ± 33.4 nmol/L) and MGUS (353.4 ± 16.5 nmol/L) compared to healthy volunteers (276.7 ± 20.8 nmol/L; P < .05). In the presence of APC, endogenous thrombin potential was significantly lower in control plasma (228.6 ± 44.5 nmol/L × min) than in either myeloma (866.2 ± 241.3 nmol/L × min, P = .01) or MGUS plasma (627 ± 91.5 nmol/L × min, P = .003). Within the myeloma cohort, peak thrombin generation was significantly higher at diagnosis (353.2 ± 15.9 nmol/L) than following completion of the third cycle of therapy (282.1 ± 15.2 nmol/L; P < .005). Moreover, sensitivity to APC increased progressively with each cycle of chemotherapy. Further study of the etiology and evolving patterns of hypercoagulability among patients with these conditions is warranted and may have future implications for thromboprophylaxis strategies. © The Author(s) 2016.

  20. SPECIFIC UPTAKE OF MONOCLONAL ANTIBODY-CONJUGATED METHOTREXATE BY HUMAN LYMPHOCYTIC LEUKEMIC B CELLS

    Institute of Scientific and Technical Information of China (English)

    Zhu Zhenping; Yang Chunzheng; Tarunendu Ghose; Jaroslav Kralovec

    1998-01-01

    Objective: To analysis the uptake of free MTX and MTX conjugated to tumor specific monoclonal antibody by target and non-target cells. Methods: The folate antagonist methotrexate (MTX) was conjugated to two monoclonal antibodies (Mab) directed against human chronic lymphocytic leukemia (CLL), Dal B01 and Dal B02, by an active ester method. Both conjugates were more cytotoxic toward the target tumor cell line D10-1than to the non-target cell line MOLT-3, and Dal B02-MTX conjugate was more inhibitory to D10-1 cells than free MTX in a 6 h pulse exposure assay. Results: Drug uptake studies revealed that D10-1 cells took up much more Dal B01 and Dal B02-conjugated MTX than free MTX. The amounts of drug taken up by D10-1 cells incubated with Dal B01 and Dal B02-conjugated MTX were always 3 to 5-fold higher than that taken up by MOLT-3 cells, although the latter took up more drug when incubated with free MTX. Furthermore, tumor cells incubated with Dal B01 or Dal B02-conjugated MTX retained much larger amounts of drug for a prolonged period of time than those incubated with free MTX.Conclusion: The enhanced specific cytotoxicity of Dal B01 and Dal B02-MTX conjugates toward target tumor cells is therefore likely due to (Ⅰ) delivery of larger amounts of MTX to target cells when the drug is conjugated to Mab;(ii) longer retention of Mab-conjugated MTX by target cells; and (iii) slow, prolonged release of MTX from the surface-bound or endocytosed conjugates, rendering them into a sustained release dosage form.

  1. A novel monoclonal antibody of human stem cell factor inhibits umbilical cord blood stem cell ex vivo expansion

    Directory of Open Access Journals (Sweden)

    Fan Jie

    2012-12-01

    Full Text Available Abstract Stem cell factor (SCF activates hematopoietic stem cell (HSC self-renewal and is being used to stimulate the ex vivo expansion of HSCs. The mechanism by which SCF supports expansion of HSCs remains poorly understood. In cord blood ex vivo expansion assays, a newly produced anti-SCF monoclonal antibody (clone 23C8 was found to significantly inhibit the expansion of CD34+ cells. This antibody appears to bind directly to a part of SCF that is critical for biological activity toward expansion of CD34+ cells, which is located in the first 104 amino acids from the NH2-terminus.

  2. Production of monoclonal antibodies against the outer cell wall of Clostridium tyrobutyricum.

    Science.gov (United States)

    Talbot, F; Robreau, G; Gueguen, F; Malcoste, R

    1994-02-01

    Several hybridoma cell lines producing murine monoclonal antibodies (mAbs) directed to the Clostridium tyrobutyricum outer cell wall have been established and characterized. Whole bacteria, crude extract of cell wall, and polysaccharide fraction of crude extract have been used as immunogens. The immunizations were performed either in vivo or in vitro after priming in vivo. Amongst the clones obtained, six hybridoma cell lines were selected. Four mAbs recognized only the immunizing strain (ATCC 25755), while two mAbs recognized all the C. tyrobutyricum tested strains. Three mAbs were IgM, one IgG3, and two IgG1 isotypes. The antigens (proteins or polysaccharides) recognized by these mAbs have been characterized by Western Blot. These mAbs could be used for an early detection of C. tyrobutyricum in milk.

  3. Studying host cell protein interactions with monoclonal antibodies using high throughput protein A chromatography.

    Science.gov (United States)

    Sisodiya, Vikram N; Lequieu, Joshua; Rodriguez, Maricel; McDonald, Paul; Lazzareschi, Kathlyn P

    2012-10-01

    Protein A chromatography is typically used as the initial capture step in the purification of monoclonal antibodies produced in Chinese hamster ovary (CHO) cells. Although exploiting an affinity interaction for purification, the level of host cell proteins in the protein A eluent varies significantly with different feedstocks. Using a batch binding chromatography method, we performed a controlled study to assess host cell protein clearance across both MabSelect Sure and Prosep vA resins. We individually spiked 21 purified antibodies into null cell culture fluid generated with a non-producing cell line, creating mock cell culture fluids for each antibody with an identical composition of host cell proteins and antibody concentration. We demonstrated that antibody-host cell protein interactions are primarily responsible for the variable levels of host cell proteins in the protein A eluent for both resins when antibody is present. Using the additives guanidine HCl and sodium chloride, we demonstrated that antibody-host cell protein interactions may be disrupted, reducing the level of host cell proteins present after purification on both resins. The reduction in the level of host cell proteins differed between antibodies suggesting that the interaction likely varies between individual antibodies but encompasses both an electrostatic and hydrophobic component.

  4. Mammalian Cell Culture Clarification: A Case Study Using Chimeric Anti-CEA Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Mohamed Ali Abol Hassan

    2011-12-01

    Full Text Available The extracellular expression of monoclonal antibodies (mAbs in mammalian cell culture provides both opportunities and restrictions for the design of robust harvest and clarification operations. With advances in cell culture media and cell lines, it is now possible to achieve high titers of over 5 g/l for mAbs. However, Mammalian cells are sensitive to breakage due to shear stress that can result in release of proteases and other host cell proteins (HCPs which eventually affects product stability and purity. There is larger number of mAbs undergoing clinical development and it has placed significant importance on platform technologies of process development. Generally, Centrifugation and microfiltration are the primary harvest techniques used in the industry and depth filtration is also used as a step operation on clarification. This study compares the unit operations; centrifugation, microfiltration and depth filtration for maximum recovery of monoclonal antibodies. The results have shown that the depth filtration as more suitable operation for mammalian cell culture clarification since it gives 96% recovery of mAbs in comparison to centrifugation and microfiltration. ABSTRAK: Pengungkapan luar sel dari antibodi monoklon (monoclonal antibodies ((mAbs dalam kultur sel mamalia memberi ruang dan batasan terhadap reka bentuk penuaian yang cekap dan penerangan operasi. Dengan kemajuan dalam media sel kultur dan cell lines (produk yang berupa sel kekal yang digunakan untuk tujuan kajian biologi, kini adalah berkemungkinan untuk memperolehi titer tinggi melebihi 5g/l untuk mAbs [2]. Walaupun begitu, sel mamalia sensitif terhadap retakan disebabkan tegasan ricih yang menyebabkan pengeluaran protease dan hos sel protein yang lain, (host cell proteins (HCPs akhirnya mempengaruhi kestabilan dan keaslian produk. Terdapat mAbs dalam jumlah besar yang masih menjalani pembangunan klinikal dan sesungguhnya ini penting sebagai satu landasan teknologi dalam

  5. Novel monoclonal antibody against beta 1 integrin enhances cisplatin efficacy in human lung adenocarcinoma cells.

    Science.gov (United States)

    Kim, Min-Young; Cho, Woon-Dong; Hong, Kwon Pyo; Choi, Da Bin; Hong, Jeong Won; Kim, Soseul; Moon, Yoo Ri; Son, Seung-Myoung; Lee, Ok-Jun; Lee, Ho-Chang; Song, Hyung Geun

    2016-05-01

    The use of anti-beta 1 integrin monoclonal antibody in lung cancer treatment has proven beneficial. Here, we developed a novel monoclonal antibody (mAb), called P5, by immunizing mice with human peripheral blood mononuclear cells (PBMC). Its anti-tumor effect is now being tested, in a clinical phase III trial, in combinatorial treatments with various chemical drugs. To confirm that P5 indeed binds to beta 1 integrin, cell lysates were immunoprecipitated with commercial anti-beta 1 integrin mAb (TS2/16) and immunoblotted against P5 to reveal a 140 kDa molecular weight band, as expected. Immunoprecipitation with P5 followed by LC/MS protein sequence analysis further verified P5 antigen to be beta 1 integrin. Cisplatin treatment upregulated cell surface expression of beta 1 integrin in A549 cells, while causing inhibition of cell growth. When cells were co-treated with different concentrations of P5 mAb, the cisplatin-mediated inhibitory effect was enhanced in a dose-dependent manner. Our findings show that a combinatorial treatment of P5 mAb and cisplatin in A549 cells resulted in a 30% increase in apoptosis, compared to baseline, and significantly more when compared to either the cisplatin or P5 alone group. The entire peptide sequences in CDR from variable region of Ig heavy and light chain gene for P5 mAb are also disclosed. Together, these results provide evidence of the beneficial effect of P5 mAb in combinatorial treatment of human lung adenocarcinoma.

  6. Renal small B-cell lymphoma with plasmacytic differentiation presenting with monoclonal gammopathy and disseminated intravascular coagulation syndrome

    Directory of Open Access Journals (Sweden)

    Paula de Oliveira Pádua Prestes

    2013-10-01

    Full Text Available Primary renal lymphomas are very rare. However, the kidney may be a site of metastasis, usually from a disseminated aggressive lymphoma. A 58-year-old woman was brought to the medical facility due to acute mental confusion, severe hypotension, septic shock, and signs of disseminated intravascular coagulation. Laboratory tests showed severe leukopenia, renal failure, altered liver function, and elevated serum lactate dehydrogenase levels. Protein electrophoresis revealed hypergammaglobulinemia with a monoclonal peak of IgG lambda. The clinical outcome was fulminant and the patient died less than 24 hours after admission. Autopsy revealed an indolent B-cell lymphoma with extensive plasmacytic differentiation infiltrating the right renal sinus and involving the submandibular and sublingual glands, cervical and peri-aortic lymph nodes, multiple microscopic foci in pituitary and adrenal glands, lung, breast, liver, thyroid, and bone marrow. Numerous IgG4-positive plasma cells were detected by immunohistochemistry although other histological features of IgG4-related disease were missing. There was also extensive hemorrhagic necrosis of the adrenal glands and purulent cystitis, which was probably responsible for the septic shock. The authors concluded that the kidney was most likely the primary site of the indolent lymphoma, as that was the site with the largest tumor mass. Infiltration of other organs was considered as dissemination of the disease. The differential diagnosis with mucosa-associated lymphoid tissue and lymphoplasmacytic lymphoma is discussed.

  7. A human monoclonal antibody to high-frequency red cell antigen Jra.

    Science.gov (United States)

    Miyazaki, T; Kwon, K W; Yamamoto, K; Tone, Y; Ihara, H; Kato, T; Ikeda, H; Sekiguchi, S

    1994-01-01

    A human-mouse heterohybridoma (HMR0921) secreting human monoclonal IgG3, lambda antibody was produced from peripheral blood lymphocytes of a healthy blood donor with serum antibody to Jra, by EBV transformation and hybridization with mouse myeloma cell line P3X63Ag8.653. The reactivity of HMR0921 antibody was assessed by antiglobulin test with a panel of red cells including 14 different rare blood types. Only Jr(a-) red cells were negative. The strict specificity of this antibody to Jra antigen was further confirmed by absorption test with fluorescence flow cytometry. On screening of 28,744 blood donor samples by HMR0921 antibody, we detected 19 agglutination-negative samples, which were confirmed as Jr(a-) by conventional anti-Jra antisera. Therefore, our HMR0921 antibody is extremely useful for detecting rare Jr(a-) blood.

  8. Identification and purification of human erythroid progenitor cells by monoclonal antibody to the transferrin receptor (TU 67).

    Science.gov (United States)

    Herrmann, F; Griffin, J D; Sabbath, K D; Oster, W; Wernet, P; Mertelsmann, R

    1988-04-01

    Anti-TU 67 is a murine monoclonal antibody that recognizes the transferrin receptor. With respect to hematopoietic cells TU 67 is expressed by human multipotent colony-forming cells (CFU-Mix), erythroid progenitor cells (BFU-E and CFU-E) and a fraction of granulocyte/monocyte colony forming cells, but is not expressed by mature hematopoietic cells including erythrocytes, platelets, lymphocytes, and peripheral blood myeloid cells. The TU 67-positive fraction of normal bone marrow, separated by fluorescence-activated cell sorting (FACS) or immune rosettes, contained 87% of the erythroid progenitor cells. Erythroid progenitor cells were enriched up to 50-fold by using a combination of monoclonal antibodies to deplete mature hematopoietic cells, followed by positive selection of BFU-E and CFU-E by TU 67 antibody.

  9. The monoclonal antibody ER-BMDM1 recognizes a macrophage and dendritic cell differentiation antigen with aminopeptidase activity

    NARCIS (Netherlands)

    P.J. Leenen (Pieter); M.L. Melis (Marleen); G. Kraal (Georg); A.T. Hoogeveen (Andre); W. van Ewijk (Willem)

    1992-01-01

    markdownabstractAbstract Here we describe the reactivity of monoclonal antibody (mAb) ER-BMDM1, directed against a 160-kDa cell membrane-associated antigen (Ag) with aminopeptidase activity. The aminopeptidase recognized by ER-BMDM1 is present on various mouse macrophage (MΦ) and dendritic cell (D

  10. C-src enriched serum microvesicles are generated in malignant plasma cell dyscrasia.

    Directory of Open Access Journals (Sweden)

    Giuseppe Di Noto

    Full Text Available Plasma cell dyscrasias are immunosecretory disorders that can lead to hematological malignancies such as Multiple Myeloma (MM. MM accounts for 15% of all hematologic cancers, and those diagnosed with MM typically become severely ill and have a low life expectancy. Monoclonal immunoglobulin Free Light Chains (FLC are present in the serum and urine of many patients with plasma cell diseases. The biological differences between monoclonal FLCs, produced under malignant or benign dyscrasias, has not yet been characterized. In the present study, we show that endothelial and heart muscle cell lines internalize kappa and lambda FLCs. After internalization, FLCs are rerouted in the extracellular space via microvesicles and exosomes that can be re-internalized in contiguous cells. Only FLCs secreted from malignant B Lymphocytes were carried in Hsp70, annexin V, and c-src positive vesicles. In both MM and AL Amyloidosis patients we observed an increase in microvesicle and exosome production. Isolated serum vesicles from MM, AL Amyloidosis and monoclonal gammopathy of undetermined significance (MGUS patients contained FLCs. Furthermore MM and AL amyloidosis vesicles were strongly positive for Hsp70, annexin V, and c-src compared to MGUS and control patients. These are the first data implying that FLCs reroute via microvesicles in the blood stream, and also suggest a potential novel mechanism of c-src activation in plasma cell dyscrasia.

  11. Monoclonal gammopathy with both nemaline myopathy and amyloid myopathy.

    Science.gov (United States)

    Wang, Min; Lei, Lin; Chen, Hai; Di, Li; Pang, Mi; Lu, Yan; Lu, Lu; Shen, Xin-Ming; Da, Yuwei

    2017-10-01

    Monoclonal gammopathies due to plasma cell dyscrasias can induce diverse rare neuromuscular disorders. Deposition of monoclonal antibody light chains in skeletal muscle causes amyloid myopathy. Monoclonal gammopathy is occasionally associated with sporadic late-onset nemaline myopathy. Here we report a monoclonal gammopathy patient with both sporadic late-onset nemaline myopathy and amyloid myopathy. The diagnoses were based on immunofixation electrophoresis of urine, and serum for free light chain assay, Congo red staining and Thioflavin S staining of muscle biopsies, as well as immunohistochemical staining and electron-microscopic observation. Nemaline myopathy and amyloid myopathy can present in the same patient with monoclonal gammopathy. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Targeting the replisome with transduced monoclonal antibodies triggers lethal DNA replication stress in cancer cells.

    Science.gov (United States)

    Desplancq, Dominique; Freund, Guillaume; Conic, Sascha; Sibler, Annie-Paule; Didier, Pascal; Stoessel, Audrey; Oulad-Abdelghani, Mustapha; Vigneron, Marc; Wagner, Jérôme; Mély, Yves; Chatton, Bruno; Tora, Laszlo; Weiss, Etienne

    2016-03-15

    Although chemical inhibition of the DNA damage response (DDR) in cancer cells triggers cell death, it is not clear if the fork blockade achieved with inhibitors that neutralise proteins of the replisome is sufficient on its own to overcome the DDR. Monoclonal antibodies to PCNA, which block the DNA elongation process in vitro, have been developed. When these antibodies were transduced into cancer cells, they are able to inhibit the incorporation of nucleoside analogues. When co-delivered with anti-PCNA siRNA, the cells were flattened and the size of their nuclei increased by up to 3-fold, prior to cell death. Analysis of these nuclei by super-resolution microscopy revealed the presence of large numbers of phosphorylated histone H2AX foci. A senescence-like phenotype of the transduced cells was also observed upon delivery of the corresponding Fab molecules or following PCNA gene disruption or when the Fab fragment of an antibody that neutralises DNA polymerase alpha was used. Primary melanoma cells and leukaemia cells that are resistant to chemical inhibitors were similarly affected by these antibody treatments. These results demonstrate that transduced antibodies can trigger a lethal DNA replication stress, which kills cancer cells by abolishing the biological activity of several constituents of the replisome.

  13. Stoichiometry of monoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1

    DEFF Research Database (Denmark)

    Schønning, Kristian; Lund, O; Lund, O S

    1999-01-01

    In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes are coexpr......In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes...

  14. Stoichiometry of monoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1

    DEFF Research Database (Denmark)

    Schønning, Kristian; Lund, O; Lund, O S;

    1999-01-01

    In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes are coexpr......In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes...

  15. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  16. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  17. Main Quality Attributes of Monoclonal Antibodies and Effect of Cell Culture Components

    Science.gov (United States)

    Torkashvand, Fatemeh; Vaziri, Behrouz

    2017-05-01

    The culture media optimization is an inevitable part of upstream process development in therapeutic monoclonal antibodies (mAbs) production. The quality by design (QbD) approach defines the assured quality of the final product through the development stage. An important step in QbD is determination of the main quality attributes. During the media optimization, some of the main quality attributes such as glycosylation pattern, charge variants, aggregates, and low-molecular-weight species, could be significantly altered. Here, we provide an overview of how cell culture medium components affects the main quality attributes of the mAbs. Knowing the relationship between the culture media components and the main quality attributes could be successfully utilized for a rational optimization of mammalian cell culture media for industrial mAbs production.

  18. Entrapment of animal cells for the production of biomolecules such as monoclonal antibodies.

    Science.gov (United States)

    Scheirer, W; Nilsson, K; Merten, O W; Katinger, H W; Mosbach, K

    1983-01-01

    An important problem in the production of monoclonal antibodies is the large-scale cultivation of hybridoma cells in vitro. Fragility of cells and suboptimal in vitro cultivation methods have led to poor results in larger scale production up to now. To lower the mechanical stress on the cells we tried to entrap the cells into microspheres made of polymer material. In addition to other materials, agarose as embedding medium was investigated and results with hybridoma and other, non anchorage-dependent cell lines are given. The conclusion of the results is that encapsulation of living cells is possible and entrapped cells remain viable and continue to produce the desired substance for at least several weeks. The substances are secreted through the polymer matrix. Handling of microspheres is shown to be easy and simple fermentation apparatus may be used for the production on a reliable technical scale. Some problems remain unsolved, such as the determination of viable cell count within the microspheres and cultivation in columns which seems to be the simplest form of continuous production process.

  19. An automated robotic platform for rapid profiling oligosaccharide analysis of monoclonal antibodies directly from cell culture.

    Science.gov (United States)

    Doherty, Margaret; Bones, Jonathan; McLoughlin, Niaobh; Telford, Jayne E; Harmon, Bryan; DeFelippis, Michael R; Rudd, Pauline M

    2013-11-01

    Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. [The expression of antigens, detectable with ICO-series monoclonal antibodies, on the surfaces of cells forming granulocyte and macrophage colonies in semiliquid agar].

    Science.gov (United States)

    Savel'eva, E V; Kharlamova, L A; Baryshnikov, A Iu; Agafonov, V A; Tupitsyn, N N

    1989-01-01

    The expression of antigens on granulocyte-macrophagal colony-forming cells of patients with nonhematological diseases was studied. Treatment of bone marrow cells with murine monoclonal antibodies ICO-1 and ICO-11 led to statistically significant inhibition of the number of growing colonies. Monoclonal antibodies ICO-02, ICO-10, ICO-GM-1 and ICO-G-2 had no such effect.

  1. Reactional Plasmacytosis In Plasma Cell Orificial mucositis In A Patient Of Pulmonary Tuberculosis

    Directory of Open Access Journals (Sweden)

    Bose Sumit Kumar

    1997-01-01

    Full Text Available Skin biopsy of a 50 year old Moroccan male patient with labial and oro-pharyngeal plasmocytosis showed hyperplastic, with papillomatous eroded epithelium. Dense infiltrates of plasma cells were seen in the dermis, with perivascular prominence. Hypopharynx, epiglottis, adenoids, and tonsils showed the same type of infiltration. Immunofluorescence (IF and peroxidase antiperoxidase (PAP techniques demonstrated the presence of mostly and infiltrate of plasma cells showing IgA (30 â€" 40%, IgM (20-30%, IgG(10-20% after staining with polyclonal antibodies along with T4 & T8 Iymphocytes with monoclonal staining. Electron microscopy showed absence of atypical plasma cells with abundant endoplasmic reticulum. Patient’s symptoms of stomtitis, dysphonia and pharyngitis were temporarily relieved by systemic corticosteroids of plasma cells suggesting a reactive type of benign plasmocytosis.

  2. Patterning processes in aggregates of Hydra cells visualized with the monoclonal antibody, TS19.

    Science.gov (United States)

    Sato, M; Bode, H R; Sawada, Y

    1990-10-01

    The monoclonal antibody, TS19, (Heimfeld et al., 1985), labels the apical surface of ectodermal epithelial cells of tentacles and lower peduncles in Hydra. To investigate the patterning process in a tissue whose original pattern was completely destroyed, the TS19 staining pattern was examined in developing aggregates of Hydra cells. Two types of aggregates were prepared. G-aggregates were made from tissue of the gastric portion of animals and RG-aggregates from gastric tissue allowed to regenerate for 24 hr before making aggregates. G-aggregates were initially TS19-negative, and later dim and uniformly TS19-positive. Thereafter, TS19 staining broke up into brightly stained and unstained regions. The brightly staining regions developed into head or foot structures. The TS19 pattern in RG-aggregates developed differently. Since the initial aggregates contained cells of regenerating tips, they started with TS19-positive cells as well as TS19-negative cells. The numbers of brightly staining TS19-positive cells increased with time. Some patches of these cells developed into head or foot structures, while others did not. These results and a simulation using a reaction-diffusion model suggest that the changes in activation levels affected the temporal changes in the pattern of TS19 staining, and that the de novo pattern formation in hydra can be explained in terms of a process involving activation and inhibition properties.

  3. Dysmegakaryocytopoiesis and maintaining platelet count in patients with plasma cell neoplasm

    Directory of Open Access Journals (Sweden)

    Yasmin Mair

    2013-01-01

    Full Text Available Background: Dysmegakaryocytopoiesis in patients with the plasma cell neoplasm (PCN is rarely discussed in the literature. The puzzling phenomenon, which PCN patients maintaining normal platelet count even when the marrow is mostly replaced by plasma cells, is hardly explored. Aim: This study was aimed to determine the frequency of dysmegakaryocytopoiesis in PCN and the relationships between bone marrow (BM plasma cell percentage, plasma cell immunomarkers, the severity of dysmegakaryocytopoiesis, and peripheral blood platelet count in PCN. Materials and Methods: We randomly selected 16 cases of PCN, among which 4 were with monoclonal gammopathy of undetermined significance and 12 were with plasma cell myeloma. Results: Our study showed that: (1 Dysmegakaryocytopoiesis was present in all the selected cases of PCN and its severity was not correlated with the percentage of the plasma cells in BM; (2 almost all patients maintained normal platelet count even when BM was mostly replaced by plasma cells; (3 immunomarkers of the neoplastic plasma cells were not associated with dysmegakaryocytopoiesis or maintaining of platelet count. The possible mechanisms behind dysmegakaryocytopoiesis and maintaining of platelet count were also discussed. Conclusion: Despite the universal presence of dysmegakaryocytopoiesis in PCN, the platelet count is maintained at normal range.

  4. Detection of lymphocystis disease virus infection to flounder gill cells in vitro by monoclonal antibodies

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Flounder gill (FG) cells were used to isolate lymphocystis disease virus (LCDV) and two monoclonal antibodies (Mabs) (1A8 and 3G3) against LCDV were used to trace LCDV infection to FG cells. FG monolayer cells was inoculated with LCDV supernatant, obtained from lymphocystis cells of diseased flounder, Paralichthys olivaceus. LCDV infection was detected with Mabs employing immunocytochemical assay (ICA) and indirect immunofluorescence assay test (ⅡFAT) technique. Detected by ⅡFAT, they were specific for LCDV. The results of experimental infection illustrated that FG cells was sensitive to LCDV, and showed virus-infection positive detected by ICA. Cytopathic effect (CPE) occurred 1-2 days post inoculation (PI), and half tissue culture infection dosage (TCID50) of virus supernatant was 22.57 per 40μl. Tracing by ⅡFAT showed that LCDV positive signal first appeared at the cell membrane immediately PI, and then in cytoplasm at 24h PI, it reached the strongest positive at 48-72 h PI, and began to decrease at 96h PI.

  5. Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm.

    Science.gov (United States)

    Gadue, Paul; Gouon-Evans, Valerie; Cheng, Xin; Wandzioch, Ewa; Zaret, Kenneth S; Grompe, Markus; Streeter, Philip R; Keller, Gordon M

    2009-09-01

    The development of functional cell populations such as hepatocytes and pancreatic beta cells from embryonic stem cell (ESC) is dependent on the efficient induction of definitive endoderm early in the differentiation process. To monitor definitive endoderm formation in mouse ESC differentiation cultures in a quantitative fashion, we generated a reporter cell line that expresses human CD25 from the Foxa3 locus and human CD4 from the Foxa2 locus. Induction of these reporter ESCs with high concentrations of activin A led to the development of a CD25-Foxa3+CD4-Foxa2+ population within 4-5 days of culture. Isolation and characterization of this population showed that it consists predominantly of definitive endoderm that is able to undergo hepatic specification under the appropriate conditions. To develop reagents that can be used for studies on endoderm development from unmanipulated ESCs, from induced pluripotent stem cells, and from the mouse embryo, we generated monoclonal antibodies against the CD25-Foxa3+CD4-Foxa2+ population. With this approach, we identified two antibodies that react specifically with endoderm from ESC cultures and from the early embryo. The specificity of these antibodies enables one to quantitatively monitor endoderm development in ESC differentiation cultures, to study endoderm formation in the embryo, and to isolate pure populations of culture- or embryo-derived endodermal cells.

  6. Inhibition of Insulin Degradation by Hepatoma Cells after Microinjection of Monoclonal Antibodies to a Specific Cytosolic Protease

    Science.gov (United States)

    Shii, Kozui; Roth, Richard A.

    1986-06-01

    Four monoclonal antibodies were identified by their ability to bind to 125I-labeled insulin covalently linked to a cytosolic insulin-degrading enzyme from human erythrocytes. All four antibodies were also found to remove more than 90% of the insulin-degrading activity from erythrocyte extracts. These antibodies were shown to be directed to different sites on the enzyme by mapping studies and by their various properties. Two antibodies recognized the insulin-degrading enzyme from rat liver; one inhibited the erythrocyte enzyme directly; and two recognized the enzyme after gel electrophoresis and transfer to nitrocellulose filters. By this latter procedure and immunoprecipitation from metabolically labeled cells, the enzyme from a variety of tissues was shown to be composed of a single polypeptide chain of apparent Mr 110,000. Finally, these monoclonal antibodies were microinjected into the cytoplasm of a human hepatoma cell line to assess the contribution of this enzyme to insulin degradation in the intact cell. In five separate experiments, preloading of cells with these monoclonal antibodies resulted in an inhibition of insulin degradation of 18-54% (average 39%) and increased the amount of 125I-labeled insulin associated with the cells. In contrast, microinjection of control antibody or an extraneous monoclonal antibody had no effect on insulin degradation or on the amount of insulin associated with the cells. Moreover, the monoclonal antibodies to the insulin-degrading enzyme caused no significant inhibition of degradation of another molecule, low density lipoprotein. Thus, these results support a role for this enzyme in insulin degradation in the intact cell.

  7. Monoclonal antibodies to intermediate filament proteins of human cells: unique and cross-reacting antibodies.

    Science.gov (United States)

    Gown, A M; Vogel, A M

    1982-11-01

    Monoclonal antibodies were generated against the intermediate filament proteins of different human cells. The reactivity of these antibodies with the different classes of intermediate filament proteins was determined by indirect immunofluorescence on cultured cells, immunologic indentification on SDS polyacrylamide gels ("wester blot" experiments), and immunoperoxidase assays on intact tissues. The following four antibodies are described: (a) an antivimentin antibody generated against human fibroblast cytoskeleton; (b), (c) two antibodies that recognize a 54-kdalton protein in human hepatocellular carcinoma cells; and (d) an antikeratin antibody made to stratum corneum that recognizes proteins of molecular weight 66 kdaltons and 57 kdaltons. The antivimentin antibody reacts with vimentin (58 kdaltons), glial fibrillary acidic protein (GFAP), and keratins from stratum corneum, but does not recognize hepatoma intermediate filaments. In immunofluorescence assays, the antibody reacts with mesenchymal cells and cultured epithelial cells that express vimentin. This antibody decorates the media of blood vessels in tissue sections. One antihepatoma filament antibody reacts only with the 54 kdalton protein of these cells and, in immunofluorescence and immunoperoxidase assays, only recognizes epithelial cells. It reacts with almost all nonsquamous epithelium. The other antihepatoma filament antibody is much less selective, reacting with vimentin, GFAP, and keratin from stratum corneum. This antibody decorates intermediate filaments of both mesenchymal and epithelial cells. The antikeratin antibody recognizes 66-kdalton and 57-kdalton proteins in extracts of stratum corneum and also identifies proteins of similar molecular weights in all cells tested. However, by immunofluorescence, this antibody decorates only the intermediate filaments of epidermoid carcinoma cells. When assayed on tissue sections, the antibody reacts with squamous epithelium and some, but not all

  8. Monoclonal gammopathy and spurious hypophosphatemia.

    Science.gov (United States)

    Weisbord, Steven D; Chaudhuri, Anita; Blauth, Kathleen; DeRubertis, Frederick R

    2003-02-01

    Spuriously low levels of plasma phosphate have been reported previously in patients with multiple myeloma and polyclonal gammopathy. We report 2 cases of spurious hypophosphatemia in patients with elevated concentrations of serum monoclonal immunoglobulins, 1 of whom had monoclonal gammopathy of undetermined significance and the other multiple myeloma. Plasma phosphate concentrations were measured using nondeproteinized and deproteinized plasma samples from patients with monoclonal gammopathies. In 2 patients with monoclonal gammopathy, the levels of plasma inorganic phosphate were reported as <1.0 mg/dL when the phosphate concentration was determined using an analyzer that employs nondeproteinized plasma. When the samples were reanalyzed using a laboratory method that removes serum proteins, normal or elevated concentrations of phosphate were found. Plasma levels of phosphate in 4 other patients with monoclonal gammopathy were normal by both methods. These data confirm previous reports that spurious hypophosphatemia occurs in some patients with increased levels of serum monoclonal immunoglobulins when laboratory methods using nondeproteinized samples are employed. The occurrence of unusually low plasma phosphate concentrations in patients without symptoms or clinically apparent causes of hypophosphatemia should alert physicians to search for monoclonal gammopathy.

  9. Understanding Transcriptional Enhancement in Monoclonal Antibody-Producing Chinese Hamster Ovary Cells

    Science.gov (United States)

    Nicoletti, Sarah E.

    With the demand for monoclonal antibody (mAB) therapeutics continually increasing, the need to better understand what makes a high productivity clone has gained substantial interest. Monoclonal antibody producing Chinese hamster ovary (CHO) cells with different productivities were provided by a biopharmaceutical company for investigation. Gene copy numbers, mRNA levels, and mAb productivities were previously determined for two low producing clones and their amplified progeny. These results showed an increase in mRNA copy number in amplified clones, which correlated to the observed increases in specific productivity of these clones. The presence of multiple copies of mRNA per one copy of DNA in the higher productivity clones has been coined as transcriptional enhancement. The methylation status of the CMV promoter as well as transcription factor/promoter interactions were evaluated to determine the cause of transcriptional enhancement. Methylation analysis via bisulfite sequencing revealed no significant difference in overall methylation status of the CMV promoter. These data did, however, reveal the possibility of differential interactions of transcription factors between the high and low productivity cell clones. This finding was further supported by chromatin immunoprecipitations previously performed in the lab, as well as literature studies. Transcription activator-like effector (TALE) binding proteins were constructed and utilized to selectively immunoprecipitate the CMV promoter along with its associated transcription factors in the different CHO cell clones. Cells were transfected with the TALE proteins, harvested and subjected to a ChIP-like procedure. Results obtained from the TALE ChIP demonstrated the lack of binding of the protein to the promoter and the need to redesign the TALE. Overall, results obtained from this study were unable to give a clear indication as to the causes of transcriptional enhancement in the amplified CHO cell clones. Further

  10. Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals.

    Directory of Open Access Journals (Sweden)

    Davide Corti

    Full Text Available BACKGROUND: The isolation of human monoclonal antibodies (mAbs that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. METHODS AND FINDINGS: We immortalized IgG(+ memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16 specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194 bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20 with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. CONCLUSIONS: This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

  11. Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry.

    Science.gov (United States)

    Kurki, P; Ogata, K; Tan, E M

    1988-04-22

    Proliferating cell nuclear antigen (PCNA)/cyclin is an intranuclear polypeptide antigen that is found in both normal and transformed proliferating cells. We have recently described two mouse monoclonal antibodies reacting with PCNA. In this report we describe the application of these antibodies to the study of proliferating human cells by indirect immunofluorescence microscopy and by flow cytometry. A fixation/permeation procedure was developed in order to obtain satisfactory binding of monoclonal PCNA-specific antibodies to proliferating cells. This method involved fixation with 1% paraformaldehyde followed by methanol treatment. For the staining of cells in suspension with the IgM type monoclonal antibodies lysolecithin was added to the paraformaldehyde solution to achieve a better permeation by the antibody molecules. This procedure gave a good ratio of specific staining relative to the background staining. It also preserved the shape and normal architecture of the cells as judged by visual microscopic observation and by light scatter measurements using a flow cytometer. Furthermore, this fixation technique permits simultaneous labeling of DNA by propidium iodide and PCNA by monoclonal antibodies. PCNA was detected in various types of normal and transformed proliferating cells by indirect immunofluorescence. Quiescent peripheral blood mononuclear cells were PCNA-negative whereas a fraction of lectin-stimulated lymphocytes became PCNA-positive. Similarly, early passages of fetal skin fibroblasts were PCNA-positive but non-proliferating senescent fibroblasts of later passages were PCNA-negative. The association of PCNA-staining by monoclonal antibodies with cell proliferation was confirmed by flow cytometry. Simultaneous labeling of PCNA and DNA showed that the PCNA signal increased during the G1 phase of the cell cycle, reached its maximum in the S-phase, and declined during the G2/M phase. Using cell sorting we demonstrated that mitotic cells had a very low PCNA

  12. Plasma cells negatively regulate the follicular helper T cell program

    OpenAIRE

    2010-01-01

    B lymphocytes differentiate into antibody-secreting cells under the antigen-specific control of follicular helper T (TFH) cells. Here, we demonstrate that isotype-switched plasma cells expressed MHCII, CD80 and CD86 and intracellular machinery required for antigen presentation. Antigen-specific plasma cells could access, process and present sufficient antigen in vivo to induce multiple TH cell functions. Importantly, antigen-primed plasma cells failed to induce interleukin 21 or Bcl-6 in naïv...

  13. Cytotoxic murine monoclonal antibody LAM8 with specificity for human small cell carcinoma of the lung.

    Science.gov (United States)

    Stahel, R A; O'Hara, C J; Mabry, M; Waibel, R; Sabbath, K; Speak, J A; Bernal, S D

    1986-04-01

    The reactivity of the murine immunoglobulin monoclonal antibody LAM8 directed against a membrane antigen of human small cell carcinoma (SCC) of the lung was investigated on human cell lines and tissues. Indirect immunofluorescence staining, radioimmunoassays, and cytotoxicity assays showed LAM8 antibody to selectively react with SCC but not with non-SCC lung cancer cell lines and extrapulmonary tumor cell lines. Unlike other SCC antibodies, including those we have previously described, highly preferential reactivity with SCC tissues was also demonstrated by immunoperoxidase staining of deparaffinized formalin-fixed tissue sections. Membrane and cytoplasmic staining was seen in of 9 of 12 SCC tissues. No significant staining was seen in non-SCC lung cancer and a wide range of other tumors, including mesothelioma and bronchial carcinoids. Significant LAM8 reactivity was also absent in normal tissues of all major organs. Few tumors and epithelial tissues, including bronchial epithelium had rare LAM8 positive cells which were always less than 2% of the entire cell population. In vitro treatment with antibody and human complement was highly cytotoxic to SCC cells, but had not effect on bone marrow progenitor cells. Immunoblotting of membrane extracts separated on sodium dodecyl sulfate-polyacrylamide gels showed the LAM8 antigen to have a band of an approximate molecular weight of 135,000 and a cluster of bands with approximate molecular weights of 90,000. This reactivity was lost after incubation of the extracts with periodate. LAM8 antibody shows a highly preferential reactivity with SCC cell lines and formalin-fixed paraffin-embedded SCC tissues and is selectively cytotoxic to cells expressing LAM8 antigen.

  14. In vitro photothermal destruction of neuroblastoma cells using carbon nanotubes conjugated with GD2 monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Chung-Hao; Huang, Yao-Jhang; Chang, Chia-Wei; Peng, Ching-An [Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan (China); Hsu, Wen-Ming, E-mail: cpeng@mtu.ed [Department of Surgery, National Taiwan University, Taipei, Taiwan (China)

    2009-08-05

    Despite aggressive multimodality therapy, most neuroblastoma-bearing patients relapse and survival rate remains poor. Exploration of alternative therapeutic modalities is needed. Carbon nanotubes (CNTs), revealing optical absorbance in the near-infrared region, warrant their merits in photothermal therapy. In order to specifically target disialoganglioside (GD2) overexpressed on the surface of neuroblastoma stNB-V1 cells, GD2 monoclonal antibody (anti-GD2) was conjugated to acidified CNTs. To examine the fate of anti-GD2 bound CNTs after incubation with stNB-V1 cells, rhodamine B was labeled on carboxylated CNTs functionalized with and without anti-GD2. Our results illustrated that anti-GD2-linked CNTs were extensively internalized by neuroblastoma cells via GD2-mediated endocytosis. In addition, we showed that anti-GD2 bound CNTs were not ingested by PC12 cells without GD2 expression. After anti-GD2 conjugated CNTs were incubated with neuroblastoma cells for 6 h and endocytosed by the cells, CNT-laden neuroblastoma cells were further irradiated with an 808 nm near-infrared (NIR) laser with intensity ramping from 0.6 to 6 W cm{sup -2} for 10 min which was then maintained at 6 W cm{sup -2} for an additional 5 min. Post-NIR laser exposure, and after being examined by calcein-AM dye, stNB-V1 cells were all found to undergo necrosis, while non-GD2 expressing PC12 cells all remained viable. Based on the in vitro study, CNTs bound with anti-GD2 have the potential to be utilized as a therapeutic thermal coupling agent that generates heat sufficient to selectively kill neuroblastoma cells under NIR laser light exposure.

  15. Gas Plasma Effects on Living Cells

    Science.gov (United States)

    Stoffels, E.; Sladek, R. E. J.; Kieft, I. E.

    This paper surveys the research activities at the Eindhoven University of Technology (The Netherlands) in the area of biomedical applications of gas discharge plasmas. A non-thermal atmospheric plasma source (the plasma needle) has been developed, and its interactions with living mammalian cells and bacteria are studied. It is concluded that plasma can efficiently kill bacteria without harming the cells, and also influence the cells without causing cell death (necrosis). In future it will lead to applications like skin (wound) and caries treatment.

  16. Generation of Rat Monoclonal Antibodies Against Human Pancreatic Ductal Adenocarcinoma Cells.

    Science.gov (United States)

    Higashi, Kiyoshi; Fujii, Nobuaki; Kushida, Masahiko; Yamada, Keita; Suzuki, Noriyuki; Saito, Koichi; Tachibana, Taro

    2016-06-01

    Pancreatic ductal adenocarcinoma is an aggressive tumor with a poor prognosis. Biomarkers that can detect the tumor in its early stages when it may be amenable to curative resection might improve prognosis. To discover novel markers expressed in primary pancreatic cancer, we generated a panel of monoclonal antibodies against pancreatic ductal adenocarcinoma cell line BxPC3 using a rat medial iliac lymph node method. The antigen recognized by 1B5A5 was expressed on the cell surface and secreted into the conditioned medium of BxPC3 cells, and characterized as glycoproteins with molecular mass between 60 and 90 kDa. A wide range of molecular weights of 1B5A5 antigen in several pancreatic cancer cell lines were observed. Immunohistochemistry using a human multiple organ tumor tissue array showed an enhanced expression of 1B5A5 antigen in pancreas, lung, stomach, breast, urinary bladder, colon, and cervix uteri cancers. Immunoprecipitation followed by proteomic analyses identified CEACAM6 as a 1B5A5 antigen. In addition, western blot analysis results indicated that the 1B5A5 epitope is located within an amino-terminal domain of CEACAM6. These results raised the possibility that our approach could lead to discovery of novel biomarkers for the early stage of cancers in a relatively short period of time.

  17. Ontology driven decision support systems for medical diagnosis - an interactive form for consultation in patients with plasma cell disease.

    Science.gov (United States)

    Donfack Guefack, Valéry; Bertaud Gounot, Valérie; Duvauferrier, Régis; Bourde, Annabel; Morelli, John; Lasbleiz, Jérémy

    2012-01-01

    Multiple myeloma (MM) is a malignant disorder characterized by the monoclonal proliferation of B cell derived plasma cells in the bone marrow. The diagnosis depends on the identification of abnormal monoclonal marrow plasma cells, monoclonal protein in the serum or urine, evidence of end-organ damage, and a clinical picture consistent with MM. The distinction between MM stages- monoclonal gammopathy of undetermined significance or indolent myeloma-is critical in guiding therapy. This paper describes how to produce ontology-driven semiological rules base (SRB) and a consultation form to aid in the diagnosis of plasma cells diseases. We have extracted the MM sub-ontology from the NCI Thesaurus. Using Protégé 3.4.2 and owl1, criteria in the literature for the diagnosis and staging of MM have been added to the ontology. All quantitative parameters have been transformed to a qualitative format. A formal description of MM variants and stages has been given. The obtained ontology has been checked by a reasoner and instantiated to obtain a SRB. The form created has been tested and evaluated utilizing 63 clinical medical reports. The likelihood for a disease being the correct diagnosis is determined by computing a ratio. The resulting tool is relevant for MM diagnosis and staging.

  18. General Information about Plasma Cell Neoplasms (Including Multiple Myeloma)

    Science.gov (United States)

    ... Cell Neoplasms Treatment Research Plasma Cell Neoplasms (Including Multiple Myeloma) Treatment (PDQ®)–Patient Version General Information About Plasma ... the throat can make it hard to swallow. Multiple myeloma In multiple myeloma , abnormal plasma cells ( myeloma cells ) ...

  19. Stages of Plasma Cell Neoplasms (Including Multiple Myeloma)

    Science.gov (United States)

    ... Cell Neoplasms Treatment Research Plasma Cell Neoplasms (Including Multiple Myeloma) Treatment (PDQ®)–Patient Version General Information About Plasma ... the throat can make it hard to swallow. Multiple myeloma In multiple myeloma , abnormal plasma cells ( myeloma cells ) ...

  20. Treatment Options for Plasma Cell Neoplasms (Including Multiple Myeloma)

    Science.gov (United States)

    ... Cell Neoplasms Treatment Research Plasma Cell Neoplasms (Including Multiple Myeloma) Treatment (PDQ®)–Patient Version General Information About Plasma ... the throat can make it hard to swallow. Multiple myeloma In multiple myeloma , abnormal plasma cells ( myeloma cells ) ...

  1. Treatment Option Overview (Plasma Cell Neoplasms Including Multiple Myeloma)

    Science.gov (United States)

    ... Cell Neoplasms Treatment Research Plasma Cell Neoplasms (Including Multiple Myeloma) Treatment (PDQ®)–Patient Version General Information About Plasma ... the throat can make it hard to swallow. Multiple myeloma In multiple myeloma , abnormal plasma cells ( myeloma cells ) ...

  2. (Investigations into the use of radiolabeled monoclonal antibodies for selective cell labeling in whole blood):

    Energy Technology Data Exchange (ETDEWEB)

    Thakur, M.L.

    1987-01-01

    Seventeen monoclonal antibodies (MAbs), 7 specific for human platelets and 10 specific for human polumorphonuclear leukocytes (PMNs) have been evaluated. One MAb has been identified as the antibody most suitable for canine platelets and another has been evaluted as the best among the group, for human neutrophil studies. Indium-111, Tc-99m, and I-125 have been used as the tracers. Six bifunctional chelating agents (BFCAs) were evaluated in order to determine the most efficient agent for maximal cell labeling efficiency. Among these, the DTPA has given us the best results. (4) To botain maximum In-111 chelation and minimum loss of the MAb affinity, the optimal BFCA to MAb ratios for both IgG and IgM type of MAbs were determined. Four different substances, stannous chloride, ascorbic acid, sodium dithionite and sodium borohydride, were evaluated as reducing agents for Tc-99m reduction and its optimal binding to MAbs. Dithionite at the concentration of 200 ug/ml DTPA-MAb solution provides greater than 50% Tc-99m labeling efficiency and maintains its immunospecificity equal to that of In-111-DTPA-MAb. The ability of radiolabeled MAb to interact with blood cells selectively in whole blood and with isolated blood cells was assessed and compared.

  3. Prognostic value of miR-155 in individuals with monoclonal B-cell lymphocytosis and patients with B chronic lymphocytic leukemia.

    Science.gov (United States)

    Ferrajoli, Alessandra; Shanafelt, Tait D; Ivan, Cristina; Shimizu, Masayoshi; Rabe, Kari G; Nouraee, Nazila; Ikuo, Mariko; Ghosh, Asish K; Lerner, Susan; Rassenti, Laura Z; Xiao, Lianchun; Hu, Jianhua; Reuben, James M; Calin, Steliana; You, M James; Manning, John T; Wierda, William G; Estrov, Zeev; O'Brien, Susan; Kipps, Thomas J; Keating, Michael J; Kay, Neil E; Calin, George A

    2013-09-12

    Noncoding RNAs play a pivotal role in the pathogenesis of chronic lymphocytic leukemia (CLL). We hypothesized that microRNAs (miRs) are involved in the transition from monoclonal B-cell lymphocytosis (MBL) to CLL and tested miR-15a/16-1 cluster, miR-21, and miR-155 expression in purified B cells of normal individuals, individuals with MBL, and patients with CLL. When we analyzed 224 samples from 2 independent training and validation cohorts, we found that miR-155 was overexpressed in B cells from individuals with MBL, and even more so in B cells from patients with CLL, when compared with B cells from normal individuals. Furthermore, we were able to identify miR-155 in circulating microvesicles from both individuals with MBL and patients with CLL. Next, to examine the prognostic role of miR-155, we measured its expression level in plasma samples collected before treatment initiation in 228 patients with CLL. We found significantly higher miR-155 expression levels in patients who failed to achieve a complete response compared with those who experienced complete response. Our findings support the use of cellular and plasma levels of miR-155 as biomarkers for the risk of progression in individuals with MBL, as well as to identify patients with CLL who may not respond well to therapy.

  4. T10B9 monoclonal antibody: A short-acting nonstimulating monoclonal antibody that spares γδ T-cells and treats and prevents cellular rejection

    Directory of Open Access Journals (Sweden)

    Thomas H Waid

    2009-06-01

    Full Text Available Thomas H Waid1, John S Thompson1, Maria Siemionow2, Stephen A Brown1 1Department of Internal Medicine, University of Kentucky, Lexington, Kentucky, USA; 2Cleveland Clinic, Cleveland, Ohio, USAAbstract: T10B9.1A-31/MEDI-500 is a nonmitogenic immunoglobulin M kappa murine monoclonal antibody (mAb directed against the alpha-beta (αβ heterodimer of the T-lymphocyte receptor complex. The hybridoma was first produced by fusing spleen cells from BALB/C mice immunized with human peripheral blood T-lymphocytes with SP2/O-Ag14 mutant myeloma cells. The mAb is produced and purified using multistep ion exchange and molecular sieve chromatography protocols. T10B9 has been used successfully to treat acute cellular rejection in renal transplantation and as an immunosuppression induction agent in heart and simultaneous kidney-pancreas transplantation. Because T10B9 is nonmitogenic and causes minimal cytokine release, both treatment of rejection and induction of immunosuppression were accomplished with significantly fewer and milder untoward effects (cytokine release syndrome than its comparator OKT3. Since T10B9 is directed against the αβ heterodimer of the CD3 epitope, it spares the gamma delta (γδ region. These gamma delta (γδ T cells have a unique role in the immune response controlling many serious human diseases and perhaps facilitating the development of immunologic tolerance. T10B9 has a relatively short duration of action, depleting T cells for only 10 to 14 days, unlike the protracted depletion seen with thymoglobulin and Campath-1H. There is no B-lymphocyte depletion with T10B9 as there is with both of the aforementioned reagents. The lack of prolonged lymphocyte depletion may account for less infection observed with T10B9 treatment.Keywords: T10B9.1A-31, γδ T-cell, monoclonal antibody, Campath-1H, thymoglobulin, OKT3

  5. Evolution of Excited Convective Cells in Plasmas

    DEFF Research Database (Denmark)

    Pécseli, Hans; Juul Rasmussen, Jens; Sugai, H.

    1984-01-01

    Convective cells are excited externally in a fully ionized magnetized plasma and their space-time evolution is investigated by two-dimensional potential measurements. A positive cell is excited externally by control of the end losses in the 'scrape off' layer of a plasma column produced by surface...

  6. Detection of acrolein-derived cyclic DNA adducts in human cells by monoclonal antibodies

    Science.gov (United States)

    Pan, Jishen; Awoyemi, Bisola; Xuan, Zhuoli; Vohra, Priya; Wang, Hsiang-Tsui; Dyba, Marcin; Greenspan, Emily; Fu, Ying; Creswell, Karen; Zhang, Lihua; Berry, Deborah; Tang, Moon-Shong; Chung, Fung-Lung

    2013-01-01

    Acrolein (Acr) is a ubiquitous environmental pollutant found in cigarette smoke and automobile exhaust. It can also be produced endogenously by oxidation of polyunsaturated fatty acids. The Acr-derived 1,N2-propanodeoxyguanosine (Acr-dG) adducts in DNA are mutagenic lesions that are potentially involved in human cancers. In this study, monoclonal antibodies were raised against Acr-dG adducts and characterized using ELISA. They showed strong reactivity and specificity towards Acr-dG, weaker reactivity towards crotonaldehyde- and trans-4-hydroxy-2-nonenal-derived 1,N2-propanodeoxyguanosines, and weak or no reactivity towards 1,N6-ethenodeoxyadenosine and 8-oxo-deoxyguanosine. Using these novel antibodies, we developed assays to detect Acr-dG in vivo: First, a simple and quick FACS-based assay for detecting these adducts directly in cells; Second, a highly sensitive direct ELISA assay for measuring Acr-dG in DNA of cells and tissues using only one μg DNA without DNA digestion and sample enrichment; And third, a competitive ELISA for better quantitative measurement of Acr-dG levels in DNA samples. The assays were validated using Acr-treated HT29 cell DNA samples or calf thymus DNA and the results were confirmed by LC-MS/MS-MRM. An immunohistochemical assay was also developed to detect and visualize Acr-dG in HT29 cells as well as in human oral cells. These antibody-based methods provide useful tools for the studies of Acr-dG as a cancer biomarker and of the molecular mechanisms by which cells respond to Acr-dG as a ubiquitous DNA lesion. PMID:23126278

  7. Detection of acrolein-derived cyclic DNA adducts in human cells by monoclonal antibodies.

    Science.gov (United States)

    Pan, Jishen; Awoyemi, Bisola; Xuan, Zhuoli; Vohra, Priya; Wang, Hsiang-Tsui; Dyba, Marcin; Greenspan, Emily; Fu, Ying; Creswell, Karen; Zhang, Lihua; Berry, Deborah; Tang, Moon-Shong; Chung, Fung-Lung

    2012-12-17

    Acrolein (Acr) is a ubiquitous environmental pollutant found in cigarette smoke and automobile exhaust. It can also be produced endogenously by oxidation of polyunsaturated fatty acids. The Acr-derived 1,N(2)-propanodeoxyguanosine (Acr-dG) adducts in DNA are mutagenic lesions that are potentially involved in human cancers. In this study, monoclonal antibodies were raised against Acr-dG adducts and characterized using ELISA. They showed strong reactivity and specificity toward Acr-dG, weaker reactivity toward crotonaldehyde- and trans-4-hydroxy-2-nonenal-derived 1,N(2)-propanodeoxyguanosines, and weak or no reactivity toward 1,N(6)-ethenodeoxyadenosine and 8-oxo-deoxyguanosine. Using these antibodies, we developed assays to detect Acr-dG in vivo: first, a simple and quick FACS-based assay for detecting these adducts directly in cells; second, a highly sensitive direct ELISA assay for measuring Acr-dG in cells and tissues using only 1 μg of DNA without DNA digestion and sample enrichment; and third, a competitive ELISA for better quantitative measurement of Acr-dG levels in DNA samples. The assays were validated using Acr-treated HT29 cell DNA samples or calf thymus DNA, and the results were confirmed by LC-MS/MS-MRM. An immunohistochemical assay was also developed to detect and visualize Acr-dG in HT29 cells as well as in human oral cells. These antibody-based methods provide useful tools for the studies of Acr-dG as a cancer biomarker and of the molecular mechanisms by which cells respond to Acr-dG as a ubiquitous DNA lesion.

  8. The clinical relevance and management of monoclonal gammopathy of undetermined significance and related disorders

    DEFF Research Database (Denmark)

    van de Donk, Niels W C J; Palumbo, Antonio; Johnsen, Hans Erik

    2014-01-01

    Monoclonal gammopathy of undetermined significance is one of the most common pre-malignant disorders. IgG and IgA monoclonal gammopathy of undetermined significance are precursor conditions of multiple myeloma; light-chain monoclonal gammopathy of undetermined significance of light-chain multiple...... myeloma; and IgM monoclonal gammopathy of undetermined significance of Waldenström's macroglobulinemia and other lymphoproliferative disorders. Clonal burden, as determined by bone marrow plasma cell percentage or M-protein level, as well as biological characteristics, including heavy chain isotype...... and light chain production, are helpful in predicting risk of progression of monoclonal gammopathy of undetermined significance to symptomatic disease. Furthermore, alterations in the bone marrow microenvironment of monoclonal gammopathy of undetermined significance patients result in an increased risk...

  9. Selecting B cells and plasma cells to memory

    OpenAIRE

    2005-01-01

    Humoral immunity appears to be based on immunological memory provided by memory plasma cells, which secrete protective antibodies, and memory B cells, which react to antigen challenge by differentiating into plasma cells. How these differentiation pathways relate to each other, how cells are selected into these memory populations, and how these populations are maintained remains enigmatic.

  10. Immunocytochemical analysis of intermediate filaments in embryonic heart cells with monoclonal antibodies to desmin.

    Science.gov (United States)

    Danto, S I; Fischman, D A

    1984-06-01

    Monoclonal antibodies ( McAbs ) have been generated against a preparation of intermediate filament proteins (IFP) from adult chicken gizzard. Two antibodies, D3 and D76 , have been characterized in detail. They bind specifically to desmin but recognize different epitopes. In the adult chicken, both McAbs produced equivalent immunofluorescent staining patterns, reacting in frozen sections with all forms of muscle tissue, including vascular smooth muscle, but with no other tissue types. In isolated skeletal myofibrils and in longitudinal frozen sections of cardiac and skeletal muscle, desmin was detected with both McAbs at the Z-band and in longitudinally-oriented filament bundles between myofibrils. In contrast to these results in the adult, the intermediate filaments (IF) of embryonic cardiac myocytes in primary cultures were decorated only with McAb D3, whereas McAb D76 was completely unreactive with these cells. Similarly, frozen sections through the heart at early stages of embryonic chick development (Hamburger-Hamilton stages 17-18) revealed regions of myocytes, identified by double immunofluorescence with myosin-specific McAbs , that were unstained with McAb D76 even though similar regions were stained by McAb D3. That McAb D76 reacted with desmin in all adult cardiac myocytes but not with all embryonic heart cells indicates that embryonic and adult cardiac IF are immunologically distinct and implies a conversion in IF immunoreactivity during cardiac development.

  11. Eliminating tyrosine sequence variants in CHO cell lines producing recombinant monoclonal antibodies.

    Science.gov (United States)

    Feeney, Lauren; Carvalhal, Veronica; Yu, X Christopher; Chan, Betty; Michels, David A; Wang, Yajun Jennifer; Shen, Amy; Ressl, Jan; Dusel, Brendon; Laird, Michael W

    2013-04-01

    Amino acid sequence variants are defined as unintended amino acid sequence changes that contribute to product variation with potential impact to product safety, immunogenicity, and efficacy. Therefore, it is important to understand the propensity for sequence variant (SV) formation during the production of recombinant proteins for therapeutic use. During the development of clinical therapeutic products, several monoclonal antibodies (mAbs) produced from Chinese Hamster Ovary (CHO) cells exhibited SVs at low levels (≤3%) in multiple locations throughout the mAbs. In these examples, the cell culture process depleted tyrosine, and the tyrosine residues in the recombinant mAbs were replaced with phenylalanine or histidine. In this work, it is demonstrated that tyrosine supplementation eliminated the tyrosine SVs, while early tyrosine starvation significantly increased the SV level in all mAbs tested. Additionally, it was determined that phenylalanine is the amino acid preferentially misincorporated in the absence of tyrosine over histidine, with no other amino acid misincorporated in the absence of tyrosine, phenylalanine, and histidine. The data support that the tyrosine SVs are due to mistranslation and not DNA mutation, most likely due to tRNA(Tyr) mischarging due to the structural similarities between tyrosine and phenylalanine.

  12. Inhibition of P-Selectin and PSGL-1 Using Humanized Monoclonal Antibodies Increases the Sensitivity of Multiple Myeloma Cells to Bortezomib.

    Science.gov (United States)

    Muz, Barbara; Azab, Feda; de la Puente, Pilar; Rollins, Scott; Alvarez, Richard; Kawar, Ziad; Azab, Abdel Kareem

    2015-01-01

    Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow. Despite the introduction of novel therapies majority of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) play a key role in proliferation of MM and using small-molecule inhibitors of P-selectin/PSGL-1 sensitized MM cells to therapy. However, these small-molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. Therefore, we tested blocking of P-selectin and PSGL-1 using functional monoclonal antibodies in order to sensitize MM cells to therapy. We have demonstrated that inhibiting the interaction between MM cells and endothelial and stromal cells decreased proliferation in MM cells and in parallel induced loose-adhesion to the primary tumor site to facilitate egress. At the same time, blocking this interaction in vivo led to MM cells retention in the circulation and delayed homing to the bone marrow, thus exposing MM cells to bortezomib which contributed to reduced tumor growth and better mice survival. This study provides a better understanding of the biology of P-selectin and PSGL-1 and their roles in dissemination and resensitization of MM to treatment.

  13. Inhibition of P-Selectin and PSGL-1 Using Humanized Monoclonal Antibodies Increases the Sensitivity of Multiple Myeloma Cells to Bortezomib

    Directory of Open Access Journals (Sweden)

    Barbara Muz

    2015-01-01

    Full Text Available Multiple myeloma (MM is a plasma cell malignancy localized in the bone marrow. Despite the introduction of novel therapies majority of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1 play a key role in proliferation of MM and using small-molecule inhibitors of P-selectin/PSGL-1 sensitized MM cells to therapy. However, these small-molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. Therefore, we tested blocking of P-selectin and PSGL-1 using functional monoclonal antibodies in order to sensitize MM cells to therapy. We have demonstrated that inhibiting the interaction between MM cells and endothelial and stromal cells decreased proliferation in MM cells and in parallel induced loose-adhesion to the primary tumor site to facilitate egress. At the same time, blocking this interaction in vivo led to MM cells retention in the circulation and delayed homing to the bone marrow, thus exposing MM cells to bortezomib which contributed to reduced tumor growth and better mice survival. This study provides a better understanding of the biology of P-selectin and PSGL-1 and their roles in dissemination and resensitization of MM to treatment.

  14. Effects of anti-vascular endothelial growth factor monoclonal antibody (bevacizumab on lens epithelial cells

    Directory of Open Access Journals (Sweden)

    Jun JH

    2016-06-01

    Full Text Available Jong Hwa Jun,1 Wern-Joo Sohn,2 Youngkyun Lee,2 Jae-Young Kim21Department of Ophthalmology, School of Medicine, Dongsan Medical Center, Keimyung University, 2Department of Oral Biochemistry, School of Dentistry, IHBR, Kyungpook National University, Daegu, South KoreaAbstract: The molecular and cellular effects of anti-vascular endothelial growth factor monoclonal antibody (bevacizumab on lens epithelial cells (LECs were examined using both an immortalized human lens epithelial cell line and a porcine capsular bag model. After treatment with various concentrations of bevacizumab, cell viability and proliferation patterns were evaluated using the water-soluble tetrazolium salt assay and 5-bromo-2'-deoxyuridine enzyme-linked immunosorbent assay, respectively. The scratch assay and Western blot analysis were employed to validate the cell migration pattern and altered expression levels of signaling molecules related to the epithelial–mesenchymal transition (EMT. Application of bevacizumab induced a range of altered cellular events in a concentration-dependent manner. A 0.1–2 mg/mL concentration demonstrated dose-dependent increase in proliferation and viability of LECs. However, 4 mg/mL decreased cell proliferation and viability. Cell migrations displayed dose-dependent retardation from 0.1 mg/mL bevacizumab treatment. Transforming growth factor-β2 expression was markedly increased in a dose-dependent manner, and α-smooth muscle actin, matrix metalloproteinase-9, and vimentin expression levels showed dose-dependent changes in a B3 cell line. Microscopic observation of porcine capsular bag revealed changes in cellular morphology and a decline in cell density compared to the control after 2 mg/mL treatment. The central aspect of posterior capsule showed delayed confluence, and the factors related to EMT revealed similar expression patterns to those identified in the cell line. Based on these results, bevacizumab modulates the proliferation

  15. Prevention of progression in monoclonal gammopathy of undetermined significance.

    Science.gov (United States)

    Rajkumar, S Vincent

    2009-09-15

    Monoclonal gammopathy of undetermined significance (MGUS) is a common premalignant plasma cell proliferative disorder with a lifelong risk of progression to multiple myeloma. Because myeloma is an incurable malignancy, strategies to delay or prevent progression in high-risk patients are of considerable importance.

  16. Prevention of Progression in Monoclonal Gammopathy of Undetermined Significance

    Science.gov (United States)

    Rajkumar, S. Vincent

    2009-01-01

    Summary Monoclonal gammopathy of undetermined significance (MGUS) is a common premalignant plasma cell proliferative disorder with a lifelong risk of progression to multiple myeloma. Since myeloma is an incurable malignancy, strategies to delay or prevent progression in high-risk patients are of considerable importance. PMID:19737944

  17. Imaging of cutaneous T cell lymphoma (CTCL) with In-111-T101 monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Carrasquillo, J.A.; Bunn, P.A.; Keenan, A.M.; Reynolds, J.C.; Schroff, R.W.; Foon, K.A.; Ming-Hsu, S.; Gazdar, A.F.; Mulshine, J.M.; Perentesis, P.

    1985-05-01

    T101 is a murine monoclonal antibody (MoAb), IgC2a, directed against a cell surface pan T-cell antigen present in high concentration in CTCL cells. In-111 labelling was performed with a modification of the Krejcarek method (Hybritech, Inc.). I mg of DTPA conjugated T101 was labeled with 5 mCi, with a mean incorporation of 95%. Immunoreactivity was preserved, mean 88%. In vivo, less than 3.6% of the injected dose was on circulating transferrin. 11 patients (pts) received 2-6h intravenous infusion of 1 mg (5 pts), 10 mg (3 pts), 50 mg (3 pts) of In-111 T101. By 24h all pts showed avid uptake in pathologically or clinically involved nodes and erythroderma including several previously unsuspected nodal regions. Skin plaques were not visualized. In addition, there was localization in liver, spleen and bone marrow. Concentration of In-111 in biopsied nodes was 0.01, 0.02 and 0.03% of the injected dose per gram. Control studies with In-111Cl/sub 3/ or a nonspecific MoAb, 9.2.27, did not concentrate in nodes or skin disease. No dose dependent differences in tumor localization was seen although blood clearance was prolonged for doses less than or equal to 10 mgs of T101. All pts receiving less than or equal to 10 mgs developed transient itching, urticaria and chills. 1 of 8 pts tested had an antimouse immune response. Modulation of the antigen from circulating T-cells, skin and nodes was seen. This study shows the feasibility of imaging CTCL pts with In-111 T101 and suggest a potential for radioimmunotherapy.

  18. Monoclonal B-cell lymphocytosis (MBL) with normal lymphocyte counts is associated with decreased numbers of normal circulating B-cell subsets.

    Science.gov (United States)

    Hauswirth, Alexander W; Almeida, Julia; Nieto, Wendy G; Teodosio, Cristina; Rodriguez-Caballero, Arancha; Romero, Alfonso; López, Antonio; Fernandez-Navarro, Paulino; Vega, Tomas; Perez-Andres, Martin; Valent, Peter; Jäger, Ulrich; Orfao, Alberto

    2012-07-01

    Monoclonal B-cell lymphocytosis (MBL) with normal lymphocyte counts is associated with decreased numbers of normal circulating B-cell subsets.Little is known about the distribution of normal lymphoid cells and their subsets in the peripheral blood (PB) of subjects with monoclonal B-cell lymphocytosis (MBL). In our study, we compared the absolute number of PB lymphoid cells and their subpopulations in 95 MBL cases with normal lymphocyte counts vs. 617 age-/sex-matched non-MBL healthy subjects (controls), using highly sensitive flow cytometry. MBL cases showed significantly reduced numbers of normal circulating B-cells, at the expense of immature and naive B-cells; in addition, CD4+CD8+ double-positive T-cells and CD8+ T-cells were significantly lower and higher vs. controls, respectively. Moreover, most normal B-cell subsets were significantly decreased in PB at >1% MBL-counts, vs. "low-count" MBL cases, and lower amounts of immature/naive B-cells were detected in biclonal (particularly in cases with coexisting CLL-like- and non-CLL-like B-cell clones) vs. monoclonal MBL subjects. In summary, our results show imbalanced (reduced) absolute numbers of recently produced normal circulating B-cells (e.g., immature and naıve B-cells) in MBL, which becomes more pronounced as the MBL cell count increases.

  19. Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

    Science.gov (United States)

    Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

    1992-09-01

    The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

  20. Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Frankfurt, O.S. (Roswell Park Memorial Institute, Buffalo, NY (USA))

    1987-06-01

    A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to the drug dose. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.

  1. Inhibition of lipoxygenase activity in lentil protoplasts by monoclonal antibodies introduced into the cells via electroporation

    OpenAIRE

    J. F. G. Vliegenthart; Maccarrone, M.; Veldink, G.A.

    1992-01-01

    The isolation of lentil protoplasts and the transfer of anti-lipoxygenase monoclonal antibodies into plant protoplasts by electroporation is reported. The dependence of the efficiency of monoclonal antibody incorporation on the field strength is shown as well. The transferred immunoglobulins retained their functional and structural integrity and were able to inhibit the intracellular target enzyme, with a linear relationship between inhibition of lipoxygenase activity and amount of incorporat...

  2. CD20 monoclonal antibody targeted nanoscale drug delivery system for doxorubicin chemotherapy: an in vitro study of cell lysis of CD20-positive Raji cells.

    Science.gov (United States)

    Jiang, Shuang; Wang, Xiaobo; Zhang, Zhiran; Sun, Lan; Pu, Yunzhu; Yao, Hongjuan; Li, Jingcao; Liu, Yan; Zhang, Yingge; Zhang, Weijing

    A monoclonal antibody targeted nanoscale drug delivery system (NDDS) for chemotherapy was evaluated in CD20-positive Raji cells in vitro. Nanoparticles were formed by the assembly of an amphiphilic polymer consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxypolyethyleneglycol-2000 (DSPE-PEG2000). Active carbon nanoparticles (ACNP) were conjugated to the chemotherapeutic agent, doxorubicin (DOX), and the nanoliposome carrier, DSPE-PEG2000 and DSPE-PEG2000-NH2 conjugated to the human anti-CD20 monoclonal antibody that targets B-lymphocytes. This monoclonal antibody targeted nanoparticle delivery system for chemotherapy formed the active NDDS complex, ACNP-DOX-DSPE-PEG2000-anti-CD20. This active NDDS was spherical in morphology and had good dispersion in the culture medium. When compared with the effects on CD20-negative YTS cells derived from natural killer/T-cell lymphoma, the active NDDS, ACNP-DOX-DSPE-PEG2000-anti-CD20, demonstrated DOX delivery to CD20-positive Raji cells derived from Burkitt's lymphoma (B cell lymphoma), resulting in increased cell killing in vitro. The intracellular targeting efficiency of the ACNP-DOX-DSPE-PEG2000-anti-CD20 complex was assessed by confocal laser microscopy and flow cytometry. The findings of this in vitro study have shown that the DSPE-PEG2000 polymeric liposome is an effective nanocarrier of both a monoclonal antibody and a chemotherapy agent and can be used to target chemotherapy to specific cells, in this case to CD20-positive B-cells. Future developments in this form of targeted therapy will depend on the development of monoclonal antibodies that are specific for malignant cells, including antibodies that can distinguish between lymphoma cells and normal lymphocyte subsets.

  3. Computer Simulation of Convective Plasma Cells

    OpenAIRE

    Carboni, Rodrigo; Frutos-Alfaro, Francisco

    2015-01-01

    Computer simulations of plasmas are relevant nowadays, because it helps us understand physical processes taking place in the sun and other stellar objects. We developed a program called PCell which is intended for displaying the evolution of the magnetic field in a 2D convective plasma cell with perfect conducting walls for different stationary plasma velocity fields. Applications of this program are presented. This software works interactively with the mouse and the users can create their ow...

  4. Characterization of a monoclonal antibody cell culture production process using a quality by design approach.

    Science.gov (United States)

    Horvath, Brian; Mun, Melissa; Laird, Michael W

    2010-07-01

    The goal of quality by design (QbD) in cell culture manufacturing is to develop manufacturing processes which deliver products with consistent critical quality attributes (CQAs). QbD approaches can lead to better process understanding through the use of process parameter risk ranking and statistical design of experiments (DOE). The QbD process starts with an analysis of process parameter risk with respect to CQAs and key performance indicators (KPIs). Initial DOE study designs and their factor test ranges are based on the outcomes of the process parameter risk ranking exercises. Initial DOE studies screen factors for significant influences on CQAs as well as characterize responses for process KPIs. In the case study provided here, multifactor process characterization studies using a scale-down model resulted in significant variation in charge heterogeneity of a monoclonal antibody (MAb) as measured by ion-exchange chromatography (IEC). Iterative DOE studies, using both screening and response surface designs, were used to narrow the operating parameter ranges so that charge heterogeneity could be controlled to an acceptable level. The data from the DOE studies were used to predict worst-case conditions, which were then verified by testing at those conditions. Using the approach described here, multivariate process parameter ranges were identified that yield acceptable CQA levels and that still provide operational flexibility for manufacturing.

  5. Identification and characterization of host cell protein product-associated impurities in monoclonal antibody bioprocessing.

    Science.gov (United States)

    Levy, Nicholas E; Valente, Kristin N; Choe, Leila H; Lee, Kelvin H; Lenhoff, Abraham M

    2014-05-01

    Downstream processing of monoclonal antibodies (mAbs) has evolved to allow the specific process for a new product to be developed largely by empirical specialization of a platform process that enables removal of impurities of different kinds. A more complete characterization of impurities and the product itself would provide insights into the rational design of efficient downstream processes. This work identifies and characterizes host cell protein (HCP) product-associated impurities, that is, HCP species carried through the downstream processes via direct interactions with the mAb. Interactions between HCPs and mAbs are characterized using cross-interaction chromatography under solution conditions typical of those used in downstream processing. The interacting species are then identified by two-dimensional gel electrophoresis and mass spectrometry. This methodology has been applied to identify product-associated impurities in one particular purification step, namely protein A affinity chromatography, for four therapeutic mAbs as well as the Fab and Fc domains of one of these mAbs. The results show both the differences in HCP-mAb interactions among different mAbs, and the relative importance of product association compared to co-elution in protein A affinity chromatography. © 2013 Wiley Periodicals, Inc.

  6. Plasma cell gingivitis: treatment with chlorpheniramine maleate.

    Science.gov (United States)

    Ranganathan, Aravindhan Thiruputkuzhi; Chandran, Chitraa R; Prabhakar, Priya; Lakshmiganthan, Mahalingam; Parthasaradhi, Thakkalapati

    2015-01-01

    Plasma cell gingivitis is a benign lesion of unknown etiology characterized by massive and diffuse infiltration of plasma cells into the gingival connective tissue. Clinically, it can be seen as a diffuse, erythematous, and edematous swelling involving the marginal gingiva and extending into the attached gingiva. Although usually painless, the lesion can be esthetically unappealing, especially when anterior gingiva is involved. Although the usual line of management is removal of the offending agent, this report describes the treatment of plasma cell gingivitis with the topical application of chlorpheniramine maleate (25 mg) for a period of 10 days.

  7. Mechanisms of action of the anti-VEGF monoclonal antibody bevacizumab on chronic lymphocytic leukemia cells

    Directory of Open Access Journals (Sweden)

    Jakub Bogusz

    2013-03-01

    Full Text Available Introduction: Chronic lymphocytic leukemia (CLL remains incurable; therefore searching for new therapeutic strategies in this disease is necessary. An important mechanism of tumor development is neoangiogenesis. A potent antiangiogenic factor, bevacizumab (Avastin, AVA, has been poorly explored in CLL so far. In the current study we assessed cytotoxic activity of AVA alone or in combinations with drugs routinely used in this disease.Matherials and Methods: Cells isolated from 60 CLL patients were treated with AVA alone or in combination with anti-CD20 monoclonal antibody (MoAb, rituximab (RIT, anti-CD52 MoAb, alemtuzumab (ALT, 2-CdA (2-chlorodeoxyadenosine, FA (fludarabine, MAF (mafosfamide or RAPA (rapamycin. Cytotoxicity was assessed by propidium iodide staining. Apoptosis was evaluated using annexin-V and TUNEL assays. Additionally, a drop of mitochondrial potential (DYm as well as expression of apoptosis-regulating proteins Bax, Bak, Bid, Bad, Bcl-2, Mcl-2, XIAP, FLIP, Akt and Bcl-2-A1 were determined by flow cytometry.Results: At the dose of 40 μg/ml, after 48 hours of incubation, AVA induced significant cytotoxicity against CLL cells. The drug triggered apoptosis, with activation of caspase-3 and -9, but not caspase-8, along with a drop of DYm. Incubation with AVA induced significant overexpression of proapoptotic Bak and Bad as well as downregulation of antiapoptotic Mcl-2 and Akt proteins. Combination of AVA with RIT, ALT or RAPA significantly increased cytotoxicity when compared with the effects of single drugs.Discussion: In conclusion, this is the first report showing proapoptotic activity of AVA against CLL cells. Combination of AVA with RIT, ALT or RAPA may be a promising therapeutic strategy, which requires confirmation in further studies.

  8. Nonthermal-plasma-mediated animal cell death

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Wanil; Woo, Kyung-Chul; Kim, Kyong-Tai [Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, San 31, Hyoja Dong, Pohang 790-784 (Korea, Republic of); Kim, Gyoo-Cheon, E-mail: ktk@postech.ac.kr [Department of Oral Anatomy and Cell Biology, School of Dentistry, Pusan National University, Yangsan 626-810 (Korea, Republic of)

    2011-01-12

    Animal cell death comprising necrosis and apoptosis occurred in a well-regulated manner upon specific stimuli. The physiological meanings and detailed molecular mechanisms of cell death have been continuously investigated over several decades. Necrotic cell death has typical morphological changes, such as cell swelling and cell lysis followed by DNA degradation, whereas apoptosis shows blebbing formation and regular DNA fragmentation. Cell death is usually adopted to terminate cancer cells in vivo. The current strategies against tumour are based on the induction of cell death by adopting various methods, including radiotherapy and chemotherapeutics. Among these, radiotherapy is the most frequently used treatment method, but it still has obvious limitations. Recent studies have suggested that the use of nonthermal air plasma can be a prominent method for inducing cancer cell death. Plasma-irradiated cells showed the loss of genomic integrity, mitochondrial dysfunction, plasma membrane damage, etc. Tumour elimination with plasma irradiation is an emerging concept in cancer therapy and can be accelerated by targeting certain tumour-specific proteins with gold nanoparticles. Here, some recent developments are described so that the mechanisms related to plasma-mediated cell death and its perspectives in cancer treatment can be understood. (topical review)

  9. A B-lymphoma cell line that forms rosettes with neuraminidase-treated sheep erythrocytes through monoclonal surface immunoglobulin.

    Science.gov (United States)

    Tsutsumi, Y; Suzuki, S; Mikata, A; Suzuki, H; Kageyama, K; Watanabe, S; Minato, K; Shimoyama, M

    1982-06-01

    Undifferentiated lymphoma from a 39-year-old female became serially xenotransplantable to preirradiated nude mice. The tumor cells (KT) possessed a monoclonal surface immunoglobulin (SIg mu, kappa) and formed rosettes with neuraminidase-treated sheep erythrocytes (SEn). Precise characterizations of the SEn rosette, however, revealed the following facts: (1) Neuraminidase-untreated or 2-aminoethylisothiuronium bromide (AET) treated sheep erythrocytes were not bound to the KT cells. (2) SEn rosettes on the KT cells did not show a temperature dependency. (3) Neuraminidase-treated erythrocytes from man, horse, mouse, and rabbit were not bound to the KT cells. (4) Preincubation of the KT cells with antipolyvalent immunoglobulin or anti-kappa-chain serum abolished the SEn rosette formation. (5) Trypsinization decreased both SEn rosettes and SIg on the KT cells. (6) SEn rosettes on the KT cells were too loose to be separated from nonrosetting cells by a Percoll gradient centrifugation method. Summarizing these results, the monoclonal SIg on the KT cells recognized sheep erythrocyte antigen(s) that were exposed only after the neuraminidase treatment. Therefore, this was considered to be a case with peculiar B-lymphoma cells that bound SEn through their SIg.

  10. Natural History Study of Monoclonal B Cell Lymphocytosis (MBL), Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL), Lymphoplasmacytic Lymphoma (LPL)/Waldenstrom Macroglobulinemia (WM), and Splenic Marginal Zone Lymphoma (SMZL)

    Science.gov (United States)

    2017-10-05

    B-Cell Chronic Lymphocytic Leukemia; Monoclonal B-Cell Lymphocytosis; Lymhoma, Small Lymphocytic; Chronic Lymphocytic Leukemia; Lymphoplasmacytic Lymphoma; Waldenstrom Macroglobulinemia; Splenic Marginal Zone Lymphoma

  11. Modulation and modeling of monoclonal antibody N-linked glycosylation in mammalian cell perfusion reactors.

    Science.gov (United States)

    Karst, Daniel J; Scibona, Ernesto; Serra, Elisa; Bielser, Jean-Marc; Souquet, Jonathan; Stettler, Matthieu; Broly, Hervé; Soos, Miroslav; Morbidelli, Massimo; Villiger, Thomas K

    2017-09-01

    Mammalian cell perfusion cultures are gaining renewed interest as an alternative to traditional fed-batch processes for the production of therapeutic proteins, such as monoclonal antibodies (mAb). The steady state operation at high viable cell density allows the continuous delivery of antibody product with increased space-time yield and reduced in-process variability of critical product quality attributes (CQA). In particular, the production of a confined mAb N-linked glycosylation pattern has the potential to increase therapeutic efficacy and bioactivity. In this study, we show that accurate control of flow rates, media composition and cell density of a Chinese hamster ovary (CHO) cell perfusion bioreactor allowed the production of a constant glycosylation profile for over 20 days. Steady state was reached after an initial transition phase of 6 days required for the stabilization of extra- and intracellular processes. The possibility to modulate the glycosylation profile was further investigated in a Design of Experiment (DoE), at different viable cell density and media supplement concentrations. This strategy was implemented in a sequential screening approach, where various steady states were achieved sequentially during one culture. It was found that, whereas high ammonia levels reached at high viable cell densities (VCD) values inhibited the processing to complex glycan structures, the supplementation of either galactose, or manganese as well as their synergy significantly increased the proportion of complex forms. The obtained experimental data set was used to compare the reliability of a statistical response surface model (RSM) to a mechanistic model of N-linked glycosylation. The latter outperformed the response surface predictions with respect to its capability and reliability in predicting the system behavior (i.e., glycosylation pattern) outside the experimental space covered by the DoE design used for the model parameter estimation. Therefore, we can

  12. Diagnosis and management of neuropathies associated with plasma cell dyscrasias.

    Science.gov (United States)

    Rosenbaum, Evan; Marks, Douglas; Raza, Shahzad

    2017-04-10

    Neuropathies associated with plasma cell dyscrasias are a major cause of morbidity for patients managed by medical oncologists. Because of similarities in clinical presentation and on nerve conduction studies, identifying the underlying disease leading to a paraproteinemic neuropathy can often be difficult. In addition, the degree of neurologic deficit does not strictly correlate with the extent of abnormalities on common clinical laboratory testing. Fortunately, with increasing understanding into the biologic mechanisms of underlying hematologic diseases, additional biomarkers have recently been developed, thus improving our diagnostic capacity. Neuropathies associated with plasma cells dyscrasias are seen with Monoclonal gammopathy of undetermined significance (MGUS) particularly IgM subtype, followed by IgG and IgA MGUS, multiple myeloma, Waldenström's macroglobulinemia, amyloid, Castleman's disease, and POEMS syndrome. The mechanisms of neuronal injury associated with plasma cell dyscrasia vary based on underlying diagnosis and include malignant infiltration, immune-mediated antibody deposition, or local compression of nerve roots. The polyneuropathies are frequently demyelinating, although axonal and mixed neuropathies can also be seen. As demonstrated by the cases included in this review, patients frequently present with symmetric sensory disturbance, followed by progressive motor weakness. Unfortunately, because of the complexity of diagnostic testing, patients are frequently examined late, often after receiving several ineffective therapies. The aim of this case-based review is to provide clinicians with insight on how to properly recognize these atypical neuropathies and send the appropriate diagnostic work, increasing the likelihood of accurately classify the patient's underlying hematologic disorder. Copyright © 2017 John Wiley & Sons, Ltd.

  13. Molecular Characterization of Two Monoclonal Antibodies against the Same Epitope on B-Cell Receptor Associated Protein 31

    OpenAIRE

    Kim, Won-Tae; Shin, Saemina; Hwang, Hyo Jeong; Kim, Min Kyu; Jung, Han-Sung; Park, Hwangseo; RYU, CHUN JEIH

    2016-01-01

    Previously, we showed that B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum (ER) membrane chaperone, is also expressed on the cell surface by two monoclonal antibodies (MAbs) 297-D4 and 144-A8. Both MAbs recognize the same linear epitope on the C-terminal domain of BAP31, although they were independently established. Here, flow cytometric analysis showed that 144-A8 had additional binding properties to some cells, as compared to 297-D4. Quantitative antigen binding assa...

  14. Affinity isolation of antigen-specific circulating B cells for generation of phage display-derived human monoclonal antibodies

    DEFF Research Database (Denmark)

    Ditzel, Henrik

    2009-01-01

    A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries generated from...... peripheral blood lymphocytes caused by the low abundance of antigen-specific B cells in the circulation. The preselection of B cells is based on the specificity of the surface Ig receptor and is accomplished using the antigen of interest conjugated to magnetic beads. This method should significantly increase...

  15. A Novel Approach to Monitor Clearance of Host Cell Proteins Associated With Monoclonal Antibodies

    Science.gov (United States)

    Aboulaich, Nabila; Chung, Wai Keen; Thompson, Jenny Heidbrink; Larkin, Christopher; Robbins, David; Zhu, Min

    2014-01-01

    Co-purification of a subset of host cell proteins (HCPs) with monoclonal antibodies (mAbs) during the capture of mAbs on Protein A affinity chromatography is primarily caused by interactions of HCPs with the mAbs. To date, there is limited information about the identity of those HCPs due to the difficulty in detecting low abundance HCPs in the presence of a large amount of the mAb. Here, an approach is presented that allows identification of HCPs that specifically associate with the mAb, while avoiding interference from the mAb itself. This approach involves immobilization of purified mAb onto chromatography resin via cross-linking, followed by incubation with HCPs obtained from supernatant of non-mAb producer cells that are representative of the expression systems used in mAb manufacturing. The HCPs that bind to the mAb are recovered and identified using mass spectrometry. This approach has not only allowed a comprehensive comparison of HCP subpopulations that associate with different mAbs, but also enabled monitoring of the effects of a variety of wash modifiers on the dissociation of individual HCP–mAb interactions. The dissociation of HCPs that associated with the mAb was monitored by enzyme-linked immunosorbent assay and mass spectrometry. This approach can be utilized as a screening tool to assist the development of effective and targeted wash steps in Protein A chromatography that ensures not only reduction of HCP levels copurified with the mAb but also removal of specific HCPs that may have a potential impact on mAb structural stability and patient safety. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1114–1124, 2014 PMID:25044920

  16. Select host cell proteins coelute with monoclonal antibodies in protein A chromatography.

    Science.gov (United States)

    Nogal, Bartek; Chhiba, Krishan; Emery, Jefferson C

    2012-01-01

    The most significant factor contributing to the presence of host cell protein (HCP) impurities in Protein A chromatography eluates is their association with the product monoclonal antibodies (mAbs) has been reported previously, and it has been suggested that more efficacious column washes may be developed by targeting the disruption of the mAbs-HCP interaction. However, characterization of this interaction is not straight forward as it is likely to involve multiple proteins and/or types of interaction. This work is an attempt to begin to understand the contribution of HCP subpopulations and/or mAb interaction propensity to the variability in HCP levels in the Protein A eluate. We performed a flowthrough (FT) recycling study with product respiking using two antibody molecules of apparently different HCP interaction propensities. In each case, the ELISA assay showed depletion of select subpopulations of HCP in Protein A eluates in subsequent column runs, while the feedstock HCP in the FTs remained unchanged from its native harvested cell culture fluid (HCCF) levels. In a separate study, the final FT from each molecule's recycling study was cross-spiked with various mAbs. In this case, Protein A eluate levels remained low for all but two molecules which were known as having high apparent HCP interaction propensity. The results of these studies suggest that mAbs may preferentially bind to select subsets of HCPs, and the degree of interaction and/or identity of the associated HCPs may vary depending on the mAb.

  17. Plasma Cells For Hire: Prior Experience Required

    OpenAIRE

    2013-01-01

    Activation of IgG+ memory B cells accounts for much of the antibodies in secondary immune responses. Here, Khometani et al. (2013) demonstrate that reduced amounts of Bach2 in antigen-experienced memory B cells control the robust production of IgG1+ plasma cells.

  18. Cell Adhesion to Plasma-Coated PVC

    OpenAIRE

    Elidiane C. Rangel; Souza,Eduardo S. de; Francine S. de Moraes; Eliana A. R. Duek; Carolina Lucchesi; Schreiner, Wido H.; Durrant, Steven F.; Cruz, Nilson C.

    2014-01-01

    To produce environments suitable for cell culture, thin polymer films were deposited onto commercial PVC plates from radiofrequency acetylene-argon plasmas. The proportion of argon in the plasmas, P-Ar, was varied from 5.3 to 65.8%. The adhesion and growth of Vero cells on the coated surfaces were examined for different incubation times. Cytotoxicity tests were performed using spectroscopic methods. Carbon, O, and N were detected in all the samples using XPS. Roughness remained almost unchang...

  19. The interleukin-6 receptor alpha-chain (CD126) is expressed by neoplastic but not normal plasma cells.

    Science.gov (United States)

    Rawstron, A C; Fenton, J A; Ashcroft, J; English, A; Jones, R A; Richards, S J; Pratt, G; Owen, R; Davies, F E; Child, J A; Jack, A S; Morgan, G

    2000-12-01

    Interleukin-6 (IL-6) is reported to be central to the pathogenesis of myeloma, inducing proliferation and inhibiting apoptosis in neoplastic plasma cells. Therefore, abrogating IL-6 signaling is of therapeutic interest, particularly with the development of humanized anti-IL-6 receptor (IL-6R) antibodies. The use of such antibodies clinically requires an understanding of IL-6R expression on neoplastic cells, particularly in the cycling fraction. IL-6R expression levels were determined on plasma cells from patients with myeloma (n = 93) and with monoclonal gammopathy of undetermined significance (MGUS) or plasmacytoma (n = 66) and compared with the levels found on normal plasma cells (n = 11). In addition, 4-color flow cytometry was used to assess the differential expression by stage of differentiation and cell cycle status of the neoplastic plasma cells. IL-6R alpha chain (CD126) was not detectable in normal plasma cells, but was expressed in approximately 90% of patients with myeloma. In all groups, the expression levels showed a normal distribution. In patients with MGUS or plasmacytoma, neoplastic plasma cells expressed significantly higher levels of CD126 compared with phenotypically normal plasma cells from the same marrow. VLA-5(-) "immature" plasma cells showed the highest levels of CD126 expression, but "mature" VLA-5(+) myeloma plasma cells also overexpressed CD126 when compared with normal subjects. This study demonstrates that CD126 expression is restricted to neoplastic plasma cells, with little or no detectable expression by normal cells. Stromal cells in the bone marrow microenvironment do not induce the overexpression because neoplastic cells express higher levels of CD126 than normal plasma cells from the same bone marrow in individuals with MGUS. (Blood. 2000;96:3880-3886)

  20. The clinical relevance and management of monoclonal gammopathy of undetermined significance and related disorders: recommendations from the European Myeloma Network.

    Science.gov (United States)

    van de Donk, Niels W C J; Palumbo, Antonio; Johnsen, Hans Erik; Engelhardt, Monika; Gay, Francesca; Gregersen, Henrik; Hajek, Roman; Kleber, Martina; Ludwig, Heinz; Morgan, Gareth; Musto, Pellegrino; Plesner, Torben; Sezer, Orhan; Terpos, Evangelos; Waage, Anders; Zweegman, Sonja; Einsele, Hermann; Sonneveld, Pieter; Lokhorst, Henk M

    2014-06-01

    Monoclonal gammopathy of undetermined significance is one of the most common pre-malignant disorders. IgG and IgA monoclonal gammopathy of undetermined significance are precursor conditions of multiple myeloma; light-chain monoclonal gammopathy of undetermined significance of light-chain multiple myeloma; and IgM monoclonal gammopathy of undetermined significance of Waldenström's macroglobulinemia and other lymphoproliferative disorders. Clonal burden, as determined by bone marrow plasma cell percentage or M-protein level, as well as biological characteristics, including heavy chain isotype and light chain production, are helpful in predicting risk of progression of monoclonal gammopathy of undetermined significance to symptomatic disease. Furthermore, alterations in the bone marrow microenvironment of monoclonal gammopathy of undetermined significance patients result in an increased risk of venous and arterial thrombosis, infections, osteoporosis, and bone fractures. In addition, the small clone may occasionally be responsible for severe organ damage through the production of a monoclonal protein that has autoantibody activity or deposits in tissues. These disorders are rare and often require therapy directed at eradication of the underlying plasma cell or lymphoplasmacytic clone. In this review, we provide an overview of the clinical relevance of monoclonal gammopathy of undetermined significance. We also give general recommendations of how to diagnose and manage patients with monoclonal gammopathy of undetermined significance. Copyright© Ferrata Storti Foundation.

  1. Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

    Science.gov (United States)

    Hart, Felix; Danielczyk, Antje; Goletz, Steffen

    2017-01-01

    IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich) and hematological (CD20) cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.

  2. Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Felix Hart

    2017-05-01

    Full Text Available IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich and hematological (CD20 cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.

  3. [ICO-63 monoclonal antibodies to the differentiation antigen of hemopoietic cells suitable for immunophenotyping of human solid tumors].

    Science.gov (United States)

    Baryshnikov, A Iu; Kadyrov, Kh P; Tupitsyn, N N; Kadagidze, Z G; Petrovichev, N N

    1989-01-01

    ICO-63, new monoclonal antibodies (MCAB), used for immunodiagnosis, were produced by the standard hybridoma technique ICO-63 MCAB reacted with 50% granulocytes, 20% thrombocytes and endothelial cells, whereas they did not react with peripheral blood mononuclear cells, thymocytes and erythrocytes. The molecular weight of ICO-63 MCAB-identified antigen was about 100 kD. ICO-63 MCAB reacted with 12 out of 15 neuroblastomas, with some solid tissue sarcomas and melanomas, but did not react with tumours of the epithelial origin.

  4. High-level production of a monoclonal antibody in murine myeloma cells by perfusion culture using a gravity settler.

    Science.gov (United States)

    Choo, Chiou-Yu; Tian, Yuan; Kim, Wan-Seop; Blatter, Erich; Conary, Jon; Brady, Ciaran P

    2007-01-01

    A perfusion system is described for the production of a human monoclonal antibody in non-secreting murine myeloma (NS0) cells that was previously shown to be difficult to produce at high levels using fed-batch culture. The perfusion system was based on the use of a commercially available cell settler as the separation device to separate the cells from the culture. Separation efficiency of the cell settler was above 98%. Based on the growth and glucose consumption rates, fresh media was added to the culture and the turnover rate for the bioreactor was set at a maximum of 1.5 times the bioreactor volume per day. The perfusion process resulted in twice the maximum viable cell densities and up to three times the total protein production in a 53-day run period when compared to the fed-batch process. In addition, charge heterogeneity of the antibody as measured by ion exchange chromatography was lower for material purified from the perfusion runs compared to fed-batch. Perfusion mode of culture using a commercially available gravity settler is therefore a viable alternative to fed-batch mode for high-level production of this monoclonal antibody in NS0 cells.

  5. Does My Patient with a Serum Monoclonal Spike have Multiple Myeloma?

    OpenAIRE

    Bianchi, Giada; Ghobrial, Irene M.

    2012-01-01

    A monoclonal spike (M spike or paraprotein) on serum protein electrophoresis (SPEP) is a frequent finding in the general population and typically is pathognomonic of an asymptomatic, premalignant condition called monoclonal gammopathy of undetermined significance (MGUS). MGUS occurs in around 3% of people older than 50 and is associated with a lifelong, low, yet non negligible, risk of progression to multiple myeloma (MM) or a related plasma cell dyscrasia. It is generally an incidental diagn...

  6. Computer Simulation of Convective Plasma Cells

    CERN Document Server

    Carboni, Rodrigo

    2015-01-01

    Computer simulations of plasmas are relevant nowadays, because it helps us understand physical processes taking place in the sun and other stellar objects. We developed a program called PCell which is intended for displaying the evolution of the magnetic field in a 2D convective plasma cell with perfect conducting walls for different stationary plasma velocity fields. Applications of this program are presented. This software works interactively with the mouse and the users can create their own movies in MPEG format. The programs were written in Fortran and C. There are two versions of the program (GNUPLOT and OpenGL). GNUPLOT and OpenGL are used to display the simulation.

  7. Plasma markers of B-cell activation and clonality in pediatric liver and hematopoietic stem cell transplant recipients

    Science.gov (United States)

    Engels, Eric A.; Savoldo, Barbara; Pfeiffer, Ruth M.; Costello, Rene; Zingone, Adriana; Heslop, Helen E.; Landgren, Ola

    2012-01-01

    Introduction Transplant recipients are at risk of post-transplant lymphoproliferative disease (PTLD). Methods: Thirty-six pediatric transplant recipients were evaluated (18 hematopoietic stem cell and 18 liver recipients; 12 had PTLD). We studied 207 longitudinal plasma samples from these recipients for three markers of B-cell activation or clonality: immunoglobulin free light chains (FLCs), soluble CD30 (sCD30), and monoclonal immunoglobulins (M-proteins). Results Kappa FLCs, lambda FLCs, and sCD30 were elevated in 20.8%, 28.0%, and 94.2% of plasma specimens, respectively. FLC and sCD30 levels increased significantly 1.18–1.82 fold per log10 Epstein Barr virus (EBV) load in peripheral blood. Five PTLD cases manifested elevated FLCs with an abnormal kappa/lambda ratio, suggesting monoclonal FLC production. M-proteins were present in 91% of PTLD cases, vs. 50–67% of other recipients with high or low EBV loads (p=0.13). Concordance of FLCs, M-proteins, and PTLD tumor light chain restriction was imperfect. For example, one PTLD case with an IgG lambda M-protein had a tumor that was kappa restricted, and another case with an M-protein had a T-cell PTLD. In an additional case, an IgM kappa M-protein and excess kappa FLCs were both detected in plasma at PTLD diagnosis; while the tumor was not restricted at diagnosis, kappa restriction was present 5 years later when the PTLD relapsed. Discussion Plasma markers of B-cell dysfunction are frequent following transplantation and associated with poor EBV control. These abnormal markers may be produced by oligoclonal B-cell populations or PTLD tumor cells, and could potentially help identify recipients at high risk of PTLD. PMID:23222884

  8. Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry.

    Science.gov (United States)

    Okumura, Takeshi; Masuda, Kenji; Watanabe, Kazuhiko; Miyadai, Kenji; Nonaka, Koichi; Yabuta, Masayuki; Omasa, Takeshi

    2015-09-01

    To screen a high-producing recombinant Chinese hamster ovary (CHO) cell from transfected cells is generally laborious and time-consuming. We developed an efficient enrichment strategy for high-producing cell screening using flow cytometry (FCM). A stable pool that had possibly shown a huge variety of monoclonal antibody (mAb) expression levels was prepared by transfection of an expression vector for mAb production to a CHO cell. To enrich high-producing cells derived from a stable pool stained with a fluorescent-labeled antibody that binds to mAb presented on the cell surface, we set the cell size and intracellular density gates based on forward scatter (FSC) and side scatter (SSC), and collected the brightest 5% of fluorescein isothiocyanate (FITC)-positive cells from each group by FCM. The final product concentration in a fed-batch culture of cells sorted without FSC and SSC gates was 1.2-1.3-times higher than that of unsorted cells, whereas that of cells gated by FSC and SSC was 3.4-4.7-fold higher than unsorted cells. Surprisingly, the fraction with the highest final product concentration indicated the smallest value of FSC and SSC, and the middle value of fluorescence intensity among all fractionated cells. Our results showed that our new screening strategy by FCM based on FSC and SSC gates could achieve an efficient enrichment of high-producing cells with the smallest value of FSC and SSC.

  9. [Monoclonal autoantibodies to the epithelial basement membrane cells of human skin and thymus obtained through immunization with Rickettsia prowazekii antigens].

    Science.gov (United States)

    Drobyshevskaia, E I; Spitsyn, S V; Nedialkov, Iu A; Shchekotikhina, Iu A; Tarasevich, I V

    1989-05-01

    As the result of immunization of BALB/c mice with the commercial preparation of typhus vaccine and R. prowazekii corpuscular antigen, in 29.2% and 40.3% of cases (respectively) the appearance of hybridomas synthesizing monoclonal antibodies (McAb) to different autologous structures (skin and thymic epithelium, cell nuclei, conjunctive tissue structures and vascular endothelium) has been revealed. The McAb under test have proved to be IgM-autoantibodies. McAb M-6, active against the basal membrane of human skin and thymic epithelium, produce quite a definite picture of disturbances in the differentiation of epithelium and can be used for the diagnosis of dyskeratosis.

  10. Quantitation of soluble aggregates in recombinant monoclonal antibody cell culture by pH-gradient protein A chromatography.

    Science.gov (United States)

    Pan, Hai; Chen, Ken; Pulisic, Matt; Apostol, Izydor; Huang, Gang

    2009-05-15

    Monoclonal antibodies (mAbs) produced from mammalian cell culture may contain significant amounts of dimers and higher order aggregates. Quantitation of soluble aggregates in the cell culture is time-consuming and labor-intensive, usually involving a purification step to remove the impurities that interfere with the subsequent size exclusion chromatography (SEC) analysis. We have developed a novel pH-gradient protein A chromatography for rapid, non-size based separation of the aggregates in mAb cell culture samples. Our results demonstrate that this method has excellent correlation with SEC and can be applied to both human immunoglobulin gamma 1 (IgG1) and IgG2 antibodies. This approach can be useful in the quantitation of soluble aggregates in crude cell culture samples.

  11. Characterization of Plasminogen Binding to NB4 Promyelocytic Cells Using Monoclonal Antibodies against Receptor-Induced Binding Sites in Cell-Bound Plasminogen

    Directory of Open Access Journals (Sweden)

    Mercè Jardí

    2012-01-01

    Full Text Available The NB4 promyelocytic cell line exhibits many of the characteristics of acute promyelocytic leukemia blast cells, including the translocation (15 : 17 that fuses the PML gene on chromosome 15 to the RARα gene on chromosome 17. These cells have a very high fibrinolytic capacity. In addition to a high secretion of urokinase, NB4 cells exhibit a 10-fold higher plasminogen binding capacity compared with other leukemic cell lines. When tissue-type plasminogen activator was added to acid-treated cells, plasmin generation was 20–26-fold higher than that generated by U937 cells or peripheral blood neutrophils, respectively. We found that plasminogen bound to these cells can be detected by fluorescence-activated cell sorting using an antiplasminogen monoclonal antibody that specifically reacts with this antigen when it is bound to cell surfaces. All-trans retinoid acid treatment of NB4 cells markedly decreased the binding of this monoclonal antibody. This cell line constitutes a unique model to explore plasminogen binding and activation on cell surfaces that can be modulated by all-trans retinoid acid treatment.

  12. B lymphocyte depletion with the monoclonal antibody rituximab in Graves' disease: a controlled pilot study

    DEFF Research Database (Denmark)

    El Fassi, Daniel; Nielsen, Claus H; Bonnema, Steen J

    2007-01-01

    Graves' disease (GD) is a common TSH receptor autoantibody (TRAb)-mediated disorder. Because B lymphocytes are important self-antigen presenting cells and precursors for antibody-secreting plasma cells, temporary B-lymphocyte depletion with the monoclonal antibody rituximab (RTX) might be of bene...

  13. Construction of Larger Area Density-Uniform Plasma with Collisional Inductively Coupled Plasma Cells

    Institute of Scientific and Technical Information of China (English)

    OUYANG Liang; LIU Wandong; BAI Xiaoyan; CHEN Zhipeng; WANG Huihui; LI LUO Chen; JI Liangliang; HU Bei

    2007-01-01

    The plasma density and electron temperature of a multi-source plasma system composed of several collisional inductively coupled plasma (ICP) cells were measured by a double-probe. The discharges of the ICP cells were shown to be independent of each other. Furthermore, the total plasma density at simultaneous multi-cell discharge was observed to be approximately equal to the summation of the plasma density when the cells discharge separately. Based on the linear summation phenomenon, it was shown that a larger area plasma with a uniform density and temperature profile could be constructed with multi-collisional ICP cells.

  14. Cytology of plasma cell rich effusion in cases of plasma cell neoplasm

    Science.gov (United States)

    Gochhait, Debasis; Dey, Pranab; Verma, Neelam

    2016-01-01

    Background: Multiple myeloma or plasmacytoma resulting in malignant effusion is rarely described in literature. Aims: In this paper, we have studied the seven rare cases of plasma cell infiltration in effusion fluid. Materials and Methods: We studied six cases of pleural fluid and one case of ascetic fluid. Detailed cytological features, clinical history, bone marrow examinations, serum electrophoresis, and immunofixation data were analyzed. Result: There were two cases of plasmacytoma, four cases of multiple myeloma, and one case of plasmablastic lymphoma. On cytology, all the cases showed excess plasma cells along with mesothelial cells and lymphocytes on effusion cytology smear. Conclusion: Plasma cell rich effusion in cases of plasma cell tumor is rare. However, on cytology these cases do not pose much problem if relevant history is known.

  15. Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography.

    Science.gov (United States)

    Dutta, Amit K; Tran, Travis; Napadensky, Boris; Teella, Achyuta; Brookhart, Gary; Ropp, Philip A; Zhang, Ada W; Tustian, Andrew D; Zydney, Andrew L; Shinkazh, Oleg

    2015-11-10

    Recent studies using simple model systems have demonstrated that continuous countercurrent tangential chromatography (CCTC) has the potential to overcome many of the limitations of conventional Protein A chromatography using packed columns. The objective of this work was to optimize and implement a CCTC system for monoclonal antibody purification from clarified Chinese Hamster Ovary (CHO) cell culture fluid using a commercial Protein A resin. Several improvements were introduced to the previous CCTC system including the use of retentate pumps to maintain stable resin concentrations in the flowing slurry, the elimination of a slurry holding tank to improve productivity, and the introduction of an "after binder" to the binding step to increase antibody recovery. A kinetic binding model was developed to estimate the required residence times in the multi-stage binding step to optimize yield and productivity. Data were obtained by purifying two commercial antibodies from two different manufactures, one with low titer (∼ 0.67 g/L) and one with high titer (∼ 6.9 g/L), demonstrating the versatility of the CCTC system. Host cell protein removal, antibody yields and purities were similar to those obtained with conventional column chromatography; however, the CCTC system showed much higher productivity. These results clearly demonstrate the capabilities of continuous countercurrent tangential chromatography for the commercial purification of monoclonal antibody products.

  16. Plasma cell treatment device Plasma-on-Chip: Monitoring plasma-generated reactive species in microwells

    Science.gov (United States)

    Oh, Jun-Seok; Kojima, Shinya; Sasaki, Minoru; Hatta, Akimitsu; Kumagai, Shinya

    2017-01-01

    We have developed a plasma cell treatment device called Plasma-on-Chip that enables the real-time monitoring of a single cell culture during plasma treatment. The device consists of three parts: 1) microwells for cell culture, 2) a microplasma device for generating reactive oxygen and nitrogen species (RONS) for use in cell treatment, and 3) through-holes (microchannels) that connect each microwell with the microplasma region for RONS delivery. Here, we analysed the delivery of the RONS to the liquid culture medium stored in the microwells. We developed a simple experimental set-up using a microdevice and applied in situ ultraviolet absorption spectroscopy with high sensitivity for detecting RONS in liquid. The plasma-generated RONS were delivered into the liquid culture medium via the through-holes fabricated into the microdevice. The RONS concentrations were on the order of 10–100 μM depending on the size of the through-holes. In contrast, we found that the amount of dissolved oxygen was almost constant. To investigate the process of RONS generation, we numerically analysed the gas flow in the through-holes. We suggest that the circulating gas flow in the through-holes promotes the interaction between the plasma (ionised gas) and the liquid, resulting in enhanced RONS concentrations. PMID:28176800

  17. Enzyme immunoassay of mumps virus in cell culture with peroxidase-labelled virus specific monoclonal antibodies and its application for determination of antibodies

    NARCIS (Netherlands)

    Tiel, F.H. van; Kraaijeveld, C.A.; Baller, J.; Harmsen, T.; Oosterlaken, T.A.M.; Snippe, H.

    1988-01-01

    Mumps neutralizing monoclonal antibodies (MAs) were purified and labelled with horseradish peroxidase and used to detect virus-infected Vero cells, which were seeded as monolayers in wells of 96-well plates. This direct enzyme immunoassay (EIA) in cell culture proved to be a sensitive method for

  18. Enzyme immunoassay of mumps virus in cell culture with peroxidase-labelled virus specific monoclonal antibodies and its application for determination of antibodies

    NARCIS (Netherlands)

    Tiel, F.H. van; Kraaijeveld, C.A.; Baller, J.; Harmsen, T.; Oosterlaken, T.A.M.; Snippe, H.

    1988-01-01

    Mumps neutralizing monoclonal antibodies (MAs) were purified and labelled with horseradish peroxidase and used to detect virus-infected Vero cells, which were seeded as monolayers in wells of 96-well plates. This direct enzyme immunoassay (EIA) in cell culture proved to be a sensitive method for det

  19. Use of an Immobilized Monoclonal Antibody to Examine Integrin &agr;5&bgr;1 Signaling Independent of Cell Spreading

    Directory of Open Access Journals (Sweden)

    Bao Wenjie

    2002-01-01

    Full Text Available Cell attachment to the extracellular matrix (ECM engages integrin signaling into the cell, but part of the signaling response also stem from cell spreading (3. To analyze specific integrin signaling-mediated responses independent of cell spreading, we developed a method engaging integrin signaling by use of an immobilized anti-integrin monoclonal antibody (mab directed against the fibronectin (FN receptor integrin &agr;5&bgr;1. ECV 304 cells were plated onto FN or immobilized mab JBS5 (anti-integrin &agr;5&bgr;1 or onto poly-L-lysin (P-L-L, which mediates integrin-independent attachment. Cells attached and spread on FN, while cells on JBS5 or P-L-L attached but did not spread. Importantly, plating onto FN or mab JBS5 gave rise to identical integrin-induced responses, including a down-regulation of the cyclin-dependent kinase (Cdk2 inhibitors p21CIP1 and p27KIP1, while attachment to P-L-L did not. We conclude that engagement of the FN-receptor integrin &agr;5&bgr;1 induces integrin signaling regulating the Cdk2-inhibitors independent of cell spreading and present a method for how integrin signaling can be analyzed separate from the effects of cell spreading.

  20. A robust high throughput platform to generate functional recombinant monoclonal antibodies using rabbit B cells from peripheral blood.

    Directory of Open Access Journals (Sweden)

    Stefan Seeber

    Full Text Available We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv the recombinant expression and purification of IgG antibodies. The fully integrated and to a large degree automated platform (demonstrated in this paper using IL1RL1 immunized rabbits yielded clonal and very diverse IL1RL1-specific and functional IL1RL1-inhibiting rabbit antibodies. These functional IgGs from individual animals were obtained at a short time range after immunization and could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. Early availability of sequence information permits one to select early-on function- and sequence-diverse antibodies for further characterization. In summary, this powerful technology platform has proven to be an efficient and robust method for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal.

  1. A robust high throughput platform to generate functional recombinant monoclonal antibodies using rabbit B cells from peripheral blood.

    Science.gov (United States)

    Seeber, Stefan; Ros, Francesca; Thorey, Irmgard; Tiefenthaler, Georg; Kaluza, Klaus; Lifke, Valeria; Fischer, Jens André Alexander; Klostermann, Stefan; Endl, Josef; Kopetzki, Erhard; Pashine, Achal; Siewe, Basile; Kaluza, Brigitte; Platzer, Josef; Offner, Sonja

    2014-01-01

    We have developed a robust platform to generate and functionally characterize rabbit-derived antibodies using B cells from peripheral blood. The rapid high throughput procedure generates a diverse set of antibodies, yet requires only few animals to be immunized without the need to sacrifice them. The workflow includes (i) the identification and isolation of single B cells from rabbit blood expressing IgG antibodies, (ii) an elaborate short term B-cell cultivation to produce sufficient monoclonal antigen specific IgG for comprehensive phenotype screens, (iii) the isolation of VH and VL coding regions via PCR from B-cell clones producing antigen specific and functional antibodies followed by the sequence determination, and (iv) the recombinant expression and purification of IgG antibodies. The fully integrated and to a large degree automated platform (demonstrated in this paper using IL1RL1 immunized rabbits) yielded clonal and very diverse IL1RL1-specific and functional IL1RL1-inhibiting rabbit antibodies. These functional IgGs from individual animals were obtained at a short time range after immunization and could be identified already during primary screening, thus substantially lowering the workload for the subsequent B-cell PCR workflow. Early availability of sequence information permits one to select early-on function- and sequence-diverse antibodies for further characterization. In summary, this powerful technology platform has proven to be an efficient and robust method for the rapid generation of antigen specific and functional monoclonal rabbit antibodies without sacrificing the immunized animal.

  2. Characterisation of CD4 T cells in healthy and diseased koalas (Phascolarctos cinereus) using cell-type-specific monoclonal antibodies.

    Science.gov (United States)

    Mangar, Chandan; Armitage, Charles W; Timms, Peter; Corcoran, Lynn M; Beagley, Kenneth W

    2016-07-01

    The koala (Phascolarctos cinereus) is an arboreal herbivorous marsupial that is an Australian icon. Koalas in many parts of Australia are under multiple threats including habitat destruction, dog attacks, vehicular accidents, and infectious diseases such as Chlamydia spp. and the koala retrovirus (KoRV), which may contribute to the incidence of lymphoma and leukaemia in this species. Due to a lack of koala-specific immune reagents and assays there is currently no way to adequately analyse the immune response in healthy, diseased or vaccinated animals. This paper reports the production and characterisation of the first anti-koala CD4 monoclonal antibody (mAb). The koala CD4 gene was identified and used to develop recombinant proteins for mAb production. Fluorochrome-conjugated anti-CD4 mAb was used to measure the levels of CD4(+) lymphocytes collected from koala spleens (41.1%, range 20-45.1%) lymph nodes (36.3%, range 19-55.9%) and peripheral blood (23.8%, range 17.3-35%) by flow cytometry. Biotin-conjugated anti-CD4 mAb was used for western blot to determine an approximate size of 52 kDa for the koala CD4 molecule and used in immunohistochemistry to identify CD4(+) cells in the paracortical region and germinal centres of spleen and lymph nodes. Using the anti-CD4 mab we showed that CD4 cells from vaccinated, but not control, koalas proliferated following in vitro stimulation with UV-inactivated Chlamydia pecorum and recombinant chlamydial antigens. Since CD4(+) T cells have been shown to play a pivotal role in clearing chlamydial infection in both human and mouse infections, using this novel antibody will help determine the role CD4(+) T cells play in protection against chlamydial infection in koalas and also enhance our knowledge of how KoRV affects the koala immune system. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. The Antigen Presenting Cells Instruct Plasma Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Wei eXu

    2014-01-01

    Full Text Available The professional antigen presenting cells (APCs, including many subsets of dendritic cells and macrophages, not only mediate prompt but nonspecific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells, which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only signal 1 (the antigen, but also signal 2 to directly instruct the differentiation process of plasma cells in a T cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

  4. Stoichiometry of monoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1

    DEFF Research Database (Denmark)

    Schønning, Kristian; Lund, O; Lund, O S

    1999-01-01

    In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes...... neutralization of T-cell line-adapted HIV-1 is incremental rather than all or none and that each MAb binding an Env oligomer reduces the likelihood of infection....... are coexpressed. By the coexpression of Env glycoproteins that either can or cannot bind a neutralizing MAb in an env transcomplementation assay, virions were generated in which the proportion of MAb binding sites could be regulated. As the proportion of MAb binding sites in Env chimeric virus increased, MAb...

  5. Inhibition of enzyme activity of Rhipicephalus (Boophilus) microplus triosephosphate isomerase and BME26 cell growth by monoclonal antibodies.

    Science.gov (United States)

    Saramago, Luiz; Franceschi, Mariana; Logullo, Carlos; Masuda, Aoi; Vaz, Itabajara da Silva; Farias, Sandra Estrazulas; Moraes, Jorge

    2012-10-12

    In the present work, we produced two monoclonal antibodies (BrBm37 and BrBm38) and tested their action against the triosephosphate isomerase of Rhipicephalus (Boophilus) microplus (RmTIM). These antibodies recognize epitopes on both the native and recombinant forms of the protein. rRmTIM inhibition  by BrBm37 was up to 85% whereas that of BrBrm38 was 98%, depending on the antibody-enzyme ratio. RmTIM activity was lower in ovarian, gut, and fat body tissue extracts treated with BrBm37 or BrBm38 mAbs. The proliferation of the embryonic tick cell line (BME26) was inhibited by BrBm37 and BrBm38 mAbs. In summary, the results reveal that it is possible to interfere with the RmTIM function using antibodies, even in intact cells.

  6. Inhibition of Enzyme Activity of Rhipicephalus (Boophilus microplus Triosephosphate Isomerase and BME26 Cell Growth by Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Jorge Moraes

    2012-10-01

    Full Text Available In the present work, we produced two monoclonal antibodies (BrBm37 and BrBm38 and tested their action against the triosephosphate isomerase of Rhipicephalus (Boophilus microplus (RmTIM. These antibodies recognize epitopes on both the native and recombinant forms of the protein. rRmTIM inhibition  by BrBm37 was up to 85% whereas that of BrBrm38 was 98%, depending on the antibody-enzyme ratio. RmTIM activity was lower in ovarian, gut, and fat body tissue extracts treated with BrBm37 or BrBm38 mAbs. The proliferation of the embryonic tick cell line (BME26 was inhibited by BrBm37 and BrBm38 mAbs. In summary, the results reveal that it is possible to interfere with the RmTIM function using antibodies, even in intact cells.

  7. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies

    Science.gov (United States)

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu

    2016-01-01

    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  8. Laboratory testing for monoclonal gammopathies: Focus on monoclonal gammopathy of undetermined significance and smoldering multiple myeloma.

    Science.gov (United States)

    Willrich, Maria A V; Murray, David L; Kyle, Robert A

    2017-05-04

    Monoclonal gammopathies (MG) are defined by increased proliferation of clonal plasma cells, resulting in a detectable abnormality called monoclonal component or M-protein. Detection of the M-protein as either narrow peaks on protein electrophoresis and discrete bands on immunofixation is the defining feature of MG. MG are classified as low-tumor burden disorders, pre-malignancies and malignancies. Since significant disease can be present at any level, several different tests are employed in order to encompass the inherent diverse nature of the M-proteins. In this review, we discuss the main characteristics and limitations of clinical assays to detect M-proteins: protein electrophoresis, immunofixation, immunoglobulin quantitation, serum free light chains and heavy-light chain assays, as well as the newly developed MALDI-TOF mass spectrometric methods. In addition, the definitions of the pre-malignancies monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), as well as monoclonal gammopathy of renal significance (MGRS) are presented in the context of the 2014 international guidelines for definition of myeloma requiring treatment, and the role of the laboratory in test selection for screening and monitoring these conditions is highlighted. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  9. Novel immunohistochemical monoclonal antibody against rat B cell receptor Associated Protein 31 (BAP31).

    Science.gov (United States)

    Song, Chaojun; Yan, Binyuan; Chen, Lihua; Li, Yongming; Wei, Yuying; Sun, Yuanjie; Yang, Angang; Yang, Kun; Jin, Boquan

    2009-10-01

    BAP31 is an evolutionarily conserved polytopic integral protein of the endoplasmic reticulum (ER) membrane implicated in regulating the export of selected membrane proteins from the ER to downstream compartments of the secretory pathway. BAP31 interacts with mIgD, cellubrevin, major histocompatibility complex class I, and BCL-2/BCL-X(L) and plays an important role in regulating the egress of these proteins and in apoptosis. Although BAP31 RNA is ubiquitous, the protein's anatomic localization in rat tissues has not been determined. This is partially because production of high affinity antibodies, especially monoclonal antibodies (MAbs) suitable for immunohistochemical staining, has lagged. To gain further insight into its possible functions, we generated a novel MAb specific for rat BAP31 in immunocytochemistry and immunohistochemistry and localized BAP31 in some rat tissues. Immunoreactivity of BAP31 was prominent in fundic glands, colon, pancreatic acinuses, and liver but not in skeleton muscle and lung. Thus, successful production of rat BAP31 monoclonal antibodies provides a new powerful tool for investigation of BAP31 function in the rat model.

  10. Polyclonal and monoclonal antibodies in clinic.

    Science.gov (United States)

    Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

    2014-01-01

    Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic.

  11. Diagnosis of cutaneous T-cell lymphoma detecting T-cell receptor gamma chain gene monoclonality by denaturing gradient gel electrophoresis.

    Science.gov (United States)

    Lapière, K; Dhaene, K; Matthieu, L; Hübner, R; Lambert, J; Van Marck, E

    1999-04-01

    Cutaneous T-cell lymphomas represent a group of malignant lymphoproliferative disorders characterised by the occurrence of a monoclonal population of T-lymphocytes. Diagnosis of early stages of this disease is a difficult challenge for both the dermatologist and the dermatopathologist. With the aid of the polymerase chain reaction it is possible to amplify specific regions of the T-cell receptor gamma gene. The amplification products can then be separated by denaturing gradient gel electrophoresis in order to detect a monoclonal population of T-lymphocytes in the infiltrate. We studied 4 patients with the clinicopathologic diagnosis of mycosis fungoides and 2 patients diagnosed as large plaque parapsoriasis. A monoclonal population was detected in 3 of the 4 mycosis fungoides cases and in 1 of the patients with large plaque parapsoriasis. This indicates that our analysis can help us establishing a diagnosis, and it can also help us to identify patients with a possible early stage of the disease, which clinically or histologically is not yet recognised as such.

  12. Epithelial cell-cell junctions and plasma membrane domains

    NARCIS (Netherlands)

    Giepmans, Ben N. G.; van Ijzendoorn, Sven C. D.

    Epithelial cells form a barrier against the environment, but are also required for the regulated exchange of molecules between an organism and its surroundings. Epithelial cells are characterised by a remarkable polarization of their plasma membrane, evidenced by the appearance of structurally,

  13. Epithelial cell-cell junctions and plasma membrane domains

    NARCIS (Netherlands)

    Giepmans, Ben N. G.; van Ijzendoorn, Sven C. D.

    2009-01-01

    Epithelial cells form a barrier against the environment, but are also required for the regulated exchange of molecules between an organism and its surroundings. Epithelial cells are characterised by a remarkable polarization of their plasma membrane, evidenced by the appearance of structurally, comp

  14. Non-secretory immunoglobulin E myeloma associated with immunoglobulin G monoclonal gammopathy of undetermined significance

    Directory of Open Access Journals (Sweden)

    Masako Yasuyama

    2012-07-01

    Full Text Available A 68-year old woman came to our hospital with a severe case of anemia. Serum immunoelectropheresis identified a monoclonal immunoglobulin (Ig G and κ protein. The serum IgE level was within the nomal range and the amounts of remaining immuno - globlins were low. On bone marrow aspirate, plasma cells made up 55.5% of nucleated cells and the plasma cells showed positive readings for IgE κ and IgG by immunohistochemistry. Serum immunofixation did not reveal the IgE monoclonal band. She was diagnosed as having non-secretory IgE myeloma with IgG monoclonal gammopathy of undetermined significance. The nature of this rare myeloma will be discussed.

  15. PDADMAC flocculation of Chinese hamster ovary cells: enabling a centrifuge-less harvest process for monoclonal antibodies.

    Science.gov (United States)

    McNerney, Thomas; Thomas, Anne; Senczuk, Anna; Petty, Krista; Zhao, Xiaoyang; Piper, Rob; Carvalho, Juliane; Hammond, Matthew; Sawant, Satin; Bussiere, Jeanine

    2015-01-01

    High titer (>10 g/L) monoclonal antibody (mAb) cell culture processes are typically achieved by maintaining high viable cell densities over longer culture durations. A corresponding increase in the solids and sub-micron cellular debris particle levels are also observed. This higher burden of solids (≥15%) and sub-micron particles typically exceeds the capabilities of a continuous centrifuge to effectively remove the solids without a substantial loss of product and/or the capacity of the harvest filtration train (depth filter followed by membrane filter) used to clarify the centrate. We discuss here the use of a novel and simple two-polymer flocculation method used to harvest mAb from high cell mass cell culture processes. The addition of the polycationic polymer, poly diallyldimethylammonium chloride (PDADMAC) to the cell culture broth flocculates negatively-charged cells and cellular debris via an ionic interaction mechanism. Incorporation of a non-ionic polymer such as polyethylene glycol (PEG) into the PDADMAC flocculation results in larger flocculated particles with faster settling rate compared to PDADMAC-only flocculation. PDADMAC also flocculates the negatively-charged sub-micron particles to produce a feed stream with a significantly higher harvest filter train throughput compared to a typical centrifuged harvest feed stream. Cell culture process variability such as lactate production, cellular debris and cellular densities were investigated to determine the effect on flocculation. Since PDADMAC is cytotoxic, purification process clearance and toxicity assessment were performed.

  16. Immunohistochemical Analysis Using Antipodocalyxin Monoclonal Antibody PcMab-47 Demonstrates Podocalyxin Expression in Oral Squamous Cell Carcinomas.

    Science.gov (United States)

    Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Harada, Hiroyuki; Kato, Yukinari

    2017-09-05

    Podocalyxin is a CD34-related type I transmembrane protein that is highly glycosylated with N-glycan, O-glycan, and keratan sulfate. Podocalyxin was originally found in the podocytes of rat kidney and is reportedly expressed in many types of tumors, including brain tumors, colorectal cancers, and breast cancers. Overexpression of podocalyxin is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human podocalyxin, which was produced using LN229 glioblastoma cells, and produced a novel antipodocalyxin monoclonal antibody (mAb), PcMab-47, which reacts with endogenous podocalyxin-expressing cancer cell lines and normal cell lines independent of glycosylation in Western blot, flow cytometry, and immunohistochemical analyses. In this study, we performed immunohistochemical analysis against oral cancers using PcMab-47. PcMab-47-stained oral squamous cell carcinoma cells in a cytoplasmic pattern and detected 26/38 (68.4%) of oral squamous cell carcinoma cells on tissue microarrays. These results indicate that PcMab-47 is useful in detecting podocalyxin of oral cancers for immunohistochemical analysis.

  17. Characterization of the co-elution of host cell proteins with monoclonal antibodies during protein A purification.

    Science.gov (United States)

    Zhang, Qingchun; Goetze, Andrew M; Cui, Huanchun; Wylie, Jenna; Tillotson, Ben; Hewig, Art; Hall, Michael P; Flynn, Gregory C

    2016-05-01

    Protein A chromatography is commonly used as the initial step for purifying monoclonal antibody biotherapeutics expressed in mammalian tissue culture cells. The purpose of this step, as well as later chromatography steps, is, in part, to remove host cell proteins (HCPs) and other related impurities. Understanding the retention mechanism for the subset of HCPs retained during this step is of great interest to monoclonal antibody (mAb) process developers because it allows formation of a guided HCP clearance strategy. However, only limited information is available about the specific HCPs that co-purify with mAbs at this step. In this study, a comprehensive comparison of HCP subpopulations that associated with 15 different mAbs during protein A chromatography was conducted by a 2D-LC-HDMS(E) approach. We found that a majority of CHO HCPs binding to and eluting with the mAbs were common among the mAbs studied, with only a small percentage (∼10% on average) of a mAb's total HCP content in the protein A (PrA) eluate specific for a particular antibody. The abundance of these HCPs in cell culture fluids and their ability to interact with mAbs were the two main factors determining their prevalence in protein A eluates. Potential binding segments for HCPs to associate with mAbs were also studied through their co-purification with individual Fc and (Fab')2 antibody fragments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:708-717, 2016. © 2016 American Institute of Chemical Engineers.

  18. Plasma Cell Gingivitis: An Occasional Case Report.

    Science.gov (United States)

    Mishra, M B; Sharma, Swati; Sharma, Alok

    2015-01-01

    Plasma cell gingivitis, an infrequently observed oral condition, has been clinically characterized by diffuse gingival enlargement, erythema and sometimes desquamation. These lesions are usually asymptomatic, but invariably the patient will complain of a burning sensation in the gingiva and bleeding from the mouth. The diagnosis requires hematological screening in addition to clinical and histopathological examinations. This case report outlines one such case of plasma cell gingivitis in a 15-year-old female caused by use of an herbal, homemade toothpowder. The case presented here highlights the adverse effects and irrational use of herbal agents in dentifrices. At the same time, it emphasizes the need for comprehensive history taking, careful clinical examination and appropriate diagnostic tests in order to arrive at a definitive diagnosis and treatment plan for gingival conditions that are refractory to conventional therapy and to exclude certain malignancies and oral manifestations of systemic diseases.

  19. Cyberknife radiosurgery for cranial plasma cell tumor.

    Science.gov (United States)

    Alafaci, Cetty; Grasso, Giovanni; Conti, Alfredo; Caffo, Mariella; Salpietro, Francesco Maria; Tomasello, Francesco

    2014-01-01

    Cranial and intracranial involvement by myelomatous disease is relatively uncommon. Furthermore, systemic manifestations of multiple myeloma are present in the majority of these cases at the time of symptom onset. The authors report the case of a patient with serial appearance of multiple intracranial plasma cell tumor localizations as the first manifestations of a multiple myeloma. The patient was treated with CyberKnife radiosurgery for a lesion localized at the clivus and sella turcica with complete local control. With such a technique, based on high-dose conformality, the tumor was centered with an ablative dose of radiation and, at the same time, with a low dose spreading to the surrounding critical structures. The radiosensitivity of plasma cell tumors renders this treatment modality particularly advantageous for their localized manifestation. A technical description of this case is provided. To our knowledge, this is the first case of successful Cyberknife radiosurgery of multifocal intracranial plasmacytoma.

  20. Human plasma cells express granzyme B.

    Science.gov (United States)

    Xu, Wei; Narayanan, Priya; Kang, Ning; Clayton, Sandra; Ohne, Yoichiro; Shi, Peiqing; Herve, Marie-Cecile; Balderas, Robert; Picard, Capucine; Casanova, Jean-Laurent; Gorvel, Jean-Pierre; Oh, Sangkon; Pascual, Virginia; Banchereau, Jacques

    2014-01-01

    While studying the plasma cell (PC) compartment in human tonsils, we identified that immunoglobulin kappa or lambda chain-expressing PCs are the main cells expressing granzyme B (GrzB). In vitro studies revealed that activated B cells differentiated into GrzB-expressing PCs when co-cultured with macrophages and follicular helper T cells. This effect could be reproduced on combined stimulation of IL-15 (produced by macrophages) and IL-21 (produced by T follicular helper cells) in a STAT3-dependent manner. Whereas IL-21 triggers the transcription of mRNA of GrzB, IL-15 synergizes the translation of GrzB proteins. The precise role of GrzB in PC biology remains to be understood and studies in mice will not help as their PCs do not express GrzB.

  1. In ovo injection of anti-chicken CD25 monoclonal antibodies depletes CD4+CD25+ T cells in chickens.

    Science.gov (United States)

    Shanmugasundaram, Revathi; Selvaraj, Ramesh K

    2013-01-01

    The CD4(+)CD25(+) cells have T regulatory cell properties in chickens. This study investigated the effect of in ovo injection of anti-chicken CD25 monoclonal antibodies (0.5 mg/egg) on CD4(+)CD25(+) cell depletion and on amounts of interleukin-2 mRNA and interferon-γ mRNA in CD4(+)CD25(-) cells posthatch. Anti-chicken CD25 or PBS (control) was injected into 16-d-old embryos. Chicks hatched from eggs injected with anti-chicken CD25 antibodies had a lower CD4(+)CD25(+) cell percentage in the blood until 25 d posthatch. The anti-chicken CD25 antibody injection nearly depleted CD4(+)CD25(+) cells in the blood until 16 d posthatch. At 30 d posthatch, the CD4(+)CD25(+) cell percentage in the anti-CD25-antibody-injected group was comparable with the percentage in the control group. At 16 d posthatch, the anti-chicken CD25 antibody injection decreased CD4(+)CD25(+) cell percentages in the thymus, spleen, and cecal tonsils. Chickens hatched from anti-CD25-antibody-injected eggs had approximately 25% of CD4(+)CD25(+) cells in the cecal tonsils and thymus compared with those in the cecal tonsils and thymus of the control group. The CD4(+)CD25(-) cells from the spleen and cecal tonsils of chicks hatched from anti-chicken-CD25-injected eggs had higher amounts of interferon-γ and interleukin-2 mRNA than CD4(+)CD25(-) cells from the control group. It could be concluded that injecting anti-chicken CD25 antibodies in ovo at 16 d of incubation nearly depleted the CD4(+)CD25(+) cells until 25 d posthatch.

  2. Photothermolysis mediated by gold nanorods modified with EGFR monoclonal antibody induces Hep-2 cells apoptosis in vitro and in vivo.

    Science.gov (United States)

    Zhang, Shiwen; Li, Yunlong; He, Xiaoguang; Dong, Shouan; Huang, Yunchao; Li, Xiaojiang; Li, Yuxiao; Jin, Congguo; Zhang, Yingying; Wang, Yuanling

    2014-01-01

    Gold nanorods (AuNRs) have been used in plasmonic photothermal therapy (PPTT), which is thought to be more efficient and selective than conventional photothermal therapy. The efficiency and safety of PPTT can be improved by functionally modifying the gold nanorods with proteins or biomolecules. In this study, AuNRs were modified with anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), and the apoptotic potential of EGFRmAb-AuNR was assessed in Hep-2 cells in vitro and in vivo. The EGFRmAb modification had no obvious influence on the original optical property of the AuNRs, but it significantly increased the entry of AuNRs into Hep-2 cells. EGFRmAb-AuNRs, with appropriate laser irradiation, resulted in higher Hep-2 cells apoptosis than AuNRs did alone, in vitro, and was accompanied by alteration of reactive oxygen species (ROS) production, Ca(2+) release, change in mitochondrial membrane potential (ΔΨm), cytochrome c (Cyt-c) release, active caspase-3 expression, and level of B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma 2 protein-associated X protein (Bax). EGFRmAb-AuNR-mediated apoptosis in Hep-2 cells was also observed in vivo and had an inhibitive effect on growth of Hep-2 tumor xenografts. Our data suggest that the EGFRmAb modification improves AuNR-mediated apoptosis and may have the potential to be used clinically.

  3. Progression of cutaneous plasmacytoma to plasma cell leukemia in a dog.

    Science.gov (United States)

    Rout, Emily D; Shank, Alba Maria M; Waite, Angharad H K; Siegel, Andrea; Avery, Anne C; Avery, Paul R

    2017-02-10

    A 5-year-old male neutered Bernese Mountain Dog was presented for cutaneous plasmacytoma, which was treated by surgical excision. Four months later, the dog developed multiple skin masses, hyphema, pericardial and mild bicavitary effusions, myocardial masses, and marked plasmacytosis in the peripheral blood. Circulating plasma cells expressed CD34 and MHC class II by flow cytometry. Immunocytochemistry demonstrated that these cells were strongly positive for multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM-1) and weakly to moderately positive for Pax5. The dog was hypoglobulinemic but had a monoclonal IgA gammopathy detected by serum immunofixation electrophoresis. The PCR analysis of antigen receptor gene rearrangements (PARR) by fragment analysis using GeneScan methodology revealed that plasmacytoid cells in the original cutaneous plasmacytoma and peripheral blood had an identical immunoglobulin heavy chain gene (IgH) rearrangement, indicating that both populations were derived from the same neoplastic clone. Canine cutaneous plasmacytoma rarely progresses to a malignant form and plasma cell leukemia is rarely diagnosed in the dog. This report describes a case of cutaneous plasmacytoma progressing to plasma cell leukemia with a rapid and aggressive clinical course. This report also highlights the utility of flow cytometry, immunocytochemistry, immunofixation electrophoresis, and PARR by fragment analysis using GeneScan methodology in the diagnosis of this hematopoietic neoplasm.

  4. Homotypic aggregation of human cell lines by HLA class II-, class Ia- and HLA-G-specific monoclonal antibodies

    DEFF Research Database (Denmark)

    Odum, Niels; Ledbetter, J A; Martin, P

    1991-01-01

    adhesion between T and B cells by activating the CD18/CD11a (LFA-1) adhesion pathway. Here we report that monoclonal antibodies (mAb) against HLA-DR (L243, p4.1, HB10a, VI15) and certain broad class II reacting mAb (TU35, TU39), but not anti-DQ (TU22, Leu-10) mAb, induced homotypic aggregation of human...... and two anti-beta 2-microglobulin mAb had variable, weak effects. The aggregation response was an active, temperature-sensitive process which was almost totally abrogated by azide and by cytochalasins B and E, but unaffected by colchicine, EDTA, aphidicolin, actinomycin D and protein tyrosine kinase...

  5. Dual functions of a monoclonal antibody against cell surface F1F0 ATP synthase on both HUVEC and tumor cells

    Institute of Scientific and Technical Information of China (English)

    Xia ZHANG; Feng GAO; Li-li YU; Yan PENG; Hong-hai LIU; Jin-ying LIU; Ming YIN; Jian NI

    2008-01-01

    Aim: To generate a monoclonal antibody (McAb) against cell surface FI F0 ATP synthase (ATPase) and observe its antitumoral activity on both human umbilical vein endothelial cells (HUVEC) and tumor cells. Methods: Hybridoma cells secret- ing McAb against ATPase were produced by polyethylene glycol-mediated fu- sions and screened by ELISA. The specificity of McAb was demonstrated by immunofluorescence and confocal imaging, as well as flow cytometry analysis. After the blockade of surface ATPase with McAb on HUVEC and human breast adenocarcinoma MDA-MB-231 cells, an ATP determination kit and CellTiter96 Aqueous Assay (MTS) assay were used to detect the effect of the antibody on extracellular ATP modification and cell proliferation. A cellular cytotoxicity assay in combination with doxorubicin, and a cell migration assay on MDA-MB-231 cells were used to determine the antitumoral activity. Finally, a HUVEC tube formation assay was used to detect the antiangiogenic effect of McAb178-5G10. Results: A monoclonal antibody (McAb178-SG10) against the β-subunit of ATPase was generated, and its reactivity toward HUVEC and tumor cells was studied. We demonstrate that McAb178-SG10 binds to ATPase at the cell surface, where it is able to inhibit ATP synthesis. This antibody also prevents the proliferation of HUVEC and MDA-MB-231 cells. Furthermore, McAb 178-5G l0 enhances the tumoricidal effects of doxorubicin (P<0.05), inhibits the migration of MDA-MB- 231 in transwell assays (P<0.01), and disrupts HUVEC tube formation on Matrigel (P<0.01). Conclusion: McAb178-5GI0 binds preferentially to cell surface ATPase, blocks ATP synthesis, and exhibits both antiangiogenic and antitumorigenic effects.

  6. In vitro and in vivo evaluation of direct rhenium-188-labeled anti-CD52 monoclonal antibody alemtuzumab for radio immunotherapy of B-cell chronic lymphocytic leukemia

    NARCIS (Netherlands)

    De Decker, Mario; Bacher, Klaus; Thierens, Hubert; Slegers, Guido; Dierckx, Rudi A.; De Vos, Filip

    2008-01-01

    Alemtuzumab (Campath, Berlex) is a humanized IgG1 rat monoclonal antibody directed against the cell surface CD52 antigen, found on lymphocytes and monocytes. It is being developed for the treatment of chronic lymphocytic leukemia (CLL), autoinumme disease and for the prevention of transplant rejecti

  7. Optimization of radioimmunotherapy of renal cell carcinoma: labeling of monoclonal antibody cG250 with 131I, 90Y, 177Lu, or 186Re.

    NARCIS (Netherlands)

    Brouwers, A.H.; Eerd-Vismale, J.E.M. van; Frielink, C.; Oosterwijk, E.; Oyen, W.J.G.; Corstens, F.H.M.; Boerman, O.C.

    2004-01-01

    Radioimmunotherapy (RIT) can be performed with various radionuclides. We tested the stability, biodistribution, and therapeutic efficacy of various radioimmunoconjugates ((131)I, (88/90)Y, (177)Lu, and (186)Re) of chimeric antirenal cell cancer monoclonal antibody G250 (mAb cG250) in nude mice with

  8. CELL TYPE-DEPENDENT AND DIFFERENTIATION STAGE-DEPENDENT EXPRESSION OF PML DOMAINS IN RAT, DETECTED BY MONOCLONAL-ANTIBODY HIS55

    NARCIS (Netherlands)

    LAM, YW; AMMERLAAN, W; O, WS; KROESE, F; OPSTELTEN, D

    1995-01-01

    Using mouse monoclonal antibody (mAb) HIS55, we identified a nuclear antigen (ag) that exhibited a staining pattern of discrete foci. Such foci could be detected in cells of many mammalian species. These nuclear foci were not associated with nuclear membrane, nucleoli, or mitotic chromosomes. In iso

  9. Effect of tyrosine kinase inhibitor treatment of renal cell carcinoma on the accumulation of carbonic anhydrase IX-specific chimeric monoclonal antibody cG250

    NARCIS (Netherlands)

    Oosterwijk-Wakka, J.C.; Kats-Ugurlu, G.; Leenders, W.P.J.; Kiemeney, L.A.L.M.; Old, L.J.; Mulders, P.F.A.; Oosterwijk, E.

    2011-01-01

    OBJECTIVE: To investigate the effect of three different tyrosine kinase inhibitors (TKIs) on the biodistribution of chimeric monoclonal antibody (mAb) cG250, which identifies carbonic anhydrase IX (CAIX), in nude mice bearing human renal cell carcinoma (RCC) xenografts. TKIs represent the best, but

  10. Optimization of radioimmunotherapy of renal cell carcinoma: labeling of monoclonal antibody cG250 with 131I, 90Y, 177Lu, or 186Re.

    NARCIS (Netherlands)

    Brouwers, A.H.; Eerd-Vismale, J.E.M. van; Frielink, C.; Oosterwijk, E.; Oyen, W.J.G.; Corstens, F.H.M.; Boerman, O.C.

    2004-01-01

    Radioimmunotherapy (RIT) can be performed with various radionuclides. We tested the stability, biodistribution, and therapeutic efficacy of various radioimmunoconjugates ((131)I, (88/90)Y, (177)Lu, and (186)Re) of chimeric antirenal cell cancer monoclonal antibody G250 (mAb cG250) in nude mice with

  11. Diagnosis and follow-up of monoclonal gammopathies of undetermined significance; information for referring physicians.

    Science.gov (United States)

    Caers, Jo; Vekemans, Marie-Christiane; Bries, Greet; Beel, Karolien; Delrieu, Vanessa; Deweweire, Anne; Demuynck, Hilde; De Prijck, Bernard; De Samblanx, Hadewijch; Kentos, Alain; Meuleman, Nathalie; Mineur, Philippe; Offner, Fritz; Vande Broek, Isabelle; Van Droogenbroeck, Jan; Vande Velde, Ann; Wu, Ka Lung; Delforge, Michel; Schots, Rik; Doyen, Chantal

    2013-09-01

    The prevalence of monoclonal gammopathy of undetermined significance (MGUS) is generally estimated at 3.4% in the general population over 50 years, and its incidence increases with age. MGUS represents a preneoplastic entity that can transform into multiple myeloma or other lymphoproliferative disorders. The risk of malignant transformation is estimated at 1% per year and persists over time. Predictors of malignant transformation have been identified such as the heavy chain isotype, The level of monoclonal proteins, increasing levels of the monoclonal component during the first years off follow-up, the percentage of bone marrow plasmocytosis, the dosage of serum free light chains, the presence of immunophenotypically abnormal plasma cells, aneuploidy, and the presence of circulating plasma cells. Prognostic scores that combine certain of these factors have been proposed and allow the identification of high-risk patients. Their use could assist in tailoring the care for each patient, based on his/her risk profile.

  12. Laboratory Characterizations on 2007 Cases of Monoclonal Gammopathies in East China

    Institute of Scientific and Technical Information of China (English)

    Hao Wang; Chunfang Gao; Lingling Xu; Zaixing Yang; Wenjing Zhao

    2008-01-01

    Monoclonal gammopathies are characterized by the presence of monoclonal immunoglobulin in patients with or without evidence of multiple myeloma (MM), macroglobulinemia, amyloidosis (AL), or a related plasma cell proliferative disorder. This study aims to evaluate laboratory diagnostic characters of monoclonal gammopathies and investigates the correlation between monoclonal gammopathies and transforming growth factor β1 (TGFβ1). Immunofixation electrophoresis (IFE), serum protein electrophoresis (SPE), nephelometry and urine light chain ELISA were used for laboratory identification of monoclonal immunoglobulins. Plasma TGFβ1 was detected with double-antibodies ELISA. Lightcycler was used for single nucleotide polymorphism (SNP) analysis. Totally 2,007 cases of monoclonai immunogiobulin (M protein) were identified in 10,682 samples. The isotypes of M protein were IgG type 47.1%, IgA 23.0%, IgM 8.7%, IgD 5.3%, free light chain κ 6.1%, λ 9.8%. In reference to IFE, the coherency of diagnosis was serum light chain ratio (κ/λ) 94.4%, quantitation of lgs 83%, light chain quantitation 80.9%, and urine light chain ratio (κ/λ) 58.0%. Plasma TGFβ1 was elevated significantly compared to normal control. The allelic frequency of codon 10 (C > T) was neither associated with the existence of the M protein nor with the M protein isotype. Monoclonal gammopathies can be identified with the combination of IFE, SPE, Igs quantitaion and urine light chain determination. Although TGFβ1, an important cytokine in immune regulation, was elevated in monoclonal gammopathies, the SNPs in coding region of TGFβ1 gene did not confer susceptibility to the development of monoclonal gammopathies in this study. Cellular & Molecular Immunology. 2008;5(4): 293-298.

  13. NF-κB2 mutation targets survival, proliferation and differentiation pathways in the pathogenesis of plasma cell tumors

    Directory of Open Access Journals (Sweden)

    McCarthy Brian A

    2012-05-01

    Full Text Available Abstract Background Abnormal NF-κB2 activation has been implicated in the pathogenesis of multiple myeloma, a cancer of plasma cells. However, a causal role for aberrant NF-κB2 signaling in the development of plasma cell tumors has not been established. Also unclear is the molecular mechanism that drives the tumorigenic process. We investigated these questions by using a transgenic mouse model with lymphocyte-targeted expression of p80HT, a lymphoma-associated NF-κB2 mutant, and human multiple myeloma cell lines. Methods We conducted a detailed histopathological characterization of lymphomas developed in p80HT transgenic mice and microarray gene expression profiling of p80HT B cells with the goal of identifying genes that drive plasma cell tumor development. We further verified the significance of our findings in human multiple myeloma cell lines. Results Approximately 40% of p80HT mice showed elevated levels of monoclonal immunoglobulin (M-protein in the serum and developed plasma cell tumors. Some of these mice displayed key features of human multiple myeloma with accumulation of plasma cells in the bone marrow, osteolytic bone lesions and/or diffuse osteoporosis. Gene expression profiling of B cells from M-protein-positive p80HT mice revealed aberrant expression of genes known to be important in the pathogenesis of multiple myeloma, including cyclin D1, cyclin D2, Blimp1, survivin, IL-10 and IL-15. In vitro assays demonstrated a critical role of Stat3, a key downstream component of IL-10 signaling, in the survival of human multiple myeloma cells. Conclusions These findings provide a mouse model for human multiple myeloma with aberrant NF-κB2 activation and suggest a molecular mechanism for NF-κB2 signaling in the pathogenesis of plasma cell tumors by coordinated regulation of plasma cell generation, proliferation and survival.

  14. Production of Nfa1-specific monoclonal antibodies that influences the in vitro cytotoxicity of Naegleria fowleri trophozoites on microglial cells.

    Science.gov (United States)

    Lee, Yang-Jin; Kim, Jong-Hyun; Jeong, Seok-Ryoul; Song, Kyoung-Ju; Kim, Kyongmin; Park, Sun; Park, Moon-Sung; Shin, Ho-Joon

    2007-10-01

    Naegleria fowleri, agent of fatal primary amoebic meningoencephalitis, appears to induce cytotoxicity mechanically through its contact with the cell. The nfa1 gene cloned from a cDNA library of pathogenic N. fowleri by immunoscreening consists of 360 bp and expresses a 13.1-kDa recombinant protein (rNfa1) that demonstrated localization in the pseudopodia when examined using immunocytochemistry. To study the mechanisms involved in N. fowleri cytotoxicity, we developed a large volume of rNfa1-specific monoclonal antibody (McAb) against a 17-kDa His-tag fusion rNfa1 protein using a cell fusion technique. We established eight McAb-producing hybridoma cells. The antibodies were all immunoglobulin G2b and reacted strongly with a 17-kDa band representing the rNfa1 fusion protein in Western blotting, demonstrating immunoreactivity to the Nfa1 protein in pseudopodia (especially in the food cups) of N. fowleri trophozoites. A 51Cr-release assay indicated N. fowleri cytotoxicity by demonstrating that it eliminated 37.8, 60.6, and 98.8% of the target (microglial) cells 6, 12, and 24 h after co-incubation, respectively. When an anti-Nfa1 McAb was added to the coculture system, N. fowleri cytotoxicity decreased to 29.8, 44.1, and 66.3%, respectively.

  15. A partner monoclonal antibody to Moab 730 kills 100% of DU145 and PC3 androgen-independent cancer cells

    Indian Academy of Sciences (India)

    Hemant Kumar Vyas; Rahul Pal; Nirmal K Lohiya; G P Talwar

    2009-12-01

    A number of therapeutic options are available for patients with prostate carcinoma till the time that the tumour is hormone dependent. However, no fully effective therapy is available for the treatment of androgen-independent prostate carcinomas. Antibodies directed at epitopes unique to or overexpressed on the cancer cells could be of therapeutic utility. A monoclonal antibody (Moab) 2C4 has been generated, which binds with cells of two androgenindependent prostate cancers, DU145 and PC3, and does not bind to peripheral blood leukocytes (PBLs) of healthy donors. This antibody, along with the previously developed Moab 730, kills 100% of both DU145 and PC3 cells in the presence of complement and does not have a deleterious effect on PBLs of healthy males. The anti-tumour action of the two antibodies prevents the establishment of DU145 cell tumour in nude mice in vivo. Moab 2C4 in combination with 730 has potential for use as therapy for androgen-independent cancers.

  16. Generation of a monoclonal antibody against the glycosylphosphatidylinositol-linked protein Rae-1 using genetically engineered tumor cells.

    Science.gov (United States)

    Hu, Jiemiao; Vien, Long T; Xia, Xueqing; Bover, Laura; Li, Shulin

    2014-02-04

    Although genetically engineered cells have been used to generate monoclonal antibodies (mAbs) against numerous proteins, no study has used them to generate mAbs against glycosylphosphatidylinositol (GPI)-anchored proteins. The GPI-linked protein Rae-1, an NKG2D ligand member, is responsible for interacting with immune surveillance cells. However, very few high-quality mAbs against Rae-1 are available for use in multiple analyses, including Western blotting, immunohistochemistry, and flow cytometry. The lack of high-quality mAbs limits the in-depth analysis of Rae-1 fate, such as shedding and internalization, in murine models. Moreover, currently available screening approaches for identifying high-quality mAbs are excessively time-consuming and costly. We used Rae-1-overexpressing CT26 tumor cells to generate 60 hybridomas that secreted mAbs against Rae-1. We also developed a streamlined screening strategy for selecting the best anti-Rae-1 mAb for use in flow cytometry assay, enzyme-linked immunosorbent assay, Western blotting, and immunostaining. Our cell line-based immunization approach can yield mAbs against GPI-anchored proteins, and our streamlined screening strategy can be used to select the ideal hybridoma for producing such mAbs.

  17. Effect of IL-17 monoclonal antibody Secukinumab combined with IL-35 blockade of Notch signaling pathway on the invasive capability of hepatoma cells.

    Science.gov (United States)

    Li, H Ch; Zhang, Y X; Liu, Y; Wang, Q Sh

    2016-07-14

    We investigated the effect of the IL-17 monoclonal antibody Secukinumab combined with IL-35 in the blockade of the Notch signaling pathway on the invasive capability of hepatoma cells. We examined the effects of IL-17 antibody or IL-35 treatment alone or in combination on cell invasion and migration capabilities with Transwell chambers. The mRNA levels of Hes1, Hes5, and Hey1 were tested using quantitative polymerase chain reaction. The protein expression of N1ICD, Snail, and E-cadherin protein expressions were measured with western blot. The expression of Hes1, Hes5, Hey1 and N1ICD were all very high in hepatoma cell lines, and were positively correlated with the invasive migration capabilities of the cells. The combination of IL-17 monoclonal antibody Secukinumab with IL-35 could effectively inhibit the Notch signaling pathway, as well as the invasive migration of the cells. Snail and E-cadherin are involved in the migration of hepatoma cells, and it has been established that Snail can regulate the expression of E-cadherin. IL-17 monoclonal antibody Secukinumab combined with IL-35 can increase E-cadherin and decrease Snail expression, which are positively correlated with cell invasive migration capabilities. Overall, treatment with both IL-17 antibody and IL-35 is more effective than each treatment alone. Notch signaling is activated in hepatoma cell lines and increases with the enhancement of cell invasive migration capabilities. IL-17 monoclonal antibody Secukinumab combined with IL-35 can block the Notch signaling pathway, simultaneously reducing the invasive migration capability of hepatoma cells.

  18. Model-directed engineering of "difficult-to-express" monoclonal antibody production by Chinese hamster ovary cells.

    Science.gov (United States)

    Pybus, Leon P; Dean, Greg; West, Nathan R; Smith, Andrew; Daramola, Olalekan; Field, Ray; Wilkinson, Stephen J; James, David C

    2014-02-01

    Despite improvements in volumetric titer for monoclonal antibody (MAb) production processes using Chinese hamster ovary (CHO) cells, some "difficult-to-express" (DTE) MAbs inexplicably reach much lower process titers. These DTE MAbs require intensive cell line and process development activity, rendering them more costly or even unsuitable to manufacture. To rapidly and rationally identify an optimal strategy to improve production of DTE MAbs, we have developed an engineering design platform combining high-yielding transient production, empirical modeling of MAb synthesis incorporating an unfolded protein response (UPR) regulatory loop with directed expression and cell engineering approaches. Utilizing a panel of eight IgG1 λ MAbs varying >4-fold in volumetric titer, we showed that MAb-specific limitations on folding and assembly rate functioned to induce a proportionate UPR in host CHO cells with a corresponding reduction in cell growth rate. Derived from comparative empirical modeling of cellular constraints on the production of each MAb we employed two strategies to increase production of DTE MAbs designed to avoid UPR induction through an improvement in the rate/cellular capacity for MAb folding and assembly reactions. Firstly, we altered the transfected LC:HC gene ratio and secondly, we co-expressed a variety of molecular chaperones, foldases or UPR transactivators (BiP, CypB, PDI, and active forms of ATF6 and XBP1) with recombinant MAbs. DTE MAb production was significantly improved by both strategies, although the mode of action was dependent upon the approach employed. Increased LC:HC ratio or CypB co-expression improved cell growth with no effect on qP. In contrast, BiP, ATF6c and XBP1s co-expression increased qP and reduced cell growth. This study demonstrates that expression-engineering strategies to improve production of DTE proteins in mammalian cells should be product specific, and based on rapid predictive tools to assess the relative impact of

  19. Analyses of CD20 Monoclonal Antibody–Mediated Tumor Cell Killing Mechanisms: Rational Design of Dosing Strategies

    Science.gov (United States)

    Lindorfer, Margaret A.

    2014-01-01

    Since approval of rituximab for treatment of B cell non-Hodgkin lymphoma, development of monoclonal antibodies (mAbs) for cancer treatment and elucidation of their cytotoxic mechanisms have been subject to intense investigations. Compelling evidence indicates that rituximab and another CD20 mAb, ofatumumab, must use the body’s cellular and humoral immune effector functions to kill malignant cells. Other U.S. Food and Drug Administration–approved mAbs, including obinutuzumab, cetuximab, and trastuzumab, require, in part, these effector mechanisms to eliminate tumor cells. Although gram quantities of mAbs can be administered to patients, our investigations of CD20 mAb-based therapies for chronic lymphocytic leukemia (CLL), including correlative measurements in clinical trials and studies with primary cells and cell lines, indicate that effector mechanisms necessary for mAb activity can be saturated or exhausted if tumor burdens are high, thus substantially compromising the efficacy of high-dose mAb therapy. Under these conditions, another reaction (trogocytosis) predominates in which bound CD20 mAb and CD20 are removed from targeted cells by effector cells that express Fcγ receptors, thereby allowing malignant cells to escape unharmed and continue to promote disease pathology. To address this problem, we propose that a low-dose strategy, based on administering 30–50 mg of CD20 mAb three times per week, may be far more effective for CLL than standard dosing because it will minimize effector function saturation and reduce trogocytosis. This approach may have general applicability to other mAbs that use immune effector functions, and could be formulated into a subcutaneous treatment strategy that would be more accessible and possibly more efficacious for patients. PMID:24944188

  20. Circulating Regulatory T-Cells in Monoclonal Gammopathies of Uncertain Significance and Multiple Myeloma: In Search of a Role

    Directory of Open Access Journals (Sweden)

    Giovanni D’Arena

    2016-01-01

    Full Text Available The frequency and function of regulatory T-cells (Tregs in multiple myeloma (MM are still matter of debate. The percentage and absolute number of circulating Tregs (CD4+CD25+high  densityCD127-/low  density from 39 patients with untreated MM and 44 patients with monoclonal gammopathies of uncertain significance (MGUS were tested and compared with 20 healthy subjects as controls. The mean percentage number of circulating Tregs was 2.1%  ± 1.0 (range 0.75–6.1% in MM patients; 2.1%  ± 0.9 (range 0.3–4.4% in MGUS; and 1.5%  ± 0.4 (range 0.9–2.1% in controls (p ns. Mean absolute number of Tregs was 36.3/μL ± 23.7 (range 6.7–149/μL in MM; 38.8/μL ± 19.1 (range 4.3–87/μL in MGUS; and 39.4/μL ± 12.5 (range 18–63/μL in controls (p ns. After a median follow-up of 38 months, 5 MGUS and 2 smoldering MM (SMM transformed into overt MM; however Tregs number did not predict this evolution. With respect to MM patients and after a median follow-up of 33 months, Tregs did not show any significant correlation with main clinical and laboratory characteristics. Finally, from a functional point of view, Tregs displayed an effective suppressor function, irrespective of disease status. This study indicates that the number of circulating Tregs does not differ in different monoclonal gammopathies and normal subjects and do not correlate with clinical features of MM.

  1. Circulating Regulatory T-Cells in Monoclonal Gammopathies of Uncertain Significance and Multiple Myeloma: In Search of a Role.

    Science.gov (United States)

    D'Arena, Giovanni; Rossi, Giovanni; Laurenti, Luca; Statuto, Teodora; D'Auria, Fiorella; Valvano, Luciana; Simeon, Vittorio; Giudice, Aldo; Innocenti, Idanna; De Feo, Vincenzo; Filosa, Rosanna; Musto, Pellegrino

    2016-01-01

    The frequency and function of regulatory T-cells (Tregs) in multiple myeloma (MM) are still matter of debate. The percentage and absolute number of circulating Tregs (CD4(+)CD25(+high  density)CD127(-/low  density)) from 39 patients with untreated MM and 44 patients with monoclonal gammopathies of uncertain significance (MGUS) were tested and compared with 20 healthy subjects as controls. The mean percentage number of circulating Tregs was 2.1%  ± 1.0 (range 0.75-6.1%) in MM patients; 2.1%  ± 0.9 (range 0.3-4.4%) in MGUS; and 1.5%  ± 0.4 (range 0.9-2.1%) in controls (p ns). Mean absolute number of Tregs was 36.3/μL ± 23.7 (range 6.7-149/μL) in MM; 38.8/μL ± 19.1 (range 4.3-87/μL) in MGUS; and 39.4/μL ± 12.5 (range 18-63/μL) in controls (p ns). After a median follow-up of 38 months, 5 MGUS and 2 smoldering MM (SMM) transformed into overt MM; however Tregs number did not predict this evolution. With respect to MM patients and after a median follow-up of 33 months, Tregs did not show any significant correlation with main clinical and laboratory characteristics. Finally, from a functional point of view, Tregs displayed an effective suppressor function, irrespective of disease status. This study indicates that the number of circulating Tregs does not differ in different monoclonal gammopathies and normal subjects and do not correlate with clinical features of MM.

  2. Circulating Regulatory T-Cells in Monoclonal Gammopathies of Uncertain Significance and Multiple Myeloma: In Search of a Role

    Science.gov (United States)

    Rossi, Giovanni; Laurenti, Luca; Statuto, Teodora; D'Auria, Fiorella; Valvano, Luciana; Simeon, Vittorio; Giudice, Aldo; Innocenti, Idanna; De Feo, Vincenzo; Filosa, Rosanna; Musto, Pellegrino

    2016-01-01

    The frequency and function of regulatory T-cells (Tregs) in multiple myeloma (MM) are still matter of debate. The percentage and absolute number of circulating Tregs (CD4+CD25+high  densityCD127−/low  density) from 39 patients with untreated MM and 44 patients with monoclonal gammopathies of uncertain significance (MGUS) were tested and compared with 20 healthy subjects as controls. The mean percentage number of circulating Tregs was 2.1%  ± 1.0 (range 0.75–6.1%) in MM patients; 2.1%  ± 0.9 (range 0.3–4.4%) in MGUS; and 1.5%  ± 0.4 (range 0.9–2.1%) in controls (p ns). Mean absolute number of Tregs was 36.3/μL ± 23.7 (range 6.7–149/μL) in MM; 38.8/μL ± 19.1 (range 4.3–87/μL) in MGUS; and 39.4/μL ± 12.5 (range 18–63/μL) in controls (p ns). After a median follow-up of 38 months, 5 MGUS and 2 smoldering MM (SMM) transformed into overt MM; however Tregs number did not predict this evolution. With respect to MM patients and after a median follow-up of 33 months, Tregs did not show any significant correlation with main clinical and laboratory characteristics. Finally, from a functional point of view, Tregs displayed an effective suppressor function, irrespective of disease status. This study indicates that the number of circulating Tregs does not differ in different monoclonal gammopathies and normal subjects and do not correlate with clinical features of MM. PMID:27493974

  3. Antibody-Dependent Cell-Mediated Cytotoxicity Effector-Enhanced EphA2 Agonist Monoclonal Antibody Demonstrates Potent Activity against Human Tumors

    Directory of Open Access Journals (Sweden)

    Elizabeth M. Bruckheimer

    2009-06-01

    Full Text Available EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated ∼80% tumor cell killing. In a dose-dependent manner, natural killer (NK cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID mice (which have functional NK cells and monocytes and SCID nonobese diabetic (NOD mice (which largely lack functional NK cells and monocytes. Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells.

  4. The effect of unlabelled monoclonal antibody (mAb) on the biodistribution of [sup 131]I-anti-idiotype mAb in murine B cell lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Schiele, J. (Stanford University (United States). Division of Laboratory Animal Medicine); Knox, S.J. (Stanford University Medical Center (United States). Department of Radiation Oncology); Ruhl, W. (Stanford University (United States). Division of Laboratory Animal Medicine Stanford Univ., CA (United States). Dept. of Radiology); Goris, M.L. (Stanford University Medical Center (United States). Department of Diagnostic Radiology and Nuclear Medicine)

    1992-07-01

    The 38C13 murine B cell lymphoma model was used to study the effect of the pre-injection of unlabelled anti-idiotype monoclonal antibody (mAb) on the subsequent biodistribution of [sup 131]I-anti-idiotype mAb. Mice with established tumors received 0-500 [mu]g of unlabelled anti-idiotype mAb 24 h prior to the administration of [sup 131]I-anti-idiotype (specific), or both [sup 125]I-anti-idiotype and [sup 131]I-isotype-matched irrelevant control (non-specific) mAb. Mice were counted daily in a gamma counter and sacrificed at 2-144 h following injection. Mice were dissected and the weight and activity of the animals and organs were measured. Mice were bled periodically and circulating idiotype levels were measured using an ELISA assay. 500 [mu]g of unlabelled anti-idiotype mAb increased the retention time of the specific but not the nonspecific mAb in all organs and tumor. Following pretreatment with unlabelled mAb, the cumulative tumor/whole body and tumor/normal organ ratios became similar to those of the nonspecific mAb, with concentration ratios (specific/nonspecific mAb) of approximately 1, which persisted until 96 h post injection when circulating idiotype reappears in antigen excess. In the absence of unlabelled mAb there was less retention in tumor and normal tissue. This is presumed to be due in part to decreased levels of circulating [sup 131]I-mAb secondary to rapid plasma clearance of antigen-body complexes and tumor cell mediated dehalogenation, which results when the specific mAb specifically binds the target antigen. Thus, the addition of unlabelled mAb increased the retention by decreasing the specific behavior of the anti-idiotype antibody. (author). 12 refs.; 1 fig.; 3 tabs.

  5. Forced KLF4 expression increases the generation of mature plasma cells and uncovers a network linked with plasma cell stage.

    Science.gov (United States)

    Schoenhals, Matthieu; Jourdan, Michel; Seckinger, Anja; Pantesco, Véronique; Hose, Dirk; Kassambara, Alboukadel; Moreaux, Jérôme; Klein, Bernard

    2016-07-17

    A role of the transcription factor Krüppel-like factor 4 (KLF4) in the generation of mature plasma cells (PC) is unknown. Indeed, KLF4 is critical in controlling the differentiation of various cell linages, particularly monocytes and epithelial cells. KLF4 is expressed at low levels in pro-B cells and its expression increases as they mature into pre-B cells, resting naïve B cells and memory B cells. We show here that KLF4 is expressed in human bone marrow plasma cells and its function was studied using an in vitro model of differentiation of memory B cells into long lived plasma cells. KLF4 is rapidly lost when memory B cells differentiate into highly cell cycling plasmablasts, poorly cycling early plasma cells and then quiescent long-lived plasma cells. A forced expression of KLF4 in plasmablasts enhances the yield of their differentiation into early plasma cell and long lived plasma cells, by inhibiting apoptosis and upregulating previously unknown plasma cell pathways.

  6. [Monoclonal gammopathy of undetermined significance (MGUS) in Mexican mestizos: one institution's experience].

    Science.gov (United States)

    Ruiz-Delgado, Guillermo J; Gómez Rangel, J David

    2004-01-01

    Monoclonal gammopathy of undetermined significance (MGUS) is defined as presence of serum monoclonal protein at a concentration of 3 g per deciliter or less, no monoclonal protein or only moderate amounts of monoclonal light chains in urine, absence of lytic bone lesions, anemia, hypercalemia, and renal insufficiency related with monoclonal protein, and with a proportion of plasma cells in bone marrow of 10% or less. In Caucasian population, MGUS affects about 3% of individuals > 70 years of age, whereas in Mexican mestizos this figure is substantially lower (0.7%); on the other hand, MGUS represents in Mexico only 2.4% of all monoclonal gammopathies. In a total of 9081 individuals studied prospectively at the Centro de Hematología y Medicina Interna de Puebla throughout a 20-year period, 11 patients with MGUS were identified. Median age was 70 years (range 43-83 years). Patients have been followed in periods ranging from 6 to 3270 days (median, 308 days). Two patients evolved into overt multiple myeloma at 308 and 1687 days after diagnosis of MGUS. Overall median survival (SV) of the group has not been reached, whereas 3270 days overall SV is 91%. After discussing underreporting, biasing, and other confounding factors, it would seem that MGUS, like other monoclonal gammopathies, is less frequent in Mexican mestizos than in Caucasians. Routine screening studies to identify the condition should result in increased numbers of patients.

  7. Cell adhesion to plasma-coated PVC.

    Science.gov (United States)

    Rangel, Elidiane C; de Souza, Eduardo S; de Moraes, Francine S; Duek, Eliana A R; Lucchesi, Carolina; Schreiner, Wido H; Durrant, Steven F; Cruz, Nilson C

    2014-01-01

    To produce environments suitable for cell culture, thin polymer films were deposited onto commercial PVC plates from radiofrequency acetylene-argon plasmas. The proportion of argon in the plasmas, P(Ar), was varied from 5.3 to 65.8%. The adhesion and growth of Vero cells on the coated surfaces were examined for different incubation times. Cytotoxicity tests were performed using spectroscopic methods. Carbon, O, and N were detected in all the samples using XPS. Roughness remained almost unchanged in the samples prepared with 5.3 and 28.9% but tended to increase for the films deposited with P(Ar) between 28.9 and 55.3%. Surface free energy increased with increasing P(Ar), except for the sample prepared at 28.9% of Ar, which presented the least reactive surface. Cells proliferated on all the samples, including the bare PVC. Independently of the deposition condition there was no evidence of cytotoxicity, indicating the viability of such coatings for designing biocompatible devices.

  8. Cell Adhesion to Plasma-Coated PVC

    Directory of Open Access Journals (Sweden)

    Elidiane C. Rangel

    2014-01-01

    Full Text Available To produce environments suitable for cell culture, thin polymer films were deposited onto commercial PVC plates from radiofrequency acetylene-argon plasmas. The proportion of argon in the plasmas, PAr, was varied from 5.3 to 65.8%. The adhesion and growth of Vero cells on the coated surfaces were examined for different incubation times. Cytotoxicity tests were performed using spectroscopic methods. Carbon, O, and N were detected in all the samples using XPS. Roughness remained almost unchanged in the samples prepared with 5.3 and 28.9% but tended to increase for the films deposited with PAr between 28.9 and 55.3%. Surface free energy increased with increasing PAr, except for the sample prepared at 28.9% of Ar, which presented the least reactive surface. Cells proliferated on all the samples, including the bare PVC. Independently of the deposition condition there was no evidence of cytotoxicity, indicating the viability of such coatings for designing biocompatible devices.

  9. Characterization of monoclonal antibodies against hepatitis C virus nonstructural protein 3: different antigenic determinants from human B cells.

    Science.gov (United States)

    Ou-Yang, P; Chiang, B L; Hwang, L H; Chen, Y G; Yang, P M; Chi, W K; Chen, P J; Chen, D S

    1999-04-01

    The nonstructural (NS3) region protein of hepatitis C virus (HCV) possesses major B-cell epitopes that induce antibodies after infection. To elucidate further the characteristics of these B cells and their role in the immune regulation of HCV infection, T9 (portion of NS3 region, amino acids [a.a.] 1188-1493)-specific monoclonal antibodies were derived and mapped for B-cell antigenic determinants with recombinant proteins. A total of 10 T9-specific hybridomas were generated and tested for B-cell antigenic determinants. To analyze the B-cell antigenic determinants, eight recombinant proteins including NS3-e (a.a. 1175-1334), NS3-a' (a.a. 1175-1250), NS3-a (a.a. 1251-1334), NS3-b (a.a. 1323-1412), NS3-c (a.a. 1407-1499), NS3-a/b (a.a. 1251-1412), NS3-bc (a.a. 1323-1499), and NS3-abc (a.a. 1251-1499) encoded by NS3-region internal clones were expressed and tested for immunoblotting. The data suggested IgG hybridomas recognized NS3-a, NS3-a', or NS3-b protein by immunoblotting. By contrast, the NS3-e protein bears the major antigenic determinant recognized by human sera. Half of the hybridomas were found to react with protein NS3-a', which is not a major B-cell antigenic determinant in humans. These data suggested that conformational epitopes in vivo may be important for B-cell recognition.

  10. Monoclonal Antibody Therapy Before Stem Cell Transplant in Treating Patients With Relapsed or Refractory Lymphoid Malignancies

    Science.gov (United States)

    2015-12-07

    Adult Nasal Type Extranodal NK/T-cell Lymphoma; Anaplastic Large Cell Lymphoma; Angioimmunoblastic T-cell Lymphoma; Cutaneous B-cell Non-Hodgkin Lymphoma; Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Hepatosplenic T-cell Lymphoma; Intraocular Lymphoma; Nodal Marginal Zone B-cell Lymphoma; Noncutaneous Extranodal Lymphoma; Peripheral T-cell Lymphoma; Recurrent Adult Burkitt Lymphoma; Recurrent Adult Diffuse Large Cell Lymphoma; Recurrent Adult Diffuse Mixed Cell Lymphoma; Recurrent Adult Diffuse Small Cleaved Cell Lymphoma; Recurrent Adult Grade III Lymphomatoid Granulomatosis; Recurrent Adult Hodgkin Lymphoma; Recurrent Adult Immunoblastic Large Cell Lymphoma; Recurrent Adult Lymphoblastic Lymphoma; Recurrent Adult T-cell Leukemia/Lymphoma; Recurrent Cutaneous T-cell Non-Hodgkin Lymphoma; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Grade 3 Follicular Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Marginal Zone Lymphoma; Recurrent Mycosis Fungoides/Sezary Syndrome; Recurrent Small Lymphocytic Lymphoma; Refractory Hairy Cell Leukemia; Small Intestine Lymphoma; Splenic Marginal Zone Lymphoma; T-cell Large Granular Lymphocyte Leukemia; Testicular Lymphoma; Waldenström Macroglobulinemia

  11. Cell Wall Growth and Modulation Dynamics in a Model Unicellular Green Alga—Penium margaritaceum: Live Cell Labeling with Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    David S. Domozych

    2011-01-01

    Full Text Available Penium margaritaceum is a unicellular charophycean green alga that possesses cell wall polymers similar to those of land plants. Several wall macromolecules of this alga are recognized by monoclonal antibodies specific for wall polymer epitopes of land plants. Immunofluorescence protocols using these antibodies may be employed to label specific cell wall constituents of live cells. Fluorescent labeling persists for several days, and this attribute allows for tracing of wall epitopes in both long- and short-term studies of cell development. Quantitative analysis of surface area covered by cell wall polymers is also easily performed. We show that significant cell expansion caused by incubation of cells in low levels of osmotically active agents like mannitol, glucose, or sucrose results from the inability of cells to undergo cytokinesis but does not result in significant changes to the amount of new cell wall. We also demonstrate that cells can be maintained for long periods of time in culture medium supplemented with specific cell wall-degrading enzymes where notable changes to wall infrastructure occur. These results demonstrate the great potential value of Penium in elucidating fundamental events during cell wall synthesis and modulation in plant cells.

  12. Influenza vaccination-induced B cell response in monoclonal gammopathy of undetermined significance (MGUS)

    NARCIS (Netherlands)

    Tete, Sarah

    2014-01-01

    Monoklonale gammopathie van onbepaalde betekenis (MGUS) is een proliferatieve plasma cel stoornis die voornamelijk voorkomt onder ouderen. Deze stoornis kan uiteindelijk leiden tot een kwaadaardige vorm van multipel myeloom. Er is sprake van een intrinsiek defect in de productie van immunoglobulines

  13. Monoclonal antibodies to snakehead, Channa striata immunoglobulins: detection and quantification of immunoglobulin-positive cells in blood and lymphoid organs.

    Science.gov (United States)

    Sood, Neeraj; Chaudhary, Dharmendra K; Rathore, Gaurav; Singh, Akhilesh; Lakra, W S

    2011-02-01

    Snakehead Channa striata is an important freshwater food fish in many Southeast Asian countries. Three monoclonal antibodies (C9, C10 and D10) were developed against purified serum immunoglobulins of Channa striata (Cs-Ig) and characterized. C9 and D10 MAbs were specific to heavy chain, while C10 MAb detected only unreduced Cs-Ig in western blotting. In competitive ELISA, C9 and C10 MAbs were specific to C. striata Ig and showed no cross reactivity with serum Ig of other fish species i.e. Channa punctatus, Channa marulius, Clarias batrachus and Labeo rohita. D10 MAb showed reactivity to serum Ig of C. striata and C. marulius. In FACS analysis of gated lymphocytes, the percentage of Ig+ cells detected by C9 MAb was 18.2%, 27.7% and 10.3% in blood, spleen and kidney, respectively (n=3, body weight 500-600 g). However, only a few cells (0.5%) were found to be Ig+ in thymus (n=5). C9 MAb was also successfully employed to demonstrate Ig+ cells in blood smears and formalin fixed sections of spleen and kidney. These findings suggest that the spleen plays an important role in humoral immunity as compared to head kidney. Further, these MAbs can be useful immunological tool in monitoring health status of cultured C. striata.

  14. Fermentanomics: Relating quality attributes of a monoclonal antibody to cell culture process variables and raw materials using multivariate data analysis.

    Science.gov (United States)

    Rathore, Anurag S; Kumar Singh, Sumit; Pathak, Mili; Read, Erik K; Brorson, Kurt A; Agarabi, Cyrus D; Khan, Mansoor

    2015-01-01

    Fermentanomics is an emerging field of research and involves understanding the underlying controlled process variables and their effect on process yield and product quality. Although major advancements have occurred in process analytics over the past two decades, accurate real-time measurement of significant quality attributes for a biotech product during production culture is still not feasible. Researchers have used an amalgam of process models and analytical measurements for monitoring and process control during production. This article focuses on using multivariate data analysis as a tool for monitoring the internal bioreactor dynamics, the metabolic state of the cell, and interactions among them during culture. Quality attributes of the monoclonal antibody product that were monitored include glycosylation profile of the final product along with process attributes, such as viable cell density and level of antibody expression. These were related to process variables, raw materials components of the chemically defined hybridoma media, concentration of metabolites formed during the course of the culture, aeration-related parameters, and supplemented raw materials such as glucose, methionine, threonine, tryptophan, and tyrosine. This article demonstrates the utility of multivariate data analysis for correlating the product quality attributes (especially glycosylation) to process variables and raw materials (especially amino acid supplements in cell culture media). The proposed approach can be applied for process optimization to increase product expression, improve consistency of product quality, and target the desired quality attribute profile.

  15. Homotypic aggregation of human cell lines by HLA class II-, class Ia- and HLA-G-specific monoclonal antibodies

    DEFF Research Database (Denmark)

    Odum, Niels; Ledbetter, J A; Martin, P

    1991-01-01

    adhesion between T and B cells by activating the CD18/CD11a (LFA-1) adhesion pathway. Here we report that monoclonal antibodies (mAb) against HLA-DR (L243, p4.1, HB10a, VI15) and certain broad class II reacting mAb (TU35, TU39), but not anti-DQ (TU22, Leu-10) mAb, induced homotypic aggregation of human......, but not the class I-negative parental line, 221, showed homotypic aggregation in response to an HLA-G specific mAb (87G) and a broad reacting class I-specific mAb (IOT2). Both cell lines responded with aggregation to anti-class II mAb (TU35). The anti-class I mAb, W6/32, had no effect on all cell lines tested...... and two anti-beta 2-microglobulin mAb had variable, weak effects. The aggregation response was an active, temperature-sensitive process which was almost totally abrogated by azide and by cytochalasins B and E, but unaffected by colchicine, EDTA, aphidicolin, actinomycin D and protein tyrosine kinase...

  16. Production of mouse monoclonal antibody against Streptococcus dysgalactiae GapC protein and mapping its conserved B-cell epitope.

    Science.gov (United States)

    Zhang, Limeng; Zhang, Hua; Fan, Ziyao; Zhou, Xue; Yu, Liquan; Sun, Hunan; Wu, Zhijun; Yu, Yongzhong; Song, Baifen; Ma, Jinzhu; Tong, Chunyu; Zhu, Zhanbo; Cui, Yudong

    2015-02-01

    Streptococcus dysgalactiae (S. dysgalactiae) GapC protein is a protective antigen that induces partial immunity against S. dysgalactiae infection in animals. To identify the conserved B-cell epitope of S. dysgalactiae GapC, a mouse monoclonal antibody 1E11 (mAb1E11) against GapC was generated and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12). Eleven positive clones recognized by mAb1E11 were identified, most of which matched the consensus motif TGFFAKK. Sequence of the motif exactly matched amino acids 97-103 of the S. dysgalactiae GapC. In addition, the epitope (97)TGFFAKK(103) showed high homology among different streptococcus species. Site-directed mutagenic analysis further confirmed that residues G98, F99, F100 and K103 formed the core of (97)TGFFAKK(103), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1E11. Collectively, the identification of conserved B-cell epitope within S. dysgalactiae GapC highlights the possibility of developing the epitope-based vaccine.

  17. Effect of cisplatin alone or combined with monoclonal anti-programmed death ligand-1 anti-body on lung adenocarcinoma cell line SPCA-1 and T lymphocytes

    Institute of Scientific and Technical Information of China (English)

    潘雪

    2014-01-01

    Objective To observe the effect of cisplatin alone or combined with anti-programmed death ligand 1 monoclonal antibody(anti-PD-L1 mA b)on the co-culture system of lung adenocarcinoma SPCA-1 cells and T lymphocytes,and therefore to study the immunotherapeutic effect of anti-PD-L1 mA b on lung cancer.Methods Human adenocarcinoma SPCA-1 cell line was selected by

  18. [Inhibition of invasion and multiplication of Toxoplasma gondii in human colonic epithelial cells by a monoclonal antibody against protein SAG2].

    Science.gov (United States)

    Osorio, J C; Sánchez, R M; Iraola, R C; Pérez, J S

    2001-01-01

    By an bromodeoxyuridine (BrdU) incorporation assay, it was proved hat an IgG 1 subclass, murine monoclonal antibody to surface protein SAG2 of Toxoplasma gondii is capable of reducing the invasion and multiplication of the parasites in highly differentiated mucine secretory HT29-18N2 line cells from a human colon adenocarcinoma. This result shows the importance of surface protein SAG2 of T.gondii in invasion and further multiplication of parasites in the host cell.

  19. Interaction of Low Temperature Plasmas with Prokaryotic and Eukaryotic Cells

    Science.gov (United States)

    Laroussi, Mounir

    2008-10-01

    Due to promising possibilities for their use in medical applications such as wound healing, surface modification of biocompatible materials, and the sterilization of reusable heat-sensitive medical instruments, low temperature plasmas and plasma jets are making big strides as a technology that can potentially be used in medicine^1-2. At this stage of research, fundamental questions about the effects of plasma on prokaryotic and eukaryotic cells are still not completely answered. An in-depth understanding of the pathway whereby cold plasma interact with biological cells is necessary before real applications can emerge. In this paper, first an overview of non-equilibrium plasma sources (both low and high pressures) will be presented. Secondly, the effects of plasma on bacterial cells will be discussed. Here, the roles of the various plasma agents in the inactivation process will be outlined. In particular, the effects of UV and that of various reactive species (O3, O, OH) are highlighted. Thirdly, preliminary findings on the effects of plasma on few types of eukaryotic cells will be presented. How plasma affects eukaryotic cells, such as mammalian cells, is very important in applications where the viability/preservation of the cells could be an issue (such as in wound treatment). Another interesting aspect is the triggering of apoptosis (programmed cell death). Some investigators have claimed that plasma is able to induce apoptosis in some types of cancer cells. If successfully replicated, this can open up a novel method of cancer treatment. In this talk however, I will briefly focus more on the wound healing potential of cold plasmas. ^1E. A. Blakely, K. A. Bjornstad, J. E. Galvin, O. R. Monteiro, and I. G. Brown, ``Selective Neuron Growth on Ion Implanted and Plasma Deposited Surfaces'', In Proc. IEEE Int. Conf. Plasma Sci., (2002), p. 253. ^2M. Laroussi, ``Non-thermal Decontamination of Biological Media by Atmospheric Pressure Plasmas: Review, Analysis, and

  20. The potential role of curcumin (diferuloylmethane in plasma cell dyscrasias/paraproteinemia

    Directory of Open Access Journals (Sweden)

    Terry Golombick

    2008-03-01

    Full Text Available Terry Golombick, Terry DiamondDepartment of Endocrinology, St George Hospital, Kogarah, AustraliaAbstract: Plasma cell dyscrasias, most commonly associated with paraproteinemia, are a diverse group of diseases. Monoclonal gammopathy of undefined significance (MGUS can precede multiple myeloma, a progressive neoplastic disease. MGUS occurs in association with a variety of other diseases and currently no treatment is recommended but rather “watchful waiting”. Given that the size of the M-protein is a risk factor for disease progression, early intervention with the aim of reducing the paraprotein load would provide an innovative therapeutic tool. Preliminary results from our pilot study show a drop of between 5% and 30% serum paraprotein in patients taking curcumin compared with patients on placebo. Curcumin is a diferuloylmethane present in extracts of the rhizome of the Curcuma longa plant. As a natural product, this has exciting potential in the treatment of plasma cell dyscrasias.Keywords: plasma cell dyscrasias, MGUS, myeloma, curcumin, paraproteinemia

  1. Regulation of B Cell to Plasma Cell Transition within the Follicular B Cell Response.

    Science.gov (United States)

    Nera, K-P; Kyläniemi, M K; Lassila, O

    2015-09-01

    Persistent humoral immunity depends on the follicular B cell response and on the generation of somatically mutated high-affinity plasma cells and memory B cells. Upon activation by an antigen, cognately activated follicular B cells and follicular T helper (TFH ) cells initiate germinal centre (GC) reaction during which high-affinity effector cells are generated. The differentiation of activated follicular B cells into plasma cells and memory B cells is guided by complex selection events, both at the cellular and molecular level. The transition of B cell into a plasma cell during the GC response involves alterations in the microenvironment and developmental state of the cell, which are guided by cell-extrinsic signals. The developmental cell fate decisions in response to these signals are coordinated by cell-intrinsic gene regulatory network functioning at epigenetic, transcriptional and post-transcriptional levels.

  2. The CSPG4-specific monoclonal antibody enhances and prolongs the effects of the BRAF inhibitor in melanoma cells.

    Science.gov (United States)

    Yu, Ling; Favoino, Elvira; Wang, Yangyang; Ma, Yang; Deng, Xiaojuan; Wang, Xinhui

    2011-08-01

    PLX4032 is a BRAF-selective inhibitor shown to be efficacious in the treatment of melanomas presenting with the BRAF(V600E) mutation. However, favorable responses to treatment are short-lived, and complete remission is rarely observed. Therefore, it is important to identify novel therapies designed to enhance treatment responses and to increase the longevity of initial response to BRAF inhibitors. To this end, we characterized the effects of the 225.28 chondroitin sulfate proteoglycan 4 (CSPG4)-specific monoclonal antibody (mAb) capable of blocking multiple signaling pathways important to cell growth, migration, and survival. Addition of 225.28 to the treatment regimen enhanced the in vitro response magnitude and the duration efficacy of PLX4032 in treating CSPG4(+), BRAF(V600E) melanoma cells (melanoma(BRAF(V600E)/CSPG4+) cells). Data presented in this report demonstrated that (1) treatments comprised of PLX4032 and mAb 225.28 were more effective at inhibiting melanoma(BRAF(V600E)/CSPG4+) cell growth than either agent alone, (2) mAb 225.28 prevented/delayed the development of resistance in melanoma(BRAF(V600E)/CSPG4+) cells to PLX4032, and (3) the mechanism of action of the combination therapy caused a down-regulation in multiple signaling pathways. This study provides a foundation for future investigations designed to improve BRAF inhibitor effectiveness in vitro and in vivo for treating melanoma(BRAF(V600E)/CSPG4+) cells in combination with a CSPG4-specific mAb.

  3. Characterization of monoclonal antibodies directed against the gp46 of human T-cell leukemia virus type I.

    Science.gov (United States)

    Edouard, E; Legrand, E; Astier-Gin, T; Dalibart, R; Geoffre, S; Dalbon, P; Guillemain, B; Londos-Gagliardi, D

    1994-04-01

    Essential HTLV-I biological functions depend on the structural motives of the surface glycoprotein (gp46). Monoclonal antibodies (mAbs) have been generated in order to identify functional regions of gp46. We obtained three monoclonal antibodies (3F3F10, 4F5F6 and 7G5D8) by immunizing Balb/c mice with beta-propiolactone inactivated HTLV-I producing cells and partially purified gp46. The mAbs are of the IgG 1 subclass. They have been characterized by western blot analysis, reactivity with HTLV-I and HTLV-II producing cells and ELISA binding assays using synthetic peptides. The immunoblot analysis performed with sheets prepared with the virus released by HUT 102 and 2060 cells (an HTLV-I virus producing cell line established in our laboratory) indicate that the three mAbs recognize a 46 kDa product as did the anti -gp46 mAb 0.5 alpha (18). Reactivity of the three mAbs with various cell lines was examined by indirect immunofluorescence assay. The mAb 7G5D8 stained strongly the membrane of all HTLV-I producing cells (MT2, C91/PL, HUT102 and cells of seven lines established in our laboratory and by A. Gessain); uninfected lymphoid cells (HSB-2, MOLT 4, CEM and PHA activated lymphocytes from normal donors) were negative. Interestingly cells of a HTLV-II producing line (344 MO) were positive. The mAbs 3F3F10 and 4F5F6 reacted with the same cells as did 7G5D8 but the fluorescence intensity was much lower than that observed with this later. A long synthetic peptide corresponding to the immunodominant region of the gp46 defined by the amino acids 175-199 and 10-mer peptides overlapping this region were used in an approach to identify the recognized epitope(s). The long 175-199 peptide was recognized by the three mAbs. 3F3F10 and 4F5F6 recognized none of the 10-mer peptides whereas 7G5D8 bound to 186-195 and 182-191 peptides. In addition 7G5D8 did not inhibit either syncytia formation or virus infection. In view of the data concerning the previously described mAbs 0.5 alpha

  4. Sensitivity of Breast Cancer Stem Cells to TRA-8 Anti-DR5 Monoclonal Antibody

    Science.gov (United States)

    2013-08-01

    helminthic drug niclosamide. Experimental parasitology. 2008;118:637-40. 30. Jin Y, Lu Z, Ding K, Li J, Du X, Chen C, et al. Antineoplastic ...adherent cells (Fig. 1B) (p=0.01). NAAE cells were generated as a tool to analyze the effect of drugs on CSC enriched populations. Because CSCs...blot and TOPflash reporter assay that require a large number of cells. The isolation of NAAE cells allowed us to test these novel drug combinations

  5. Effects of ICOS+ T cell depletion via afucosylated monoclonal antibody MEDI-570 on pregnant cynomolgus monkeys and the developing offspring.

    Science.gov (United States)

    Nicholson, Simone M; Carlesso, Gianluca; Cheng, Lily I; Cook, Halie; DaCosta, Karma; Leininger, Joel; McKeever, Kathleen; Scott, Stephen Weasel; Taylor, Devon; Streicher, Katie; Eck, Steve; Reed, Molly; Faggioni, Raffaella; Herbst, Ronald; Dixit, Rakesh; Ryan, Patricia C

    2017-09-13

    MEDI-570 is a fully human afucosylated monoclonal antibody (MAb) against Inducible T-cell costimulator (ICOS), highly expressed on CD4+ T follicular helper (TFH) cells. Effects of MEDI-570 were evaluated in an enhanced pre-postnatal development toxicity (ePPND) study in cynomolgus monkeys. Administration to pregnant monkeys did not cause any abortifacient effects. Changes in hematology and peripheral blood T lymphocyte subsets in maternal animals and infants and the attenuated infant IgG immune response to keyhole limpet hemocyanin (KLH) were attributed to MEDI-570 pharmacology. Adverse findings included aggressive fibromatosis in one dam and two infant losses in the high dose group with anatomic pathology findings suggestive of atypical lymphoid hyperplasia. The margin of safety relative to the no observed adverse effect level (NOAEL) for the highest planned clinical dose in the Phase 1a study was 7. This study suggests that women of child bearing potential employ effective methods of contraception while being treated with MEDI-570. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. The role of antigen specificity in the binding of murine monoclonal anti-DNA antibodies to microparticles from apoptotic cells.

    Science.gov (United States)

    Ullal, Anirudh J; Marion, Tony N; Pisetsky, David S

    2014-10-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and markers of underlying immune system disturbances. These antibodies bind to both single-stranded and double-stranded DNA, mediating pathogenesis by forming immune complexes. As shown recently, DNA in blood exists in both free and particulate forms, with DNA representing an important component of microparticles. Microparticles are membrane-bound vesicles containing nuclear molecules, released by membrane blebbing during cell death and activation. A panel of monoclonal NZB/NZW F1 anti-DNA antibodies was tested for binding to microparticles generated from apoptotic THP-1 and Jurkat cells. These studies showed that only certain anti-DNA antibodies in the panel, specific for double-stranded DNA, bound to microparticles. Binding to particles was reduced by soluble DNA or DNase treatment. Together, these results indicate that particle binding is a feature of only certain anti-DNA antibodies, reflecting immunochemical properties of the antibodies and the nature of the exposed DNA antigens.

  7. Elucidating the effects of arginine and lysine on a monoclonal antibody C-terminal lysine variation in CHO cell cultures.

    Science.gov (United States)

    Zhang, Xintao; Tang, Hongping; Sun, Ya-Ting; Liu, Xuping; Tan, Wen-Song; Fan, Li

    2015-08-01

    C-terminal lysine variants are commonly observed in monoclonal antibodies (mAbs) and found sensitive to process conditions, especially specific components in culture medium. The potential roles of media arginine (Arg) and lysine (Lys) in mAb heavy chain C-terminal lysine processing were investigated by monitoring the lysine variant levels under various Arg and Lys concentrations. Both Arg and Lys were found to significantly affect lysine variant level. Specifically, lysine variant level increased from 18.7 to 31.8 % when Arg and Lys concentrations were increased from 2 to 10 mM. Since heterogeneity of C-terminal lysine residues is due to the varying degree of proteolysis by basic carboxypeptidases (Cps), enzyme (basic Cps) level, pH conditions, and product (Arg and Lys) inhibition, which potentially affect the enzymatic reaction, were investigated under various Arg and Lys conditions. Enzyme level and pH conditions were found not to account for the different lysine variant levels, which was evident from the minimal variation in transcription level and intracellular pH. On the other hand, product inhibition effect of Arg and Lys on basic Cps was evident from the notable intracellular and extracellular Arg and Lys concentrations comparable with Ki values (inhibition constant) of basic Cps and further confirmed by cell-free assays. Additionally, a kinetic study of lysine variant level during the cell culture process enabled further characterization of the C-terminal lysine processing.

  8. T cell nature of exocytic and dermal lymphoid cells in atrophic parapsoriasis demonstrated by monoclonal Leu 1 and affinity isolated antibodies.

    Science.gov (United States)

    McMillan, E M; Wasik, R; Martin, D; Everett, M A

    1981-10-01

    Tissues from atrophic large plaque parapsoriasis were examined using the indirect immunoperoxidase technique with a monoclonal T cell antibody as primary antibody, and affinity isolated peroxidase conjugated goat anti-mouse IgG as second stage antibody. The "T" cell nature of the dermal infiltrate and of single exocytic epidermal lymphocytes was demonstrated. The results obtained suggest that the method utilized will be useful for the demonstration of lymphoid cell subpopulations in a variety of cutaneous disorders in which a lymphocytic infiltrate forms a significant component of the histopathology observed. This technique also has the potential of providing information on the topographic localization and differentiation of lymphocytes within the various levels of the skin. Certain technical manipulations which may result in an improvement in the quality of results obtained are also discussed.

  9. INDUCTION OF APOPTOSIS IN HUMAN GASTRIC CARCINOMA CELL LINE MGC-803 BY MONOCLONAL ANTIBODY PD4

    Institute of Scientific and Technical Information of China (English)

    Xiao Hai; Dong Zhiwei; Shou Chengchao

    1998-01-01

    Objective: To study the effect of McAb PD4 on the cell cycle and on cell injury in the gastric carcinoma cell line,MGC-803. Methods: The effects of McAb PD4 on cell proliferation cycle and cell injury of MGC-803 cells were examined by flow cytometry analysis, DNA electrophoresis and terminal deoxynucle otidyl transferase assay. Fas antigen was investigated by ELISA.Results: McAb PD4 inhibited tumor-growth of MGC-803cells in nude mice by inducing apoptosis. Conclusion:P40 is a tumor-associated antigen distinct from the Fas antigen. Molecular cloning of P40 will define the pathway and mechanism of apoptosis induced by McAb PD4.Induction of apoptosis by McAb PD4 may be a useful therapeutic approach in treating cancer.

  10. Localized Castleman disease of plasma cell type in the abdomen

    Institute of Scientific and Technical Information of China (English)

    LU Zhi-hua; WU Mei

    2011-01-01

    Castleman disease is a relatively rare entity,with the hyaline-vascular type the predominant form.Although the plasma cell type is uncommon,it still comprises approximately 10% of cases of localized diseases.In addition,the abdomen is a rare site for involvement and localized Castleman disease of the plasma cell type in the abdomen is rare.The radiologic features of localized plasma cell type in the abdomen are mostly limited to case reports.In addition to the conventional imaging findings,we present some new imaging findings of localized plasma cell type in the abdomen.

  11. Anti-CD137 monoclonal antibodies and adoptive T cell therapy: a perfect marriage?

    Science.gov (United States)

    Weigelin, Bettina; Bolaños, Elixabet; Rodriguez-Ruiz, Maria E; Martinez-Forero, Ivan; Friedl, Peter; Melero, Ignacio

    2016-05-01

    CD137(4-1BB) costimulation and adoptive T cell therapy strongly synergize in terms of achieving maximal efficacy against experimental cancers. These costimulatory biological functions of CD137 have been exploited by means of introducing the CD137 signaling domain in clinically successful chimeric antigen receptors and to more efficiently expand T cells in culture. In addition, immunomagnetic sorting of CD137-positive T cells among tumor-infiltrating lymphocytes selects for the fittest antitumor T lymphocytes for subsequent cultures. In mouse models, co-infusion of both agonist antibodies and T cells attains marked synergistic effects that result from more focused and intense cytolytic activity visualized under in vivo microscopy and from more efficient entrance of T cells into the tumor through the vasculature. These several levels of dynamic interaction between adoptive T cell therapy and CD137 offer much opportunity to raise the efficacy of current cancer immunotherapies.

  12. Characterization of a Novel Anti-DR5 Monoclonal Antibody WD1 with the Potential to Induce Tumor Cell Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Jing Wang; Beifen Shen; Yuanfang Ma; Yan Li; Zhou Lin; Chunxia Qiao; Ming Lv; Ming Yu; He Xiao; Qingyang Wang; Liyan Wang; Jiannan Feng

    2008-01-01

    TNF-related apoptosis. inducing ligand (TRAIL) is a TNF family member capable of inducing apoptosis. Death receptor 5(DR 51 is a key receptor of TRAIL and plays an important role in TRAIL-induced apoptosis. To prepare monoclonal antibodies (mAbs) against DR5, cDNA encoding soluble DR5(sDR5)was firstly amplified by revere transcriptase. polymerase chain reaction (RT-PCR) with specific primers, and then inserted into a prokaryotic expression vector pET-30a. The recombinant plasmid Was expressed in Escherichia coil strain BL21(DE3), and sDR5 was purified by nickel affinity chromatography. As an antigen. sDR5 Was used to immunize mice. Hybridomas secreting antibodies against sDR5 were identified. One positive clone Was selected to produce antibody, WD1. ELISA and immunofluorescence demonstrated that WD1 could bind recombinant sDR5 and membrane bound DR5 (mDR5)on Jurkat and Molt-4 cells. ATPLite assays showed that Jurkat and Molt-4 cells were sensitive to the antibody in a dose dependent manner. The Annexin V/PI assays and Giemsa's staining both showed that WD1 could induce Jurkat cell apoptosis efficiently. Transient transfection of 293T cells and indirect immunofluorescence assay demonstrated that mAb(WD1)couldn't cross. react with DR4.Our findings indicated that the novel antibody, WD1 could act as a direct agonist, bind DR5 characteristically, and initiate efficient apoptotic signaling and tumor regression. Thus, WD1 would be a leading candidate for potential cancer therapeutics. Cellular & Molecular Immunology. 2008;5(1):55-60.

  13. MAXIMIZATION OF DNA DAMAGE TO MGMT(+ EGFR(+ GBM CELLS USING OPTIMAL COMBINATION OF TEMOZOLOMIDE-ANTI EGFR MONOCLONAL ANTIBODY NIMOTUZUMAB

    Directory of Open Access Journals (Sweden)

    M. A. M. Inggas

    2015-09-01

    Full Text Available Background: Glioblastoma multiforme (GBM is the most aggressive primary brain tumor in adultswith dismal prognosis due to the unavailability of an effective therapy. Up to now, there had been no definitive studies published on EGFR inhibition therapy as a chemosensitizer for GBM therapy using Temozolomide (TMZ. This study aims to reveal the most effective method and timing to administer TMZ-anti EGFR targeted therapy which causes maximal DNA damage on GBM cells.Methods: Various regimens of anti EGFR monoclonal antibody Nimotuzumab (NMZ was administered in different combinations with TMZ, performed on U87MG MGMT(+ EGFR(+ cells. The effectiveness of the combinations were evaluated by measuring yH2AX levels which reflects the degree of DNA damage. One-way Anova and LSD tests were performed to determine the effects of each treatment with p<0.05. Results and discussion: the mean SD of yH2AX of each treatment was: 11,90±1,25 for the control group; 29.33±1.91 for NMZ alone; 28.13±1.58 for TMZ alone; 41.53±3.51 for concurrent use; 35.67 ±2.65 for NMZ after 24 hours TMZ; 31.87±2.94 for NMZ after 48 hours TMZ; 39.57±4.2 for TMZ after 24 hours NMZ; and 35.93 ±3.56 for TMZ after 48 hours NMZ. The administration of TMZ concurrent with or after 24 hours NMZ gives the highest amount of DNA damage to GBM cells. Conclusion: The administration of Nimotuzumab targeted therapy up to 24 hours before Temozolomide chemotherapy has been proven to be effective in maximizing the amount of DNA damage done to GBM cells in vitro. 

  14. Capillary size exclusion chromatography with picogram sensitivity for analysis of monoclonal antibodies purified from harvested cell culture fluid.

    Science.gov (United States)

    Rea, Jennifer C; Moreno, G Tony; Vampola, Lisa; Lou, Yun; van Haan, Bjorn; Tremintin, Guillaume; Simmons, Laura; Nava, Adrian; Wang, Yajun Jennifer; Farnan, Dell

    2012-01-06

    Size exclusion chromatography (SEC) is widely used in the characterization and quality control of therapeutic proteins to detect aggregates. Aggregation is a carefully monitored quality attribute from the earliest stages of clinical development owing to the possibility of eliciting an immunogenic response in the patient. During early stage molecule assessment for cell culture production, small-scale screening experiments are performed to permit rapid turn-around of results so as to not delay timelines. We report the development of a capillary SEC methodology for preliminary molecule assessment to support the evaluation of therapeutic candidates at an early stage of development. By making several key modifications to a commercially available liquid chromatography system, we demonstrate a platform process to perform capillary SEC with excellent precision, picogram sensitivity and good ruggedness. The limit of quantitation was determined to be approximately 15 pg; picogram sensitivity for SEC analysis of monoclonal antibodies had not been achieved prior to this work. In addition, we have developed a method to capture low levels of antibody (1 μg/mL) from harvested cell culture fluid (HCCF) for capillary SEC analysis. Up to 40% recovery efficiency was achieved using this micro-recovery method on 3 mL HCCF samples. Using early stage cell culture transient transfection samples, which typically have much lower titers than stable cell line samples, we demonstrate a consistent method for analyzing aggregates in low protein concentration HCCF samples for molecule assessment and early stage candidate screening. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. Plasmoacanthoma of oral cavity and plasma cell cheilitis: two sides of same disorder “oral plasma cell mucositis” ?

    Directory of Open Access Journals (Sweden)

    Gayatri Khatri

    2014-04-01

    Full Text Available Plasmoacanthoma and plasma cell cheilitis are rare disorders of obscure etiology characterized by a plasma cell infiltrate an 80-year-old woman presented with a verrucous, fleshy, skin colored plaque over lips, gingiva, and the palate and painful swallowing for over a period of 6 months. Histopathology of the lesion showed dense infiltrate of plasma cells. The lesions resolved completely after intralesional triamcinolone acetonide. Another 52-year-old male had progressively enlarging, erosive lesion over vermilion border of lower lip for 6months resembling actinic cheilitis. Histology was diagnostic of plasma cell cheilitis. Treatment with topical clobetasol propionate was effective. Plasma cell cheilitis and plasmoacanthoma perhaps represent a spectrum of oral ”plasma cell mucositis” with plasmoacanthoma being an advanced version of the former.

  16. Effects of in vivo injection of anti-chicken CD25 monoclonal antibody on regulatory T cell depletion and CD4+CD25- T cell properties in chickens.

    Science.gov (United States)

    Shanmugasundaram, Revathi; Selvaraj, Ramesh K

    2012-03-01

    Regulatory T cells (Tregs) are defined as CD4(+)CD25(+) cells in chickens. This study examined the effects of an anti-chicken CD25 monoclonal antibody injection (0.5 mg/bird) on in vivo depletion of Tregs and the properties of CD4(+)CD25(-) cells in Treg-depleted birds. The CD4(+)CD25(+) cell percentage in the blood was lower at 8 d post injection than at 0 d. Anti-CD25-mediated CD4(+)CD25(+) cell depletion in blood was maximum at 12 d post injection. The anti-CD25 antibody injection depleted CD4(+)CD25(+) cells in the spleen and cecal tonsils, but not in the thymus, at 12 d post antibody injection. CD4(+)CD25(-) cells from the spleen and cecal tonsils of birds injected with the anti-chicken CD25 antibody had higher proliferation and higher IL-2 and IFNγ mRNA amounts than the controls at 12 d post injection. At 20 d post injection, CD4(+)CD25(+) cell percentages in the blood, spleen and thymus were comparable to that of the 0 d post injection. It could be concluded that anti-chicken CD25 injection temporarily depleted Treg population and increased and IL-2 and IFNγ mRNA amounts in CD4(+)CD25(-) cells at 12d post injection.

  17. Characterization of monoclonal antibodies recognizing 130 kDa surface proteins on human embryonic stem cells and cancer cell lines.

    Science.gov (United States)

    Kim, Jum-Ji; Choi, Hong Seo; Lee, Mi-Young; Ryu, Chun Jeih

    2013-04-01

    To study cell surface proteins expressed on human embryonic stem cells (hESCs), we generated a panel of monoclonal antibodies (MAbs) against undifferentiated hESCs by a decoy immunization strategy in a previous study. Two of the MAbs, 63-B6 and 246-D7, bound to human pluripotent stem cells but not to human primary cells such as human peripheral blood mononuclear cells and human lung fibroblasts. They did not bind to either mouse embryonic stem cells or mouse embryonic fibroblasts. The two MAbs had similar binding profiles for many various cancer cells, with few exceptions. Expression of antigens recognized by the two MAbs was rapidly decreased during embryoid body formation of hESCs and gradually increased after initial decrease. The MAbs recognized approximately 130 kDa proteins on the surface of hESCs. Cloning and sequence analysis of antibody genes showed that although the MAbs had exactly the same light chain sequences, they had different heavy chain sequences. Taken together, the results suggest that the two MAbs may recognize two different epitopes of the same or different 130 kDa surface proteins involved in regulating the early differentiation of human pluripotent stem cells and cancer cells.

  18. [CXCR3 monoclonal antibody inhibits the proliferation and migration of MCF-7 cells and HepG2 cells in vitro].

    Science.gov (United States)

    Zhu, Yuqiang; Wang, Zhiyao; Zhu, Ying; Wang, Jing; Cai, Lei; Shen, Hui; Kong, Yong; Qiu, Yuhua

    2015-11-01

    To study the effects of chemokine (C-X-C motif) receptor 3 (CXCR3) monoclonal antibody (mAb) on the proliferation and migration of MCF-7 and HepG2 cells in vitro. Ascites of CXCR3 mAb was prepared at first. MCF-7 and HepG2 cells with high expressions of CXCR3 were screened by flow cytometry. MTT assay was used to detect the effects of CXCR3 mAb on the proliferation of MCF-7 and HepG2 cells in vitro in the absence/presence of interferon-inducible T-cell alpha chemoattractant (I-TAC). Transwell™ assay was performed to investigate the effects of CXCR3 mAb on the migration of MCF-7 and HepG2 cells in vitro in the absence/presence of I-TAC. The expression rate of CXCR3 on MCF-7 and HepG2 cells were 83.5% and 96.2%, respectively. 50 mg/mL CXCR3 mAb significantly inhibited the proliferation and migration of MCF-7 and HepG2 cells, and also inhibited the promoting effect of I-TAC on the proliferation and migration of MCF-7 and HepG2 cells in vitro. CXCR3 mAb can significantly inhibit the proliferation and migration of the tumor cells highly expressing CXCR3 in vitro.

  19. Effect of ambient light on monoclonal antibody product quality during small-scale mammalian cell culture process in clear glass bioreactors.

    Science.gov (United States)

    Mallaney, Mary; Wang, Szu-Han; Sreedhara, Alavattam

    2014-01-01

    During a small-scale cell culture process producing a monoclonal antibody, a larger than expected difference was observed in the charge variants profile of the harvested cell culture fluid (HCCF) between the 2 L and larger scales (e.g., 400 L and 12 kL). Small-scale studies performed at the 2 L scale consistently showed an increase in acidic species when compared with the material made at larger scale. Since the 2 L bioreactors were made of clear transparent glass while the larger scale reactors are made of stainless steel, the effect of ambient laboratory light on cell culture process in 2 L bioreactors as well as handling the HCCF was carefully evaluated. Photoreactions in the 2 L glass bioreactors including light mediated increase in acidic variants in HCCF and formulation buffers were identified and carefully analyzed. While the acidic variants comprised of a mixture of sialylated, reduced disulfide, crosslinked (nonreducible), glycated, and deamidated forms, an increase in the nonreducible forms, deamidation and Met oxidation was predominantly observed under light stress. The monoclonal antibody produced in glass bioreactors that were protected from light behaved similar to the one produced in the larger scale. Our data clearly indicate that care should be taken when glass bioreactors are used in cell culture studies during monoclonal antibody production.

  20. Development of motorized plasma lithography for cell patterning.

    Science.gov (United States)

    Deguchi, Shinji; Nagasawa, Yohei; Saito, Akira C; Matsui, Tsubasa S; Yokoyama, Sho; Sato, Masaaki

    2014-03-01

    The micropatterning of cells, which restricts the adhesive regions on the substrate and thus controls cell geometry, is used to study mechanobiology-related cell functions. Plasma lithography is a means of providing such patterns and uses a spatially-selective plasma treatment. Conventional plasma lithography employs a positionally-fixed mask with which the geometry of the patterns is determined and thus is not suited for producing on-demand geometries of patterns. To overcome this, we have manufactured a new device with a motorized mask mounted in a vacuum chamber of a plasma generator, which we designate motorized plasma lithography. Our pilot tests indicate that various pattern geometries can be obtained with the control of a shielding mask during plasma treatment. Our approach can thus omit the laborious process of preparing photolithographically microfabricated masks required for the conventional plasma lithography.

  1. Plasma cell neoplasm manifesting initially as a sub-cutaneous supra-orbital swelling

    Directory of Open Access Journals (Sweden)

    Riddhi Jaiswal

    2016-01-01

    Full Text Available Multiple myeloma is a plasma cell neoplasm seen usually in patients over 50 years of age. Some cases may be asymptomatic initially and are detected during a routine test like complete blood count. They only require a close follow-up and monitoring. However, around 1% of these monoclonal gammopathy of undetermined significance progress to multiple myeloma every year and then they need to be taken care of by chemotherapy, targeted therapy, bisphosphonates and 6 monthly urine and bone examinations. Here, we present a case of 35-year-old female with an initial symptom of a vague backache along with a left subcutaneous supra-orbital swelling which was diagnosed as multiple myeloma by aspiration cytology and confirmed by ancillary tests. She has since been on treatment with bortezomib and prednisone and is responding well.

  2. Establishment of hepatocellular carcinoma multidrug resistant monoclone cell line HepG2/mdr1

    Institute of Scientific and Technical Information of China (English)

    CHEN Yong-bing; XIE Jian-guo; YANG Jia-yin; YAN Lü-nan; YAN Mao-lin; GONG Jian-ping; XIA Ren-pin; LIU Li-xin; LI Ning; LU Shi-chun; ZHANG Jing-guang; ZENG Dao-bing

    2007-01-01

    Background The multidrug resistance (MDR) associated with the expression of the mdr1 gene and its product P-glycoprotein is a major factor in the prognosis of hepatocellular carcinoma cell (HCC) patients treated with chemotherapy. Our study was to establish a stable HCC MDR cell line where a de novo acquisition of multidrug resistance specifically related to overexpression of a transgenic mdr1.Methods The 4.5-kb mdr1 cDNA obtained from the plasmid pHaMDR1-1 was cloned into the PCI-neo mammalian expression vector, later was transferred by liposome to human hepatocarcinoma cell line HepG2. Then the transfected HepG2 cells resisting G418 were clustered and cultured and the specific fragment of mdr1 cDNA, mRNA and the P-glycoprotein (Pgp) in these HepG2 cells were detected by PCR, RT-PCR and flow cytometry, respectively. The accumulation of the daunorubicin was determinated by flow cytometry simultaneously. The nude mice model of grafting tumour was established by injecting subcutaneously HepG2/mdr1 cells in the right axilla. When the tumour diameter reached 5 mm, adriamycin was injected into peritoneal cavity. The size and growth inhibition of tumour were evaluated.Results The mdr1 expression vector was constructed successfully and the MDR HCC line HepG2/mdr1 developed.The PCR analysis showed that the specific fragment of mdr1 cDNA in HepG2/mdr1 cells, but not in the control group HepG2 cells. Furthermore, the content of the specific fragment of mdr1 mRNA and Pgp expression in HepG2/mdr1 cells were (59.7±7.9)% and (12.28±2.09)%, respectively, compared with (16.9±3.2)% and (3.07±1.06)% in HepG2 cells.In the nude mice HCC model, the tumour genes of both groups were identified. After ADM therapy, the mean size of HepG2 cell tumours was significantly smaller than HepG2/mdr1 cell tumours.Conclusion The approach using the transfer of mdr1 cDNA may be applicable to the development of MDR hepatocarcinoma cell line, whose MDR mechanism is known. This would provide the

  3. Cell Adhesion on Polycaprolactone Modified by Plasma Treatment

    Directory of Open Access Journals (Sweden)

    Nina Recek

    2016-01-01

    Full Text Available We have investigated the influence of various plasma treatments of electrospun polycaprolactone (PCL scaffolds on the adhesion and proliferation of human umbilical endothelial cells (HUVEC. The PCL scaffolds were treated in plasmas created in O2, NH3 or SO2 gas at identical conditions. Surface functionalization of plasma-treated samples was determined using X-ray photoelectron spectroscopy. Cell adhesion and morphology were investigated by scanning electron microscopy and the influence of plasma treatment on cell adhesion and viability was evaluated with cell viability assay (MTT assay. The results showed the highest metabolic activity of HUVEC on PCL samples treated with O2 and NH3 plasma. Accordingly, the cells reflected the best adhesion and morphology on O2 and NH3 plasma-treated PCL samples already at 3 h. Moreover, treatment with O2 and NH3 plasma even stimulated endothelial cell proliferation on PCL surfaces by 60% as measured at 24 h, showing significant improvement in endothelialization of this material. Contrarily, SO2 plasma appeared to be less promising in comparison with O2 and NH3 plasma; however, it was still better than without any plasma treatment. Thus, our results importantly contribute to the biocompatibility improvement of the PCL polymer, commonly used for scaffolds in tissue engineering.

  4. A Diagnostic Dilemma: Waldenström's Macroglobulinemia/Plasma Cell Leukemia

    Directory of Open Access Journals (Sweden)

    Bhawna Sethi

    2012-01-01

    Full Text Available Waldenström's macroglobulinemia is a B-cell neoplasm characterized by infiltration of the bone marrow by a lymphoplasmacytic infiltrate and an IgM monoclonal gammopathy. It is an uncommon disease with overall incidence of approximately 3 per million persons per year, accounting for approximately 1% to 2% of all hematologic cancers. It has only one-sixth the estimated prevalence of plasma cell myeloma. Disease symptoms can be due to infiltration of bone marrow and other tissue sites by malignant lymphoplasmacytic cells or due to the effects of elevated serum IgM levels. However, patients may present with constitutional symptoms only or may be asymptomatic. In our case, patient presented with chief complaints of fatigability and dyspnoea and was misdiagnosed as plasma cell leukemia on peripheral blood film and bone marrow morphology, but turned out to be a case of Waldenström's macroglobulinemia on cytoflorometry. The patient was referred for chemotherapy but expired on 10th day of admission. The suspected cause of death was cardiorespiratory failure.

  5. Characterization of Palytoxin Binding to HaCaT Cells Using a Monoclonal Anti-Palytoxin Antibody

    Directory of Open Access Journals (Sweden)

    Chiara Florio

    2013-02-01

    Full Text Available Palytoxin (PLTX is the reference compound for a group of potent marine biotoxins, for which the molecular target is Na+/K+-ATPase. Indeed, ouabain (OUA, a potent blocker of the pump, is used to inhibit some PLTX effects in vitro. However, in an effort to explain incomplete inhibition of PLTX cytotoxicity, some studies suggest the possibility of two different binding sites on Na+/K+-ATPase. Hence, this study was performed to characterize PLTX binding to intact HaCaT keratinocytes and to investigate the ability of OUA to compete for this binding. PLTX binding to HaCaT cells was demonstrated by immunocytochemical analysis after 10 min exposure. An anti-PLTX monoclonal antibody-based ELISA showed that the binding was saturable and reversible, with a Kd of 3 × 10−10 M. However, kinetic experiments revealed that PLTX binding dissociation was incomplete, suggesting an additional, OUA-insensitive, PLTX binding site. Competitive experiments suggested that OUA acts as a negative allosteric modulator against high PLTX concentrations (0.3–1.0 × 10−7 M and possibly as a non-competitive antagonist against low PLTX concentrations (0.1–3.0 × 10−9 M. Antagonism was supported by PLTX cytotoxicity inhibition at OUA concentrations that displaced PLTX binding (1 × 10−5 M. However, this inhibition was incomplete, supporting the existence of both OUA-sensitive and -insensitive PLTX binding sites.

  6. Isolation and partial characterization of antigens from basement membranes and streptococcal cell membrane (SCM) employing anti-SCM monoclonal antibody.

    Science.gov (United States)

    Zelman, M E; Lange, C F

    1989-09-01

    Monoclonal antibodies (mAb) against streptococcal cell membrane (SCM) antigen were used to identify specific cross-reactive peptides prepared by trypsin digestion of purified glomerular basement membrane (GBM) and lung basement membrane (LBM). Anti-SCM mAb-coupled HPLC columns were used to affinity isolate soluble LBM, GBM, and SCM antigens which then were sized by HPLC. Alternatively, SCM, GBM, and LBM digests were subjected to an initial separation by HPLC into component polypeptides, followed by affinity purification and ELISA of these fractions using anti-SCM mAb. Comparison of the antigenic reactivities by ELISA of the sized polypeptides on a nanomolar basis permitted the estimation of their individual relative epitope densities. The results for SCM antigens showed increasing epitope density with increasing molecular size, which suggests that intact SCM consists of repeating epitopes. Low mol. wt GBM polypeptides in nanogram amounts inhibited mAb binding to SCM, indicating that these small GBM polypeptides may similarly contain more than a single cross-reactive epitope. The identification of these cross-reactive epitopes in LBM and GBM has important implications for the etiology of post-streptococcal sequelae.

  7. Molecular Characterization of Two Monoclonal Antibodies against the Same Epitope on B-Cell Receptor Associated Protein 31.

    Science.gov (United States)

    Kim, Won-Tae; Shin, Saemina; Hwang, Hyo Jeong; Kim, Min Kyu; Jung, Han-Sung; Park, Hwangseo; Ryu, Chun Jeih

    2016-01-01

    Previously, we showed that B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum (ER) membrane chaperone, is also expressed on the cell surface by two monoclonal antibodies (MAbs) 297-D4 and 144-A8. Both MAbs recognize the same linear epitope on the C-terminal domain of BAP31, although they were independently established. Here, flow cytometric analysis showed that 144-A8 had additional binding properties to some cells, as compared to 297-D4. Quantitative antigen binding assays also showed that 144-A8 had higher antigen binding capacity than 297-D4. Affinity measurement revealed that 144-A8 had 1.54-fold higher binding affinity than 297-D4. Analysis of the heavy- and light-chain variable region sequences of two MAbs revealed that both MAbs belonged to the same heavy chain (Igh-V3660 VH3) and light chain subgroup (IGKV21) with just two amino acid differences in each framework region, indicating that both MAbs arise from the same germline origin. Seven amino acid differences were found between the complementarity determining regions (CDRs) of the two MAbs. Molecular modeling of the epitope-paratope complexes revealed that the epitope appeared to reside in closer proximity to the CDRs of 144-A8 than to those of 297-D4 with the stronger hydrogen bond interactions with the former than the latter. More interestingly, an additional hydrophobic interaction appeared to be established between the leucine residue of epitope and the paratope of 144-A8, due to the substitution of H-Tyr101 for H-Phe101 in 144-A8. Thus, the different binding specificity and affinity of 144-A8 appeared to be due to the different hydrogen bonds and hydrophobic interaction induced by the alterations of amino acids in CDRs of 144-A8. The results provide molecular insights into how the binding specificities and affinities of antibodies evolve with the same epitope in different microenvironments.

  8. Anti-CD11b monoclonal antibody reduces ischemic cell damage after transient focal cerebral ischemia in rat.

    Science.gov (United States)

    Chen, H; Chopp, M; Zhang, R L; Bodzin, G; Chen, Q; Rusche, J R; Todd, R F

    1994-04-01

    We investigated the effect of an anti-CD11b monoclonal antibody (1B6c) on ischemic cell damage after transient middle cerebral artery occlusion. We divided animals into three groups: MAb 1 group (n = 5)--rats were subjected to 2 hours of transient occlusion and 1B6c (1 mg/kg) was administered intravenously at 0 and 22 hours of reperfusion; MAb 2 group (n = 5)--same experimental protocol as MAb 1 group, except that the initial dose of 1B6c was increased to 2 mg/kg; and control group (n = 5)--same experimental protocol as MAb 2 group, except that an isotype-matched control antibody was administered. Animals were weighed and tested for neurological function before and after occlusion of the middle cerebral artery. Forty-six hours after reperfusion, brain sections were stained with hematoxylin and eosin for histology evaluation. We observed a significant reduction of weight loss and improvement in neurological function after ischemia in the MAb 2 animals compared to MAb 1 and vehicle-treated animals (p < 0.05). The lesion volume was significantly smaller in the MAb 2 group (19.5 +/- 1.9%) compared to MAb 1 (29.9 +/- 2.6%) and vehicle-treated (34.2 +/- 5.4%) groups (p < 0.01). Tissue polymorphonuclear cell numbers were reduced in both 1B6c-administered groups. Our data demonstrate that administration of anti-CD11b antibody results in a dose-dependent, significant functional improvement and reduction of ischemic cell damage after transient focal cerebral ischemia in the rat.

  9. A reappraisal of immunoglobulin variable gene primers and its impact on assessing clonal relationships between PB B cells and BM plasma cells in AL amyloidosis.

    Science.gov (United States)

    Katoh, Nagaaki; Poshusta, Tanya L; Manske, Michelle K; Dispenzieri, Angela; Gertz, Morie A; Abraham, Roshini S; Ramirez-Alvarado, Marina

    2011-12-01

    Monoclonal tumor plasma cells as well as non-terminally differentiated B cells having a clonal relationship to the tumor cells have been detected in the peripheral blood (PB) of some multiple myeloma (MM) patients but rarely in light chain (primary systemic) amyloidosis (AL) patients. Previously, our group found these peripheral clonotypic B cells in three AL patients. Here, we report detailed analysis of a larger cohort of AL patients to validate the prior findings and to investigate the effect of this cell population on clinical outcome. Fourteen AL patients were selected from a clinical prospective trial, and the relationship between immunoglobulin light chain variable gene (V(L)) representation in PB B cells and the clonal population in the bone marrow (BM) was investigated. A clonal relationship was not detected, and the present study provides important insights into the disparity with the earlier data, including clinical history of the patients and methodological analysis.

  10. A new monoclonal antibody against DNA ligase I is a suitable marker of cell proliferation in cultured cell and tissue section samples

    Directory of Open Access Journals (Sweden)

    B Vitolo

    2009-06-01

    Full Text Available The extensive characterization of the replicative human DNA ligase I (LigI undertaken in the last decade demonstrated that the level of this protein strongly correlates with the rate of cell proliferation. This may allow to expand the repertoire of clinical biomarkers for the analysis of cell proliferation.We have produced a new monoclonal antibody (5H5 against LigI and exploited it as cell proliferation marker in Western blotting and immunofluorescence as well as in immunohistochemistry on paraffin tissue sections. The Western blot analysis showed that the LigI level detected by 5H5 antibody is high in all proliferating cells. On the contrary the protein is down regulated in resting human fibroblast and peripheral blood lymphocytes. Immunofluorescence analysis on cultured HeLa cells showed that 5H5 antibody labels all proliferating cells and displays the same staining pattern of BrdU in S-phase nuclei. Finally the analysis of serial sections of inflamed tonsils and NHL lymph nodes (either frozen or paraffin embedded demonstrated that 5H5 marks the same population of cells as the Ki-67 antibody. Our results demonstrate that 5H5 antibody is a valuable tool for labeling proliferating cells that can be conveniently used in Western blotting, immunocytochemistry and immunohistochemistry.

  11. Activity of plasma membrane V-ATPases is critical for the invasion of MDA-MB231 breast cancer cells.

    Science.gov (United States)

    Cotter, Kristina; Capecci, Joseph; Sennoune, Souad; Huss, Markus; Maier, Martin; Martinez-Zaguilan, Raul; Forgac, Michael

    2015-02-06

    The vacuolar (H(+))-ATPases (V-ATPases) are a family of ATP-driven proton pumps that couple ATP hydrolysis with translocation of protons across membranes. Previous studies have implicated V-ATPases in cancer cell invasion. It has been proposed that V-ATPases participate in invasion by localizing to the plasma membrane and causing acidification of the extracellular space. To test this hypothesis, we utilized two separate approaches to specifically inhibit plasma membrane V-ATPases. First, we stably transfected highly invasive MDA-MB231 cells with a V5-tagged construct of the membrane-embedded c subunit of the V-ATPase, allowing for extracellular expression of the V5 epitope. We evaluated the effect of addition of a monoclonal antibody directed against the V5 epitope on both V-ATPase-mediated proton translocation across the plasma membrane and invasion using an in vitro Matrigel assay. The addition of anti-V5 antibody resulted in acidification of the cytosol and a decrease in V-ATPase-dependent proton flux across the plasma membrane in transfected but not control (untransfected) cells. These results demonstrate that the anti-V5 antibody inhibits activity of plasma membrane V-ATPases in transfected cells. Addition of the anti-V5 antibody also inhibited in vitro invasion of transfected (but not untransfected) cells. Second, we utilized a biotin-conjugated form of the specific V-ATPase inhibitor bafilomycin. When bound to streptavidin, this compound cannot cross the plasma membrane. Addition of this compound to MDA-MB231 cells also inhibited in vitro invasion. These studies suggest that plasma membrane V-ATPases play an important role in invasion of breast cancer cells.

  12. Photothermolysis mediated by gold nanorods modified with EGFR monoclonal antibody induces Hep-2 cells apoptosis in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Zhang S

    2014-04-01

    Full Text Available Shiwen Zhang,1,2,* Yunlong Li,3,4,* Xiaoguang He,2 Shouan Dong,5 Yunchao Huang,6 Xiaojiang Li,1 Yuxiao Li,2 Congguo Jin,7 Yingying Zhang,8 Yuanling Wang91Department of Head and Neck, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province, Kunming, 2Department of Head and Neck, The First Affiliated Hospital of Kunming Medical University, Kunming, 3Medical Faculty, Kunming University of Science and Technology, Kunming, 4The First People's Hospital of Yunnan Province (The Affiliated Hospital of Kunming University of Science and Technology, Kunming, 5Kunming Institute of Precious Metals, Kunming, 6Department of cardiothoracic surgery, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province, Kunming, 7Institute of Oncology, The Third Affiliated Hospital of Kunming Medical University (Tumor Hospital of Yunnan Province, Kunming, 8Clinical skills training center of Kunming Medical University, Kunming, 9Department of Anesthesiology, Yan An Hospital, Kunming, Yunnan, The People's Republic of China *These authors contributed equally to this workAbstract: Gold nanorods (AuNRs have been used in plasmonic photothermal therapy (PPTT, which is thought to be more efficient and selective than conventional photothermal therapy. The efficiency and safety of PPTT can be improved by functionally modifying the gold nanorods with proteins or biomolecules. In this study, AuNRs were modified with anti-epidermal growth factor receptor (EGFR monoclonal antibody (mAb, and the apoptotic potential of EGFRmAb-AuNR was assessed in Hep-2 cells in vitro and in vivo. The EGFRmAb modification had no obvious influence on the original optical property of the AuNRs, but it significantly increased the entry of AuNRs into Hep-2 cells. EGFRmAb-AuNRs, with appropriate laser irradiation, resulted in higher Hep-2 cells apoptosis than AuNRs did alone, in vitro, and was accompanied by alteration of reactive oxygen

  13. [Demonstration of β-1,2 mannan structures expressed on the cell wall of Candida albicans yeast form but not on the hyphal form by using monoclonal antibodies].

    Science.gov (United States)

    Aydın, Cevahir; Ataoğlu, Haluk

    2015-01-01

    Candida albicans is a polymorphic fungus that may be observed as both commensal and opportunistic pathogen in humans. As one of the major components of Candida cell wall structure, mannan plays an important role in the fungus-host cell interaction and in virulence. The ability to switch from yeast to hypha form of microorganism is crutial in the development of C.albicans infections. Hyphal form has different antigenic properties compared to yeast form and structural changes occur in the yeast cell wall during transition from yeast to hypha form. Although there are several factors associated with this transition process, sufficient information is not available. The aim of this study was to investigate the change of configuration in mannan structure found in C.albicans cell wall by using monoclonal antibodies. C.albicans (NIHA 207) serotype A strains were used as test strains throughout the study, together with Salmonella choleraesuis 211 and Salmonella infantis as controls with similar cell wall structures to that of C.albicans. Cultures were maintained on YPD-agar medium by incubating at 28°C for yeast forms, and on YPD-broth medium in a shaking incubator at 37°C for 3-4 hours for the growth of hyphal forms. Cells were harvested in the exponential phase, and after being washed, the mannan content from C.albicans were extracted from pellet by heating in 20 mM sodium citrate buffer for 90 minutes at 125°C. Hybridoma technique was used for the production of monoclonal antibodies. After immunizing the Balb/C mice with antigen, the splenocytes were harvested and fusion was performed between spleen cells and F0 myeloma cells. The clones grown in HAT medium were screened for the presence of antibody producing hybrid cells by ELISA method. The antibody isotypes were determined by using a commercial kit (Pierce Biotechnology, ABD). The culture supernatants which contained monoclonal antibodies were collected and purified according to the ammonium sulphate method

  14. Responses of cells in plasma-activated medium

    Science.gov (United States)

    Tanaka, Hiromasa; Mizuno, Masaaki; Ishikawa, Kenji; Takeda, Keigo; Hashizume, Hiroshi; Nakamura, Kae; Kajiyama, Hiroaki; Kano, Hiroyuki; Okazaki, Yasumasa; Toyokuni, Shinya; Maruyama, Shoichi; Kodera, Yasuhiro; Terasaki, Hiroko; Adachi, Tetsuo; Kato, Masashi; Kikkawa, Fumitaka; Hori, Masaru

    2015-09-01

    Plasma consists of electrons, ions, radicals, and lights, and produces various reactive species in gas and liquid phase. Cells receive various inputs from their circumstances, and induce several physiological outputs. Our goal is to clarify the relationships between plasma inputs and physiological outputs. Plasma-activated medium (PAM) is a circumstance that plasma provides cells and our previous studies suggest that PAM is a promising tool for cancer therapy. However, the mode of actions remains to be elucidated. We propose survival and proliferation signaling networks as well as redox signaling networks are key factors to understand cellular responses of PAM-treated glioblastoma cells.

  15. Delayed effects of cold atmospheric plasma on vascular cells

    NARCIS (Netherlands)

    Stoffels, Eva; Roks, Anton J. M.; Deelmm, Leo E.

    2008-01-01

    We investigated the long-term behaviour of vascular cells (endothelial and smooth muscle) after exposure to a cold atmospheric plasma source. The cells were treated through a gas-permeable membrane, in order to simulate intravenous treatment with a gas plasma-filled catheter. Such indirect treatment

  16. Delayed effects of cold atmospheric plasma on vascular cells

    NARCIS (Netherlands)

    Stoffels, Eva; Roks, Anton J. M.; Deelmm, Leo E.

    2008-01-01

    We investigated the long-term behaviour of vascular cells (endothelial and smooth muscle) after exposure to a cold atmospheric plasma source. The cells were treated through a gas-permeable membrane, in order to simulate intravenous treatment with a gas plasma-filled catheter. Such indirect treatment

  17. Evolution of Externally Excited Convective Cells in Plasmas

    DEFF Research Database (Denmark)

    Sugai, H.; Juul Rasmussen, Jens; Thomsen, Kenneth

    1983-01-01

    Convective cells are excited externally in a fully ionized magnetized plasma, and their space-time evolution is investigated by two-dimensional potential measurements. A positive cell is excited externally in the `scrape-off' layer of a plasma column produced by surface ionization. Its interaction...

  18. Awakening acceleration: AWAKE’s plasma cell arrive

    CERN Multimedia

    CERN Audiovisual Productions Service; Paola Catapano; Jacques Fichet; Paola Catapano

    2016-01-01

    Interview with Edda Gschwendtner, project leader of the AWAKE collaboration, on the day the AWAKE's plasma cell is transported from the surface hall were it was tested (EHN1 on CERN Prevessin) to the AWAKE tunnel in CERN's SPS decay tunnel. The Plasma Cell was built by the Max Planck Institute in Munich.

  19. Bystander apoptosis in human cells mediated by irradiated blood plasma

    Energy Technology Data Exchange (ETDEWEB)

    Vinnikov, Volodymyr, E-mail: vlad.vinnikov@mail.ru [Grigoriev Institute for Medical Radiology of the National Academy of Medical Science of Ukraine (Ukraine); Lloyd, David; Finnon, Paul [Centre for Radiation, Chemical and Environmental Hazards of the Health Protection Agency of the United Kingdom (United Kingdom)

    2012-03-01

    Following exposure to high doses of ionizing radiation, due to an accident or during radiotherapy, bystander signalling poses a potential hazard to unirradiated cells and tissues. This process can be mediated by factors circulating in blood plasma. Thus, we assessed the ability of plasma taken from in vitro irradiated human blood to produce a direct cytotoxic effect, by inducing apoptosis in primary human peripheral blood mononuclear cells (PBM), which mainly comprised G{sub 0}-stage lymphocytes. Plasma was collected from healthy donors' blood irradiated in vitro to 0-40 Gy acute {gamma}-rays. Reporter PBM were separated from unirradiated blood with Histopaque and held in medium with the test plasma for 24 h at 37 Degree-Sign C. Additionally, plasma from in vitro irradiated and unirradiated blood was tested against PBM collected from blood given 4 Gy. Apoptosis in reporter PBM was measured by the Annexin V test using flow cytometry. Plasma collected from unirradiated and irradiated blood did not produce any apoptotic response above the control level in unirradiated reporter PBM. Surprisingly, plasma from irradiated blood caused a dose-dependent reduction of apoptosis in irradiated reporter PBM. The yields of radiation-induced cell death in irradiated reporter PBM (after subtracting the respective values in unirradiated reporter PBM) were 22.2 {+-} 1.8% in plasma-free cultures, 21.6 {+-} 1.1% in cultures treated with plasma from unirradiated blood, 20.2 {+-} 1.4% in cultures with plasma from blood given 2-4 Gy and 16.7 {+-} 3.2% in cultures with plasma from blood given 6-10 Gy. These results suggested that irradiated blood plasma did not cause a radiation-induced bystander cell-killing effect. Instead, a reduction of apoptosis in irradiated reporter cells cultured with irradiated blood plasma has implications concerning oncogenic risk from mutated cells surviving after high dose in vivo irradiation (e.g. radiotherapy) and requires further study.

  20. Nonmitogenic Anti-CD3 Monoclonal Antibodies Deliver a Partial T Cell Receptor Signal and Induce Clonal Anergy

    Science.gov (United States)

    Smith, Judith A.; Tso, J. Yun; Clark, Marcus R.; Cole, Michael S.; Bluestone, Jeffrey A.

    1997-01-01

    Anti-CD3 monoclonal antibodies (mAbs) are potent immunosuppressive agents used in clinical transplantation. However, the activation-related adverse side effects associated with these mAbs have prompted the development of less toxic nonmitogenic anti-CD3 mAb therapies. At present, the functional and biochemical consequences of T cell exposure to nonmitogenic anti-CD3 is unclear. In this study, we have examined the early signaling events triggered by a nonmitogenic anti-CD3 mAb. Like the mitogenic anti-CD3 mAb, nonmitogenic anti-CD3 triggered changes in the T cell receptor (TCR) complex, including ζ chain tyrosine phosphorylation and ZAP-70 association. However, unlike the mitogenic anti-CD3 stimulation, nonmitogenic anti-CD3 was ineffective at inducing the highly phosphorylated form of ζ (p23) and tyrosine phosphorylation of the associated ZAP-70 tyrosine kinase. This proximal signaling deficiency correlated with minimal phospholipase Cγ-1 phosphorylation and failure to mobilize detectable Ca2+. Not only did biochemical signals delivered by nonmitogenic anti-CD3 resemble altered peptide ligand signaling, but exposure of Th1 clones to nonmitogenic anti-CD3 also resulted in functional anergy. Finally, a bispecific anti-CD3 × anti-CD4 F(ab)′2 reconstituted early signal transduction events and induced proliferation, suggesting that defective association of lck with the TCR complex may underlie the observed signaling differences between the mitogenic and nonmitogenic anti-CD3. PMID:9126922

  1. Exploring the capabilities of fluorometric online monitoring on chinese hamster ovary cell cultivations producing a monoclonal antibody.

    Science.gov (United States)

    Schwab, Karen; Amann, Thomas; Schmid, Jakob; Handrick, René; Hesse, Friedemann

    2016-11-01

    Online monitoring of Chinese hamster ovary fed-batch cell cultures via two-dimensional fluorescence spectroscopy (2DFS) was evaluated in this work. Particular attention was directed toward different process strategies regarding the use of nutrient-rich feed media and temperature shifts. These intentionally performed process manipulations broadened the variances in the obtained fluorescence spectra and this was suspected to hamper the generation of reliable soft sensors. Principal component analysis of the obtained fluorescence data showed that temperature shift and feeding strategy had a considerable impact on the fluorescence signals. Partial least square regression models were calculated for the prediction of glucose, lactate, monoclonal antibody (mAb), and viable cell concentrations (VCC). It was aimed to integrate all 2DFS datasets in the respective calibration models regardless of the process-strategy-dependent diversity. Contrary to the expectations, it was feasible to calibrate soft sensors for the online prediction of glucose (7 latent variables (LVs), Rcal2 = 0.97, rout mean squared error of prediction (RMSEP) = 1.1 g L(-1) ), lactate (5 LV; Rcal2 = 0.96; RMSEP = 0.5 g L(-1) ) and mAb concentrations (4 LV; Rcal2 = 0.99; RMSEP = 11.4 mg L(-1) ). Feeding and temperature shifts had the highest impact on the VCC model (3 LV; Rcal2 = 0.94; RMSEP 3.8 × 10(5) mL(-1) ), nevertheless the prediction of VCC from the fed-batch 2DFS data was feasible. The results strongly indicate that variances in the datasets due to the process strategy can be tolerated to some extent by the respective soft sensors. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1592-1600, 2016.

  2. Characterization of a Novel Monoclonal Antibody to Human Stem Cell Factor and its Determination Effect on Ex Vivo Stem Cell Expansion

    Directory of Open Access Journals (Sweden)

    Fan Jie

    2013-06-01

    Full Text Available Background: Hematopoietic stem cell (HSC transplantation can be used to treat blood and immune system disorders. Fresh umbilical cord blood (UCB, a major source of HSC for potential clinical applications, contains a limited number of HSCs. Stem cell factor (SCF activates HSC self-renewal and is being used to stimulate ex vivo expansion of HSCs for treating various hematologic diseases in clinic. Yet, the mechanism by which SCF stimulates and supports HSCs expansion remains poorly understood. Thus, the purpose of the study is to obtain novel monoclonal antibodies for structural and functional SCF characterizations, as well as for the optimization of HSCs ex vivo expansion. Methods: Recombinant human stem cell factor (rhSCF was used for producing monoclonal antibody (mAb. High-titer mAb specific to rhSCF was selected by following immunochemical screening to various mAb cell lines. HSCs with CD34+ epitope were isolated from UCB using affinity chromatography. SCF activity was tested in an ex vivo HSC expansion assay, with use of flow cytometry for detection of CD34+ cell and total mononuclear cells. Part of rhSCF that contained the antibody-binding site was identified via immunoblot analysis of rhSCF tryptic peptides, rhSCF-specific mAb, and subsequent NH2-terminal amino acid sequence analysis of the detected peptides. Results: The mAb cell line 23C8 with a high titer was found to be specific for rhSCF. In ex vivo cord blood expansion assays, the ability of rhSCF to stimulate the expansion of CD34+ cells was significantly inhibited by 23C8 in a dose-dependet fashion(?. Through peptide mapping, the binding site of 23C8 on rhSCF was mapped to the first 104 amino acids.. Conclusion: The mAb cell line 23C8 produces specific and inhibitory anti-rhSCF mAb. The mAb appears to bind directly to a part of rhSCF that is critical for biological activity. This functionallyactive site of rhSCF is located in the first 104 amino acids from the NH2-terminus. The

  3. Characterization of a Novel Monoclonal Antibody to Human Stem Cell Factor and its Determination Effect on Ex Vivo Stem Cell Expansion

    Institute of Scientific and Technical Information of China (English)

    Jie Fan; Ding Xinxin; Jiang Yongping

    2013-01-01

    Background:Hematopoietic stem cell (HSC) transplantation can be used to treat blood and immune system disorders. Fresh umbilical cord blood (UCB), a major source of HSC for potential clinical applications, contains a limited number of HSCs. Stem cell factor (SCF) activates HSC self-renewal and is being used to stimulate ex vivo expansion of HSCs for treating various hematologic diseases in clinic. Yet, the mechanism by which SCF stimulates and supports HSCs expansion remains poorly understood. Thus, the purpose of the study is to obtain novel monoclonal antibodies for structural and functional SCF characterizations, as well as for the optimization of HSCs ex vivo expansion. Methods:Recombinant human stem cell factor (rhSCF) was used for producing monoclonal antibody (mAb). High-titer mAb speciifc to rhSCF was selected by following immunochemical screening to various mAb cell lines. HSCs with CD34+ epitope were isolated from UCB using affinity chromatography. SCF activity was tested in an ex vivo HSC expansion assay, with use of flow cytometry for detection of CD34+ cell and total mononuclear cells. Part of rhSCF that contained the antibody-binding site was identified via immunoblot analysis of rhSCF tryptic peptides, rhSCF-speciifc mAb, and subsequent NH2-terminal amino acid sequence analysis of the detected peptides. Results: The mAb cell line 23C8 with a high titer was found to be speciifc for rhSCF. In ex vivo cord blood expansion assays, the ability of rhSCF to stimulate the expansion of CD34+ cells was significantly inhibited by 23C8 in a dose-dependet fashion(?). Through peptide mapping, the binding site of 23C8 on rhSCF was mapped to the ifrst 104 amino acids.. Conclusion: The mAb cell line 23C8 produces speciifc and inhibitory anti-rhSCF mAb. The mAb appears to bind directly to a part of rhSCF that is critical for biological activity. This functionally active site of rhSCF is located in the ifrst 104 amino acids from the NH2-terminus. The novel anti

  4. Plasma pharmacokinetics and biological activity of a human immunodeficiency virus type 1 neutralizing human monoclonal antibody, F105, in cynomolgus monkeys.

    Science.gov (United States)

    Cavacini, L A; Power, J; Emes, C L; Mace, K; Treacy, G; Posner, M R

    1994-05-01

    The IgG1 kappa human monoclonal antibody (HMab), F105 reacts with a discontinuous epitope on the CD4 binding site (CD4BS) of human immunodeficiency virus type 1 (HIV-1)/gp120 and has broad neutralizing activity. F105 HMab (60 mg/kg bolus) was administered intravenously to four monkeys and serum was collected at intervals to determine pharmacokinetics in a primate model. Average serum F105 concentrations, as determined by enzyme-linked immunosorbent assay, were analyzed with MINSQ software using a two-compartment, first-order model. The half-life for the alpha phase of the distribution curve is 6.7 h and for the beta elimination phase, 9.6 days. The volume of distribution is 0.65 L/kg and the rate of clearance 2 ml/kg/h. Serum levels of 1.3-1.6 mg/ml of F105 were maintained for 24 h. When monkey serum from day 15 postdose was tested, total serum F105 was 230 +/- 79 micrograms/ml and was immunoreactive with cells infected with the MN and IIIB strains of HIV-1 as determined by flow cytometry. Binding activity was identical to that obtained with stock F105 HMab. Identical neutralizing activity between the injected and uninjected antibody was also observed. Thus, serum neutralizing titers (90%) of 1:2000 at peak and 1:30 at day 15 postdose for MN virus were observed. These data indicate that high in vivo levels of HMab F105 can be attained by single bolus administration with full retention of biological activity. Of importance, levels of antibody necessary for effective neutralization can be achieved and maintained.

  5. A novel monoclonal anti-CD81 antibody produced by genetic immunization efficiently inhibits Hepatitis C virus cell-cell transmission.

    Directory of Open Access Journals (Sweden)

    Isabel Fofana

    Full Text Available BACKGROUND AND AIMS: Hepatitis C virus (HCV infection is a challenge to prevent and treat because of the rapid development of drug resistance and escape. Viral entry is required for initiation, spread, and maintenance of infection, making it an attractive target for antiviral strategies. METHODS: Using genetic immunization, we produced four monoclonal antibodies (mAbs against the HCV host entry factor CD81. The effects of antibodies on inhibition of HCV infection and dissemination were analyzed in HCV permissive human liver cell lines. RESULTS: The anti-CD81 mAbs efficiently inhibited infection by HCV of different genotypes as well as a HCV escape variant selected during liver transplantation and re-infecting the liver graft. Kinetic studies indicated that anti-CD81 mAbs target a post-binding step during HCV entry. In addition to inhibiting cell-free HCV infection, one antibody was also able to block neutralizing antibody-resistant HCV cell-cell transmission and viral dissemination without displaying any detectable toxicity. CONCLUSION: A novel anti-CD81 mAb generated by genetic immunization efficiently blocks HCV spread and dissemination. This antibody will be useful to further unravel the role of virus-host interactions during HCV entry and cell-cell transmission. Furthermore, this antibody may be of interest for the development of antivirals for prevention and treatment of HCV infection.

  6. Natural killer (NK): dendritic cell (DC) cross talk induced by therapeutic monoclonal antibody triggers tumor antigen-specific T cell immunity.

    Science.gov (United States)

    Lee, Steve C; Srivastava, Raghvendra M; López-Albaitero, Andrés; Ferrone, Soldano; Ferris, Robert L

    2011-08-01

    Tumor antigen (TA)-targeted monoclonal antibodies (mAb), trastuzumab, cetuximab, panitumumab, and rituximab, have been among the most successful new therapies in the present generation. Clinical activity is observed as a single agent, or in combination with radiotherapy or chemotherapy, against metastatic colorectal cancer, head and neck cancer, breast cancer, and follicular lymphoma. However, the activity is seen only in a minority of patients. Thus, an intense need exists to define the mechanism of action of these immunoactive mAb. Here, we discuss some of the likely immunological events that occur in treated patients: antibody-dependent cellular cytotoxicity (ADCC), cross talk among immune cells including NK cells and dendritic cells (DCs), and generation of TA-specific T lymphocyte responses. We present evidence supporting the induction of "NK:DC cross talk," leading to priming of TA-specific cellular immunity. These observations show that mAb-mediated NK cell activation can be greatly enhanced by the action of stimulatory cytokines and surface molecules on maturing DC and that NK:DC interaction facilitates the recruitment of both NK cells and DC to the tumor site(s). The cooperative, reciprocal stimulatory activity of both NK cells and DC can modulate both the innate immune response in the local tumor microenvironment and the adaptive immune response in secondary lymphoid organs. These events likely contribute to clinical activity, as well as provide a potential biomarker of response to mAb therapy.

  7. Development of novel monoclonal antibodies that define differentiation stages of human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Andersen, Ditte Caroline; Kortesidis, Angela; Zannettino, Andrew C W;

    2011-01-01

    fewer differentiated alkaline phosphatase(+) cells compared to STRO-1(+/-)/Collagen VI(+) hMSC, suggesting that Collagen VI on the cell membrane exclusively defines differentiated MSCs. In conclusion, we have generated a panel of high quality antibodies to be used for characterization of MSCs...... of clonogenic hMSC from BMMNCs as single reagents. Using mass-spectrometric analysis, we identified the antigen recognised by DJ3 as CD44, whereas DJ9 and DJ18 recognized HLA-DRB1 and Collagen VI, respectively. The identified proteins were highly expressed throughout in vitro osteogenic- and adipogenic...... mice with hMSC, and by using a panel of subsequent screening methods. Flow cytometry analysis revealed that 83.5, 1.1, and 8.5% of primary cultures of hMSC were double positive for STRO-1 and either of DJ 3, 9, and 18, respectively. However, none of the three DJ antibodies allowed enrichment...

  8. Strategies for enhancing monoclonal antibody accumulation in plant cell and organ cultures.

    Science.gov (United States)

    Sharp, J M; Doran, P M

    2001-01-01

    Various strategies aimed at improving IgG(1) antibody accumulation in transgenic tobacco cell and organ cultures were tested. The form of tissue had a significant effect on antibody levels; shooty teratomas were less productive than hairy roots or suspended cells. Although there were several disadvantages associated with hairy roots compared with suspensions, such as slower growth, slower antibody production, and formation of a greater number of antibody fragments, the roots exhibited superior long-term culture stability. Antibody accumulation in hairy root cultures was improved by increasing the dissolved oxygen tension to 150% air saturation, indicating the need for effective oxygen transfer in root reactors used for antibody production. Preventing N-linked glycosylation using tunicamycin or inhibition of subsequent glycan processing by castanospermine reduced antibody accumulation in the biomass and/or medium in cell suspensions. Loss of antibody from the cultures after its secretion and release into the medium was identified as a major problem. This effect was minimized by inhibiting protein transport in the secretory pathway using Brefeldin A, resulting in antibody accumulation levels up to 2.7 times those in untreated cells. Strategies for protecting secreted antibody, such as addition of poly(vinylpyrrolidone) and periodic harvesting from the medium using hydroxyapatite resin, also increased antibody titers. The mechanisms responsible for the disappearance of antibody from plant culture media were not clearly identified; degradation by proteases and conformational modification of the antibody, such as formation of aggregates, provided an explanation for some but not all the phenomena observed. This work demonstrates that the manipulation and control of culture conditions and metabolic processes in plant tissue cultures can be used to improve the production of foreign proteins. However, loss of secreted antibody from plant culture medium is a significant

  9. Characterization of a Dopaminergic Stimulatory Factor Derived from Monoclonal Cell Lines of Striatal Origin

    Science.gov (United States)

    2006-12-01

    clonal pheochromocytoma cells on depolarization-dependent exocytosis. J. Biol. Chem. 257 (1982) 3491-3500. Winkler, H. and Fischer-Colbrie, R. The...Lisa Wona, Nancy Bubulaa, Suzanne Hesseforta, Martin Grossb, Alfred Hellera,* aDepartment of Neurobiology, Pharmacology and Physiology , The University of...Reddyc, Martin Grossd a Department of Neurobiology, Pharmacology and Physiology , The University of Chicago, 947 East 58th Street, Chicago, IL 60637

  10. Plasma-cell-predominant B-cell pseudolymphoma.

    Science.gov (United States)

    Nervi, Stephen J; Schwartz, R A

    2008-10-15

    A 46-year-old woman with no history of foreign travel presented to the New Jersey Medical School Dermatology Clinic in July, 2007, with pruritic ulcerating facial masses that had been present since October, 2006. Clinical and histopathologic findings were most consistent with a diagnosis of cutaneous plasma cell predominant B cell pseudolymphoma. An extensive search using special stains for an etiologic organism was negative. The term cutaneous pseudolymphoma has been coined to describe the accumulation of either T or B cell lymphocytes in the skin that is caused by a nonmalignant stimulus and encompasses several different terms depending on etiology. In cases of cutaneous pseudolymphoma where a cause is identified, treatment entails removing the underlying causative agent. Idiopathic cases tend to be recalcitrant to treatment.

  11. Generation and tumor recognition properties of two human monoclonal antibodies specific to cell surface anionic phospholipids.

    Science.gov (United States)

    Bujak, Emil; Pretto, Francesca; Neri, Dario

    2015-08-01

    Phosphatidylserine (PS) and other anionic phospholipids, which become exposed on the surface of proliferating endothelial cells, tumor cells and certain leukocytes, have been used as targets for the development of clinical-stage biopharmaceuticals. One of these products (bavituximab) is currently being investigated in Phase 3 clinical trials. There are conflicting reports on the ability of bavituximab and other antibodies to recognize PS directly or through beta-2 glycoprotein 1, a serum protein that is not highly conserved across species. Here, we report on the generation and characterization of two fully human antibodies directed against phosphatidylserine. One of these antibodies (PS72) bound specifically to phosphatidylserine and to phosphatidic acid, but did not recognize other closely related phospholipids, while the other antibody (PS41) also bound to cardiolipin. Both PS72 and PS41 stained 8/9 experimental tumor models in vitro, but both antibodies failed to exhibit a preferential tumor accumulation in vivo, as revealed by quantitative biodistribution analysis. Our findings indicate that anionic phospholipids are exposed and accessible in most tumor types, but cast doubts about the possibility of efficiently targeting tumors in vivo with PS-specific reagents.

  12. Differential effects of lenalidomide during plasma cell differentiation

    Science.gov (United States)

    Jourdan, Michel; Cren, Maïlys; Schafer, Peter; Robert, Nicolas; Duperray, Christophe; Vincent, Laure; Ceballos, Patrice; Cartron, Guillaume; Rossi, Jean-François; Moreaux, Jérôme; Chopra, Rajesh; Klein, Bernard

    2016-01-01

    Thalidomide, lenalidomide and pomalidomide have greatly improved the outcome of patients with multiple myeloma. However, their effects on plasma cells, the healthy counterpart of myeloma cells, are unknown. Here, we investigated lenalidomide effects on normal human plasma cell generation using an in vitro model. Lenalidomide inhibited the generation of pre-plasmablasts and early plasma cells, while it moderately affected plasmablast production. It also reduced the expression level of Ikaros, Aiolos, and IRF4 transcription factors, in plasmablasts and early plasma cells. This suggests that their differential sensitivity to lenalidomide is not due to a difference in Ikaros or Aiolos degradation. Lenalidomide also inhibited long-lived plasma cell generation, but did not impair their long-term survival once generated. This last observation is in agreement with the finding that lenalidomide treatment for 3-18 months did not affect the bone marrow healthy plasma cell count in allografted patients with multiple myeloma. Our findings should prompt to investigate whether lenalidomide resistance in patients with multiple myeloma could be associated with the emergence of malignant plasmablasts or long-lived plasma cells that are less sensitive to lenalidomide. PMID:27057635

  13. Immunophenotype of normal and myelomatous plasma cell subsets

    Directory of Open Access Journals (Sweden)

    Nelly eRobillard

    2014-03-01

    Full Text Available Plasma-cells are essentially characterized by the co-expression of CD138 and CD38, which allows their identification in flow cytometry in bone marrow, peripheral blood or cell suspensions from tissues. These terminally differentiated B-cells may lose the expression of surface CD19 and that of CD20 while retaining CD27. When malignant, they can gain a number of other markers such as CD28, CD33, CD56 or CD117 and lose CD27. Moreover, since each plasma cell is only able to produce a single type of immunoglobulins, they display isotypic restriction and clonal malignant plasma cells can be further characterized by their homogeneous expression of either kappa or lambda light chains. In multiple myeloma (MM, such plasma cell clones produce the immunoglobulin identified in plasma as an abnormal peak. In the bone marrow where they essentially accumulate, these plasma cells may however display various immunophenotypes. The latter were explored in a two-way approach. Firstly the various subsets delineated by the selective or common expression of CD19 together with combined CD56/CD28 were explored in normal and MM bone marrow. Then other aberrant markers’ expression was investigated, i.e. CD20, CD27, CD33, CD56, CD117. These data were compared to literature information. They underline the vast heterogeneity of MM plasma cells possibly accounting for the various answers to therapy of MM patients.

  14. Differential effects of lenalidomide during plasma cell differentiation.

    Science.gov (United States)

    Jourdan, Michel; Cren, Maïlys; Schafer, Peter; Robert, Nicolas; Duperray, Christophe; Vincent, Laure; Ceballos, Patrice; Cartron, Guillaume; Rossi, Jean-François; Moreaux, Jérôme; Chopra, Rajesh; Klein, Bernard

    2016-05-10

    Thalidomide, lenalidomide and pomalidomide have greatly improved the outcome of patients with multiple myeloma. However, their effects on plasma cells, the healthy counterpart of myeloma cells, are unknown. Here, we investigated lenalidomide effects on normal human plasma cell generation using an in vitro model. Lenalidomide inhibited the generation of pre-plasmablasts and early plasma cells, while it moderately affected plasmablast production. It also reduced the expression level of Ikaros, Aiolos, and IRF4 transcription factors, in plasmablasts and early plasma cells. This suggests that their differential sensitivity to lenalidomide is not due to a difference in Ikaros or Aiolos degradation. Lenalidomide also inhibited long-lived plasma cell generation, but did not impair their long-term survival once generated. This last observation is in agreement with the finding that lenalidomide treatment for 3-18 months did not affect the bone marrow healthy plasma cell count in allografted patients with multiple myeloma. Our findings should prompt to investigate whether lenalidomide resistance in patients with multiple myeloma could be associated with the emergence of malignant plasmablasts or long-lived plasma cells that are less sensitive to lenalidomide.

  15. Imaging of non-small cell lung cancers with a monoclonal antibody, KC-4G3, which recognizes a human milk fat globule antigen

    Energy Technology Data Exchange (ETDEWEB)

    Dienhart, D.G.; Schmelter, R.F.; Lear, J.L.; Miller, G.J.; Glenn, S.D.; Bloedow, D.C.; Kasliwal, R.; Moran, P.; Seligman, P.; Murphy, J.R. (Univ. of Colorado Cancer Center, Denver (USA))

    1990-11-01

    To determine the role of lung cancer tumor imaging with monoclonal antibodies directed against high molecular weight human milk fat globule antigens, we administered i.v. 111In-KC-4G3 to 24 patients with advanced non-small cell lung cancer. One mg of 111In-KC-4G3 was mixed with 0, 9, 49, 99, or 499 mg of unlabeled KC-4G3 and infused i.v. over 1 to 5 h. The mean 111In-KC-4G3 radiochemical purity was greater than 97% and the resultant immunoreactivity averaged 62%. Successful imaging of cancer sites was accomplished in 92% of 24 patients, and 57% of 91 total lesions were visualized. Successful localization of tumor sites related to size (P less than 0.001), with 81% of lesions greater than 3.0 cm in diameter, 50% of lesions 1.5 to 3 cm, and 6% of lesions less than 1.5 cm successfully imaging, and to location (P less than 0.05), with 69% of pulmonary lesions, 80% of soft tissue lesions, and only 32% of bone metastases being visualized. Nonspecific reticulo-endothelial uptake of radioactivity was a major problem. Approximately 35% of 111In was chelated to serum transferrin by 24 and 48 h after infusion. The mean t 1/2 beta for plasma radioisotope and immunoreactive KC-4G3 was 29 and 27 h, respectively. There was no correlation between total infused antibody dose and imaging success or between total dose and effect on 111In and KC-4G3 kinetics. Circulating free KC-4 antigen was measurable in all but one patient before study. Tumor biopsy following infusion could demonstrate antibody presence but not saturable antigen binding. We conclude that (a) 111In-KC-4G3 demonstrates successful tumor localization in non-small cell lung cancers bearing generally high expression of its antigen and (b) further investigations to diminish nonspecific radioactivity for imaging and utilization of high dose radiolabeled antibody for therapeutic intent are warranted.

  16. Development of plasma apparatus for plasma irradiation to living cell model

    Science.gov (United States)

    Suda, Yoshiyuki; Kato, Ryo; Tanoue, Hideto; Takikawa, Hirofumi; Tero, Ryugo

    2012-10-01

    Atmospheric pressure plasma has been studied for the industrial applications of biotechnology and medical care. For the development of these fields, understanding the influence of atmospheric pressure plasma on living cell and the mechanism of cell death is necessary. We focus on a basic structure of cell membrane, called lipid bilayer. Lipid bilayer is composed of lipid molecules with an amphipathic property and can be formed on hydrophilic substrates. In this paper, we report the development of the plasma apparatus for the treatment of lipid bilayer. The plasma apparatus uses a typical dielectric barrier discharge (DBD) system and employs parallel plate electrodes with a gap distance of 1 mm [1]. Each electrode is covered with a quartz plate and the substrate temperature is kept constant by cooling medium. The lower quartz electrode has a dimple, in which the substrate coated with a lipid bilayer and buffer fluid are mounted. [4pt] [1] Y. Sugioka, et al, IEEE Trans. Plasma Sci., in press

  17. Enhanced process understanding and multivariate prediction of the relationship between cell culture process and monoclonal antibody quality.

    Science.gov (United States)

    Sokolov, Michael; Ritscher, Jonathan; MacKinnon, Nicola; Souquet, Jonathan; Broly, Hervé; Morbidelli, Massimo; Butté, Alessandro

    2017-05-27

    This work investigates the insights and understanding which can be deduced from predictive process models for the product quality of a monoclonal antibody based on designed high-throughput cell culture experiments performed at milliliter (ambr-15(®) ) scale. The investigated process conditions include various media supplements as well as pH and temperature shifts applied during the process. First, principal component analysis (PCA) is used to show the strong correlation characteristics among the product quality attributes including aggregates, fragments, charge variants, and glycans. Then, partial least square regression (PLS1 and PLS2) is applied to predict the product quality variables based on process information (one by one or simultaneously). The comparison of those two modeling techniques shows that a single (PLS2) model is capable of revealing the interrelationship of the process characteristics to the large set product quality variables. In order to show the dynamic evolution of the process predictability separate models are defined at different time points showing that several product quality attributes are mainly driven by the media composition and, hence, can be decently predicted from early on in the process, while others are strongly affected by process parameter changes during the process. Finally, by coupling the PLS2 models with a genetic algorithm first the model performance can be further improved and, most importantly, the interpretation of the large-dimensioned process-product-interrelationship can be significantly simplified. The generally applicable toolset presented in this case study provides a solid basis for decision making and process optimization throughout process development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

  18. Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay.

    Science.gov (United States)

    Takeda, Hiroyuki; Ogasawara, Tomio; Ozawa, Tatsuhiko; Muraguchi, Atsushi; Jih, Pei-Ju; Morishita, Ryo; Uchigashima, Motokazu; Watanabe, Masahiko; Fujimoto, Toyoshi; Iwasaki, Takahiro; Endo, Yaeta; Sawasaki, Tatsuya

    2015-06-10

    G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.

  19. Characterization of a proapoptotic antiganglioside GM2 monoclonal antibody and evaluation of its therapeutic effect on melanoma and small cell lung carcinoma xenografts.

    Science.gov (United States)

    Retter, Marc W; Johnson, Jeffrey C; Peckham, David W; Bannink, Jeannette E; Bangur, Chaitanya S; Dresser, Karen; Cai, Feng; Foy, Teresa M; Fanger, Neil A; Fanger, Gary R; Woda, Bruce; Rock, Kenneth L

    2005-07-15

    Monoclonal antibodies have begun to show great clinical promise for the treatment of cancer. Antibodies that can directly affect a tumor cell's growth and/or survival are of particular interest for immunotherapy. Previously, we described monoclonal antibody DMF10.62.3 that had antiproliferative and proapoptotic effects when it bound an antigen of unknown identity on tumor cells in vitro. In this report, we determined that DMF10.62.3 and a clonally related antibody DMF10.167.4 recognize the ganglioside GM2. These antibodies react with a GM2 epitope that is expressed on a large number of tumor cell lines, including human melanoma and small cell lung carcinoma, but not on normal primary lines or most normal tissues. Interestingly, this pattern of cellular reactivity is distinct from that reported for other previously described GM2 antibodies, a difference that is presumably due to DMF10.167.4's binding to a unique GM2-associated epitope. Additional characterization of DMF10.167.4 revealed that this antibody was able to induce apoptosis and/or block cellular proliferation when cultured in vitro with the human Jurkat T lymphoma, CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines. In vivo, DMF10.167.4 antibody was well tolerated in mice and did not detectably bind to or damage normal tissues. However, this antibody was able to prevent murine E710.2.3 lymphoma, human CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines from establishing tumors in vivo and blocked progression of established CHL-1 and SBC-3 tumors in vivo. Therefore, monoclonal antibody DMF10.167.4 has immunotherapeutic potential.

  20. The contribution of cell surface FcRn in monoclonal antibody serum uptake from the intestine in suckling rat pups

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    Philip R Cooper

    2014-10-01

    Full Text Available The neonatal Fc Receptor (FcRn in intestinal epithelium is the primary mechanism for transfer of maternal immunoglobulin G (IgG from suckled milk to serum; but the factors contributing to the rapid uptake of IgG are poorly understood. These studies help to determine the contribution of cell-surface FcRn in IgG uptake in 2-week old rat pups by varying local pH and binding conditions. Variants of a human wild-type IgG monoclonal antibody (mAb WT were assessed for binding affinity (KD to rat (rFcRn at pH6.0 and subsequent off-rate at pH7.4 (1/s by Surface Plasmon Resonance. Selected mAbs were administered intra-intestinally in isofluorane-anesthetized 2-week rat pups. Full-length mAb in serum was quantified by immunoassay, (rFcRn mRNA expression by RT-PCR, and mAb epithelial localization was visualized by immunohistochemistry. After duodenal administration, serum levels of mAb variants correlated with their rFcRn off-rate at pH7.4, but not their affinity at pH6.0. The greatest serum levels of IgG were measured when mAb was administered in the duodenum where rFcRn mRNA expression is greatest, and was increased further by duodenal administration in pH6.0 buffer. More intense human IgG immunostaining was detected in epithelium than the same variant administered at higher pH. These data suggest an increased contribution for cell-surface receptor. We conclude that, in the neonate duodenum, receptor off-rates are as important as affinities for FcRn mediated uptake, and cell surface binding of IgG to rFcRn plays contributes to IgG uptake alongside pinocytosis; both of which responsible for increased IgG uptake.

  1. Safety, biodistribution, pharmacokinetics, and immunogenicity of 99mTc-labeled humanized monoclonal antibody BIWA 4 (bivatuzumab) in patients with squamous cell carcinoma of the head and neck.

    Science.gov (United States)

    Colnot, David R; Roos, Jan C; de Bree, Remco; Wilhelm, Abraham J; Kummer, J Alain; Hanft, Gertraud; Heider, Karl-Heinz; Stehle, Gerd; Snow, Gordon B; van Dongen, Guus A M S

    2003-09-01

    Previous studies have shown the potential of murine and chimeric anti-CD44v6 monoclonal antibodies (MAbs) for radioimmunotherapy (RIT) of head and neck squamous cell carcinoma (HNSCC). A limitation of these MAbs, however, appeared to be their immunogenicity. Therefore, humanized monoclonal antibody BIWA 4 (bivatuzumab), with an intermediate affinity for CD44v6, was recently selected. As a prelude to RIT, we evaluated the safety, tumor-targeting potential, pharmacokinetics, and immunogenicity of technetium-99m-labeled BIWA 4 in patients undergoing operations for primary HNSCC in this study. Ten patients were treated at BIWA 4 dose levels of 25 mg (n=3), 50 mg (n=4), and 100 mg (n=3). Patients received 2 mg of 750 MBq 99mTc-BIWA 4, together with 23-, 48-, and 98-mg unlabeled BIWA 4, respectively. Radioimmunoscintigraphy (RIS) was performed within 1 h and after 21 h, and patients underwent surgery at 48 h after injection. Biodistribution of 99mTc-BIWA 4 was evaluated by radioactivity measurements in blood, bone marrow, and in biopsies of a surgical specimen obtained 48 h after injection. BIWA 4 concentration in blood was assessed by ELISA and high performance liquid chromatography and related to soluble CD44v6 levels in serum samples. The development of human anti-human antibody (HAHA) responses was determined. Administration of 99mTc-BIWA 4 was well tolerated by all patients and no HAHA responses were observed. A mean t1/2 in plasma of 54.8 +/- 11.5 h, 76.1 +/- 21.8 h, and 68.5 +/- 21.2 h was found for the 25-, 50-, and 100-mg dose group, respectively. No complex formation of BIWA 4 with soluble CD44v6 in blood was observed. RIS showed targeting of primary tumors and lymph node metastases in 8 of 10 and 1 of 5 patients, respectively. The highest tumor uptake and tumor to nontumor ratios were observed for the 50-mg dose group. Tumor uptake was 12.9 +/- 5.9, 26.2 +/- 3.1, and 15.4 +/- 1.9% of the injected dose (ID)/kg for the 25-, 50-, and 100-mg dose group

  2. Rheumatic masks of plasma cell dyscrasias

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    Vladimir Ivanovich Vasilyev

    2012-01-01

    Full Text Available Objective: to consider clinical practice problems in the differential diagnosis of different types of plasma cell dyscrasias (PCD. Subjects and methods. Fourteen patients (8 men and 6 women aged 52±12 years, in whom rheumatic diseases (RD were ruled out and who were diagnosed as having primary PCD: different types of myeloma in 7 patients, myeloma + AL-amyloidosis in 2, AL-amyloidosis in 3, and Waldenstrom’s macroglobulinemia in 2, were examined. Results and discussion. The most common maldiagnosed RDs in patients with PCD were seronegative rheumatoid arthritis (RA, systemic lupus erythematosus, Sjogren’s disease, and different forms of vasculitis. The most frequent masks of RD were kidney (78% and osteoarticular system (64% lesions, vascular disorders (36%, peripheral polyneuropathies (36%, and enlarged salivary glands with xerostomia (28.5%. Serum and urine immunochemical study should be performed in all patients who have clinical manifestations of seropositive RA, spondyloarthritis, intensive bone pain syndrome, ulceronecrotic vasculitis, enlarged submandibular salivary glands with macroglossia in the absence of markers of autoimmune disease for the timely diagnosis of PCD and the exclusion of RD. The paper estimates the sensitivity and specificity of main methods used to diagnose different types of PCD.

  3. Multistage aqueous two-phase extraction of a monoclonal antibody from cell supernatant.

    Science.gov (United States)

    Muendges, Jan; Zalesko, Alexej; Górak, Andrzej; Zeiner, Tim

    2015-01-01

    This article presents results of continuous multistage aqueous two-phase extraction of an immunoglobulin G1 from cell supernatant in a mixer-settler unit. An aqueous two-phase system consisting of polyethylene glycol 2000, phosphate salt, and water was applied without and with sodium chloride (NaCl). Influences of different parameters such as throughput, phase ratio, and stage number on the extraction performance were analyzed. For systems without NaCl, the extraction was carried out as a washing step. An increase of stage number from one to five stages enabled to increase the immunoglobulin G1 purity from 11.8 to 32.6% at a yield of nearly 90%. Furthermore, a reduction of product phase volume due to a higher phase ratio led to an increase of purity from 20.8 to 29.6% in a three-stage countercurrent extraction. For experiments with NaCl moderate partitioning conditions were adjusted by adding 8 wt% NaCl. In that case, the extraction was carried out as a stripping step.

  4. In-vitro inhibition of IFNγ+ iTreg mediated by monoclonal antibodies against cell surface determinants essential for iTreg function

    Directory of Open Access Journals (Sweden)

    Daniel Volker

    2012-08-01

    Full Text Available Abstract Background IFNγ-producing CD4+CD25+Foxp3+ PBL represent a subtype of iTreg that are associated with good long-term graft outcome in renal transplant recipients and suppress alloresponses in-vitro. To study the mechanism of immunosuppression, we attempted to block cell surface receptors and thereby inhibited the function of this iTreg subset in-vitro using monoclonal antibodies and recombinant proteins. Methods PBL of healthy control individuals were stimulated polyclonally in-vitro in the presence of monoclonal antibodies or recombinant proteins against/of CD178, CD152, CD279, CD28, CD95, and HLA-DR. Induction of IFNγ+ iTreg and proliferation of effector cells was determined using four-color fluorescence flow cytometry. Blockade of iTreg function was analyzed using polyclonally stimulated co-cultures with separated CD4+CD25+CD127-IFNγ+ PBL. Results High monoclonal antibody concentrations inhibited the induction of CD4+CD25+Foxp3+IFNγ+ PBL (anti-CD152, anti-CD279, anti-CD95: p +CD25+CD127-IFNγ+ PBL (anti-CD178, anti-CD152, anti-CD279, anti-CD95: p +CD25+Foxp3+IFNγ+ PBL (rCD152 and rCD95: p +CD25+CD127-IFNγ+ PBL showed lower cell proliferation than co-cultures with CD4+CD25+CD127-IFNγ- PBL (p +CD25+CD127-IFNγ- PBL-containing co-cultures in the presence of monoclonal antibody (anti-CD28, anti-CD152, anti-CD279: p +CD25+CD127-IFNγ+ PBL (with the exception anti-CD28 monoclonal antibody: p +CD25+CD127-IFNγ- PBL but do not efficiently block suppressive iTreg function in co-cultures with CD4+CD25+CD127-IFNγ+ PBL. Conclusions CD178, CD152, CD279, CD28, CD95, and HLA-DR determinants are important for induction and suppressive function of IFNγ+ iTreg.

  5. Preparation of HIV monoclonal antibody-conjugated pulchellin in order to study its intracellular trafficking pathway in HIV-infected cells by confocal microscopy

    Science.gov (United States)

    Sadraeian, M.; Tsutae, F. M.; Moreira, H. H. T.; Araujo, A. P. U.; Guimarães, F. E. G.; Pincus, S. H.

    2015-06-01

    Pulchellin is a type 2 of ribosome-inactivating proteins isolated from some seeds significantly growing in Brazil. It is a potent agent to inhibit the protein synthesis in cancer cells and also HIV-infected cells. Pulchellin can be conjugated to HIV monoclonal antibodies to specifically target the HIV-infected cells. To analyze the protein synthesis inhibition by Pulchellin, the intracellular localization of the immunoconjugate should be compared to Pulchellin. In this case, the intracellular trafficking of this protein in cells can be determined by confocal microscopy. In our study, we utilized Pulchellin to construct HIV monoclonal antibody-conjugated Pulchellin A chain in order to target HIV-infected lymphocyte cells. Afterward the conjugation was labeled with the superior Alexa Fluor 488 dye. As a subsequent step, we are interested in studying the intracellular trafficking pathway of this novel conjugation in HIV-infected cells by confocal microscopy. Moreover, possible quantitative methods for fluorescent labeling of the immunoconjugate during confocal microscopy will be investigated.

  6. The impact of cell adaptation to serum-free conditions on the glycosylation profile of a monoclonal antibody produced by Chinese hamster ovary cells.

    Science.gov (United States)

    Costa, Ana Rita; Withers, Joanne; Rodrigues, Maria Elisa; McLoughlin, Niaobh; Henriques, Mariana; Oliveira, Rosário; Rudd, Pauline M; Azeredo, Joana

    2013-06-25

    N-glycosylation is one of the most crucial parameters affecting the biological activity of therapeutic monoclonal antibodies (mAbs), and should therefore be closely monitored and controlled to guarantee a consistent and high-quality product in biopharmaceutical processes. In the present work, the effect of the time-consuming step of gradual cell adaptation to serum-free conditions on the glycosylation profile of a mAb produced by CHO-K1 cells was evaluated. High-performance liquid chromatography analysis revealed important changes in mAb glycosylation patterns in all steps of serum reduction. These changes could be grouped in two distinct phases of the process of adaptation: middle (2.5 to 0.15% serum) and final (0.075 and 0% serum). For intermediate levels of serum, a desirable increase of galactosylation and decrease of fucosylation, but an undesirable increase in sialylation were observed; while the inverse was obtained at the final stages of adaptation. These divergences may be related to the reduction of serum supplementation, to variations in the levels of cell density and viability achieved at these stages, and to the natural shift of the cell growth mode during adaptation from adherent to suspended. The divergent glycan profiles obtained in this study demonstrate a strong influence of the adaptation process on mAb glycosylation, suggesting that control and monitoring of product quality should be implemented at the early stages of process development.

  7. Expression of blood group I and i active carbohydrate sequences on cultured human and animal cell lines assessed by radioimmunoassays with monoclonal cold agglutinins

    Energy Technology Data Exchange (ETDEWEB)

    Childs, R.A.; Kapadia, A.; Feizi, T. (Clinical Research Centre, Harrow (UK))

    1980-05-01

    Human monoclonal anti-I und anti-i antibodies, reactive with known carbohydrate sequences, have been used as reagents to quantitate (by radioimmunoassay) and visualize (by immunofluorscence) the expression of the various blood group I and i antigenic determinants in a variety of cultured cell lines commonly used in laboratory investigations. It has been shown that the antigens they recognize are widely distributed on the surface of human and animal cell lines, expressed in varying amounts in different cell lines and on individual cells within a given cell line. In two cell lines, a transformation-associated increase in the expression of I antigen was observed. Because of their precise specificity for defined carbohydrate chain domains, these autoantibodies have become valuable reagents in biological chemistry.

  8. Expression of blood group I and I active carbohydrate sequences on cultured human and animal cell lines assessed by radioimmunoassays with monoclonal cold agglutinins

    Energy Technology Data Exchange (ETDEWEB)

    Childs, R.A.; Kapadia, A.; Feizi, T.

    1980-05-01

    Human monoclonal anti-I and anti-i, reactive with known carbohydrate sequences, have been used as reagents to quantitate (by radioimmunoassay) and visualize (by immunofluorescence) the expression of the various blood group I and i antigenic determinants in a variety of cultured cell lines commonly used in laboratory investigations. It has been shown that the antigens they recognize are widely distributed on the surface of human and animal cell lines, expressed in varying amounts in different cell lines and on individual cells within a given cell line. In two cell lines, a transformation-associated increase in the expression of I antigen was observed. Because of their precise specificity for defined carbohydrate chain domains, these autoantibodies have become valuable reagents in biological chemistry.

  9. Plasma cell gingivitis with severe alveolar bone loss.

    Science.gov (United States)

    Kumar, Vivek; Tripathi, Amitandra Kumar; Saimbi, Charanjit Singh; Sinha, Jolly

    2015-01-16

    Plasma cell gingivitis is a rare benign condition of the gingiva characterised by sharply demarcated erythaematous and oedematous gingiva often extending up to the muco gingival junction. It is considered a hypersensitive reaction. It presents clinically as a diffuse, erythaematous and papillary lesion of the gingiva, which frequently bleeds, with minimal trauma. This paper presents a case of a 42-year-old man who was diagnosed with plasma cell gingivitis, based on the presence of plasma cells in histological sections, and severe alveolar bone loss at the affected site, which was managed by surgical intervention.

  10. Evaluation of high-capacity cation exchange chromatography for direct capture of monoclonal antibodies from high-titer cell culture processes.

    Science.gov (United States)

    Tao, Yinying; Ibraheem, Aladein; Conley, Lynn; Cecchini, Douglas; Ghose, Sanchayita

    2014-07-01

    Advances in molecular biology and cell culture technology have led to monoclonal antibody titers in excess of 10 g/L. Such an increase can pose concern to traditional antibody purification processes due to limitations in column hardware and binding capacity of Protein A resins. Recent development of high capacity cation exchangers can make cation exchange chromatography (CEX) a promising and economic alternative to Protein A capture. This work investigates the feasibility of using CEX for direct capture of monoclonal antibodies from high titer cell culture fluids. Two resin candidates were selected from seven newer generation cation exchangers for their higher binding capacity and selectivity. Two monoclonal antibodies with widely differing pI values were used to evaluate the capability of CEX as a platform capture step. Screening of loading pH and conductivity showed both resins to be capable of directly capturing both antibodies from undiluted cell culture fluid. At appropriate acidic pH range, product loading of over 65 g/L resin was achieved for both antibodies. A systematic design of experiment (DOE) approach was used to optimize the elution conditions for the CEX step. Elution pH showed the most significant impact on clearance of host cell proteins (HCPs). Under optimal conditions, HCP reduction factors in the range of 9-44 were achieved on the CEX step based on the pI of the antibody. Apart from comparing CEX directly to Protein A as the capture method, material from either modality was also processed through the subsequent polishing steps to compare product quality at the drug substance level. Process performance and product quality was found to be acceptable using the non-affinity based process scheme. The results shown here present a cheaper and higher capacity generic capture method for high-titer antibody processes. © 2014 Wiley Periodicals, Inc.

  11. Interleukin-24 inhibits the plasma cell differentiation program in human germinal center B cells.

    Science.gov (United States)

    Maarof, Ghyath; Bouchet-Delbos, Laurence; Gary-Gouy, Hélène; Durand-Gasselin, Ingrid; Krzysiek, Roman; Dalloul, Ali

    2010-03-04

    Complex molecular mechanisms control B-cell fate to become a memory or a plasma cell. Interleukin-24 (IL-24) is a class II family cytokine of poorly understood immune function that regulates the cell cycle. We previously observed that IL-24 is strongly expressed in leukemic memory-type B cells. Here we show that IL-24 is also expressed in human follicular B cells; it is more abundant in CD27(+) memory B cells and CD5-expressing B cells, whereas it is low to undetectable in centroblasts and plasma cells. Addition of IL-24 to B cells, cultured in conditions shown to promote plasma cell differentiation, strongly inhibited plasma cell generation and immunoglobulin G (IgG) production. By contrast, IL-24 siRNA increased terminal differentiation of B cells into plasma cells. IL-24 is optimally induced by BCR triggering and CD40 engagement; IL-24 increased CD40-induced B-cell proliferation and modulated the transcription of key factors involved in plasma cell differentiation. It also inhibited activation-induced tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT-3), and inhibited the transcription of IL-10. Taken together, our results indicate that IL-24 is a novel cytokine involved in T-dependent antigen (Ag)-driven B-cell differentiation and suggest its physiologic role in favoring germinal center B-cell maturation in memory B cells at the expense of plasma cells.

  12. The RP105/MD-1 complex is indispensable for TLR4/MD-2-dependent proliferation and IgM-secreting plasma cell differentiation of marginal zone B cells.

    Science.gov (United States)

    Nagai, Yoshinori; Yanagibashi, Tsutomu; Watanabe, Yasuharu; Ikutani, Masashi; Kariyone, Ai; Ohta, Shoichiro; Hirai, Yoshikatsu; Kimoto, Masao; Miyake, Kensuke; Takatsu, Kiyoshi

    2012-06-01

    Marginal zone (MZ) B cells mount rapid T-cell-independent (T-I) immune responses against microbial components such as LPS. While Toll-like receptor 4 (TLR4) is essential for LPS responses, MZ B cells uniquely express high levels of another LPS sensor Radioprotective 105 (RP105). However, little is known about how RP105 is used by MZ B cells. In this study, we investigated TLR4- or RP105-dependent MZ B cell responses by utilizing agonistic monoclonal antibodies (mAbs) to each receptor. Cross-linking TLR4 and RP105 at the same time with the mAbs induced robust IgM-secreting plasma cell generation as lipid A moiety of LPS. In contrast, stimulation with either mAb alone did not elicit such responses. RP105-deficient MZ B cells failed to produce IgM-secreting plasma cells in response to lipid A. TLR4 or lipid A stimulation of MZ B cells up-regulated their B lymphocyte-induced maturation protein 1 (Blimp-1) and X-box-binding protein 1 (Xbp-1) mRNA expression. RP105 stimulation alone did not give these responses and in fact decreased TLR4-mediated their expression. Compared with wild-type (WT) MZ B cells, RP105-deficient MZ B cells exhibited increased levels of Blimp-1 and Xbp-1 mRNA expression in response to lipid A. Lipid A or TLR4 plus RP105 stimulation induced massive proliferation and expression of Bcl-xL and c-Myc in WT but not RP105-deficient MZ B cells. These responses contributed to TLR4-mediated anti-apoptotic responses in MZ B cells. Thus, RP105 contributes in a unique way to the TLR4-dependent survival, proliferation and plasma cell generation of MZ B cells.

  13. Rapid production of antigen-specific monoclonal antibodies from a variety of animals

    Directory of Open Access Journals (Sweden)

    Kurosawa Nobuyuki

    2012-09-01

    Full Text Available Abstract Background Although a variety of animals have been used to produce polyclonal antibodies against antigens, the production of antigen-specific monoclonal antibodies from animals remains challenging. Results We propose a simple and rapid strategy to produce monoclonal antibodies from a variety of animals. By staining lymph node cells with an antibody against immunoglobulin and a fluorescent dye specific for the endoplasmic reticulum, plasma/plasmablast cells were identified without using a series of antibodies against lineage markers. By using a fluorescently labeled antigen as a tag for a complementary cell surface immunoglobulin, antigen-specific plasma/plasmablast cells were sorted from the rest of the cell population by fluorescence-activated cell sorting. Amplification of cognate pairs of immunoglobulin heavy and light chain genes followed by DNA transfection into 293FT cells resulted in the highly efficient production of antigen-specific monoclonal antibodies from a variety of immunized animals. Conclusions Our technology eliminates the need for both cell propagation and screening processes, offering a significant advantage over hybridoma and display strategies.

  14. AWAKE’s plasma cell arrives at its destination

    CERN Multimedia

    Antonella Del Rosso

    2016-01-01

    By harnessing the power of wakefields generated by a proton beam in a plasma cell, the AWAKE project aims to produce accelerator gradients hundreds of times higher than those achieved in current machines. Far from being just a dream, the AWAKE tunnel is progressively being filled with its vital components. This week, the plasma cell has been moved to its final position.   AWAKE's 10-metre-long plasma cell in the experiment tunnel. The proof-of-principle AWAKE experiment is being installed in the tunnel previously used by the CNGS facility. In AWAKE, a beam of protons from the SPS will be travelling through a plasma cell and will generate a wakefield that, in turn, will accelerate an electron beam. A laser will ionise the gas in the plasma cell and seed the self-modulation instability that will trigger the wakefield in the plasma. The project aims to prove that the plasma wakefield can be driven with protons and that its acceleration will be extremely powerful, hundreds of times more powe...

  15. Monoclonal gammopathy of undetermined significance and risk of infections: a population-based study.

    Science.gov (United States)

    Kristinsson, Sigurdur Y; Tang, Min; Pfeiffer, Ruth M; Björkholm, Magnus; Goldin, Lynn R; Blimark, Cecilie; Mellqvist, Ulf-Henrik; Wahlin, Anders; Turesson, Ingemar; Landgren, Ola

    2012-06-01

    No comprehensive evaluation has been made to assess the risk of viral and bacterial infections among patients with monoclonal gammopathy of undetermined significance. Using population-based data from Sweden, we estimated risk of infections among 5,326 monoclonal gammopathy of undetermined significance patients compared to 20,161 matched controls. Patients with monoclonal gammopathy of undetermined significance had a 2-fold increased risk (Pundetermined significance had an increased risk (Pundetermined significance with M-protein concentrations over 2.5 g/dL at diagnosis had highest risks of infections. However, the risk was also increased (Pundetermined significance who developed infections had no excess risk of developing multiple myeloma, Waldenström macroglobulinemia or related malignancy. Our findings provide novel insights into the mechanisms behind infections in patients with plasma cell dyscrasias, and may have clinical implications.

  16. Plasma membranes from insect midgut cells

    Directory of Open Access Journals (Sweden)

    Walter R. Terra

    2006-06-01

    Full Text Available Plasma membranes from insect midgut cells are separated into apical and basolateral domains. The apical domain is usually modified into microvilli with a molecular structure similar to other animals. Nevertheless, the microvillar structure should differ in some insects to permit the traffic inside them of secretory vesicles that may budd laterally or pinch-off from the tips of microvilli. Other microvillar modifications are associated with proton-pumping or with the interplay with an ensheathing lipid membrane (the perimicrovilllar membrane observed in the midgut cells of hemipterans (aphids and bugs. The perimicrovillar membranes are thought to be involved in amino acid absorption from diluted diets. The microvillar and perimicrovillar membranes have densities (and protein content that depend on the insect taxon. The role played by the microvillar and perimicrovillar proteins in insect midgut physiology is reviewed here trying to provide a coherent picture of data and highlighting further research areas.As membranas plasmáticas das células intestinais dos insetos apresentam um domínio apical e outro basal. O domínio apical é geralmente modificado em microvilosidades com organização molecular similar a de outros animais, embora possam diferir naqueles insetos que apresentam vesículas secretoras em trânsito que brotam lateralmente ou destacam-se das extremidades das microvilosidades. Outras modificações microvilares estão associadas a bombeamento de prótons ou a interrelações com uma membrana lipídica (a membrana perimicrovilar que reveste as microvilosidades de células intestinais de hemípteros (pulgões e percevejos. Admite-se que as membranas perimicrovilares estejam envolvidas na absorção de aminoácidos a partir de dietas diluídas. As membranas microvilares e perimicrovilares tem densidades distintas (e conteúdo protéico que dependem do táxon do inseto. O papel desempenhado pelas proteínas microvilares e

  17. Enrichment and purging of human embryonic stem cells by detection of cell surface antigens using the monoclonal antibodies TG30 and GCTM-2.

    Science.gov (United States)

    Polanco, Juan Carlos; Wang, Bei; Zhou, Qi; Chy, Hun; O'Brien, Carmel; Laslett, Andrew L

    2013-12-06

    Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all somatic cell lineages. Differentiated hESC derivatives can potentially be used in transplantation therapies to treat a variety of cell-degenerative diseases. However, hESC differentiation protocols usually yield a mixture of differentiated target and off-target cell types as well as residual undifferentiated cells. For the translation of differentiated hESC-derivatives from the laboratory to the clinic, it is important to be able to discriminate between undifferentiated (pluripotent) and differentiated cells, and generate methods to separate these populations. Safe application of hESC-derived somatic cell types can only be accomplished with pluripotent stem cell-free populations, as residual hESCs could induce tumors known as teratomas following transplantation. Towards this end, here we describe a methodology to detect pluripotency associated cell surface antigens with the monoclonal antibodies TG30 (CD9) and GCTM-2 via fluorescence activated cell sorting (FACS) for the identification of pluripotent TG30(Hi)-GCTM-2(Hi) hESCs using positive selection. Using negative selection with our TG30/GCTM-2 FACS methodology, we were able to detect and purge undifferentiated hESCs in populations undergoing very early-stage differentiation (TG30(Neg)-GCTM-2(Neg)). In a further study, pluripotent stem cell-free samples of differentiated TG30(Neg)-GCTM-2(Neg) cells selected using our TG30/GCTM-2 FACS protocol did not form teratomas once transplanted into immune-compromised mice, supporting the robustness of our protocol. On the other hand, TG30/GCTM-2 FACS-mediated consecutive passaging of enriched pluripotent TG30(Hi)-GCTM-2(Hi) hESCs did not affect their ability to self-renew in vitro or their intrinsic pluripotency. Therefore, the characteristics of our TG30/GCTM-2 FACS methodology provide a sensitive assay to obtain highly

  18. Neutralization of feline infectious peritonitis virus: preparation of monoclonal antibody that shows cell tropism in neutralizing activity after viral absorption into the cells.

    Science.gov (United States)

    Kida, K; Hohdatsu, T; Kashimoto-Tokunaga, J; Koyama, H

    2000-01-01

    Feline infectious peritonitis virus (FIPV) infection of feline macro-phages is enhanced by mouse anti-FIPV monoclonal antibody (MAb). This anti-body-dependent enhancement (ADE) of FIPV infection is dependent on mouse MAb subclass, and MAb of IgG2a subclass has a strong ADE activity. Furthermore, MAb showing strong neutralizing activity in Felis catus whole fetus (fcwf-4) cells and Crandell feline kidney (CrFK) cells shows strong enhancing activity in feline macrophages, indicating that the neutralizing epitope and the enhancing epitope are closely related. In this study, we prepared MAb FK50-4 that showed a strong neutralizing activity in feline macrophages, despite the fact that the MAb belonged to the IgG2a subclass. However, MAb FK50-4 did not exhibit neutralizing activity in CrFK cells or fcwf-4 cells, thus showing a very unusual property. MAb FK50-4 recognized FIPV small integral membrane glycoprotein (M protein). Even when feline macrophages were pretreated with MAb FK50-4 prior to FIPV inoculation, this antibody prevented FIPV infection. This reaction disappeared after treatment of FK50-4 with protein A. The neutralizing activity of FK50-4 was also effective on feline macrophages after the cells were inoculated with FIPV. These findings indicated that the FIPV replication mechanism differs between feline macrophages and CrFK/fcwf-4 cells and that a neutralizing epitope that can prevent FIPV infection of feline macrophages after viral absorption is present on M protein.

  19. Stem cell responses to plasma surface modified electrospun polyurethane scaffolds.

    Science.gov (United States)

    Zandén, Carl; Hellström Erkenstam, Nina; Padel, Thomas; Wittgenstein, Julia; Liu, Johan; Kuhn, H Georg

    2014-07-01

    The topographical effects from functional materials on stem cell behavior are currently of interest in tissue engineering and regenerative medicine. Here we investigate the influence of argon, oxygen, and hydrogen plasma surface modification of electrospun polyurethane fibers on human embryonic stem cell (hESC) and rat postnatal neural stem cell (NSC) responses. The plasma gases were found to induce three combinations of fiber surface functionalities and roughness textures. On randomly oriented fibers, plasma treatments lead to substantially increased hESC attachment and proliferation as compared to native fibers. Argon plasma was found to induce the most optimal combination of surface functionality and roughness for cell expansion. Contact guided migration of cells and alignment of cell processes were observed on aligned fibers. Neuronal differentiation around 5% was found for all samples and was not significantly affected by the induced variations of surface functional group distribution or individual fiber topography. In this study the influence of argon, oxygen, and hydrogen plasma surface modification of electrospun polyurethane fibers on human embryonic stem cell and rat postnatal neural stem cell (NSC) responses is studied with the goal of clarifying the potential effects of functional materials on stem cell behavior, a topic of substantial interest in tissue engineering and regenerative medicine. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Phagocytic plasma cells in a patient with multiple myeloma

    NARCIS (Netherlands)

    P.M. Vanhagen (P.); K. de Leeuw (K.); A. Hagemeijer (Anne); B. Löwenberg (Bob)

    1995-01-01

    textabstractPhagocytosis of blood cells by malignant plasma cells in multiple myeloma is an extremely rare condition. Here we present a 39-year-old woman with multiple myeloma. Bone marrow smear showed an extensive phagocytosis of erythrocytes and platelets by myeloma cells.

  1. Phagocytic plasma cells in a patient with multiple myeloma

    NARCIS (Netherlands)

    P.M. Vanhagen (P.); K. de Leeuw (K.); A. Hagemeijer (Anne); B. Löwenberg (Bob)

    1995-01-01

    textabstractPhagocytosis of blood cells by malignant plasma cells in multiple myeloma is an extremely rare condition. Here we present a 39-year-old woman with multiple myeloma. Bone marrow smear showed an extensive phagocytosis of erythrocytes and platelets by myeloma cells.

  2. Primary Plasma Cell Leukemia: Identity Card 2016.

    Science.gov (United States)

    Musto, Pellegrino; Simeon, Vittorio; Todoerti, Katia; Neri, Antonino

    2016-04-01

    Primary plasma cell leukemia (PPCL) is an aggressive and rare variant of multiple myeloma (MM), characterized by peculiar adverse clinical and biological features. Though the poor outcome of PPCL has been slightly improved by novel treatments during the last 10 years, due to the limited number of available studies in this uncommon disease, optimal therapy remains a classic unmet clinical need. Anyway, in the real-life practice, induction with a bortezomib-based three-drug combination, including dexamethasone and, possibly, lenalidomide, or, alternatively, thalidomide, cyclophosphamide, or doxorubicin, is a reasonable first-line option. This approach may be particularly advisable for patients with adverse cytogenetics, hyperleucocytosis, and rapidly progressive disease, in whom a fast response is required, or for those with suboptimal renal function, where, however, lenalidomide should be used with caution until renal activity is restored. In younger subjects, leukemia/lymphoma-like more intensive regimens, including hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone or continue-infusion cisplatin, doxorubicin, cyclophosphamide, and etoposide, may be also combined with bortezomib +/- thalidomide. Treatment must be started immediately after a diagnosis of PPCL is made to avoid the risk of irreversible disease complications and, in such a context, the prevention of tumor lysis syndrome is mandatory. In patients eligible for autologous stem cell transplantation (AuSCT), other alkylating agents, in particular melphalan, should be initially avoided in order to allow adequate collections of CD34+ peripheral blood stem cells (PBSC). A combination of lenalidomide and dexamethasone may be a valuable alternative option to manage older or unfit patients or those with slower disease evolution or with signs of neuropathy, contraindicating the use of bortezomib. Patients not suitable for transplant procedures should continue the treatment, if a

  3. Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

    Directory of Open Access Journals (Sweden)

    Andrabi Raiees

    2012-09-01

    Full Text Available Abstract Background Analysis of human monoclonal antibodies (mAbs developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3 is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5 binding and presence of epitopes recognized by broadly neutralizing antibodies. Methods Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females within the age range of 20–57 years (median = 33 years were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. Results We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL, suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. Conclusions Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope

  4. In-vitro inhibition of IFNγ+ iTreg mediated by monoclonal antibodies against cell surface determinants essential for iTreg function

    OpenAIRE

    Daniel Volker; Sadeghi Mahmoud; Wang Haihao; Opelz Gerhard

    2012-01-01

    Abstract Background IFNγ-producing CD4+CD25+Foxp3+ PBL represent a subtype of iTreg that are associated with good long-term graft outcome in renal transplant recipients and suppress alloresponses in-vitro. To study the mechanism of immunosuppression, we attempted to block cell surface receptors and thereby inhibited the function of this iTreg subset in-vitro using monoclonal antibodies and recombinant proteins. Methods PBL of healthy control individuals were stimulated polyclonally in-vitro i...

  5. Phage display derived human monoclonal antibodies isolated by binding to the surface of live primary breast cancer cells recognize GRP78

    DEFF Research Database (Denmark)

    Jakobsen, Charlotte G; Rasmussen, Nicolaj; Laenkholm, Anne-Vibeke

    2007-01-01

    Clinical trials using monoclonal antibodies (mAb) against cell-surface markers have yielded encouraging therapeutic results in several cancer types. Generally, however, anticancer antibodies are only efficient against a subpopulation of cancers, and there is a strong need for identification of no...... therapies including mAb-based immunotherapy. Our results suggest that the human antibody Ab39 may be a useful starting point for further genetic optimization that could render it a useful diagnostic and therapeutic reagent for a variety of cancers...

  6. Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions

    Directory of Open Access Journals (Sweden)

    Anna V. Savelyeva

    2017-01-01

    Full Text Available Human peripheral blood contains RNA in cells and in extracellular membrane vesicles, microvesicles and exosomes, as well as in cell-free ribonucleoproteins. Circulating mRNAs and noncoding RNAs, being internalized, possess the ability to modulate vital processes in recipient cells. In this study, with SOLiD sequencing technology, we performed identification, classification, and quantification of RNAs from blood fractions: cells, plasma, plasma vesicles pelleted at 16,000g and 160,000g, and vesicle-depleted plasma supernatant of healthy donors and non-small cell lung cancer (NSCLC patients. It was determined that 16,000g blood plasma vesicles were enriched with cell-free mitochondria and with a set of mitochondrial RNAs. The variable RNA set of blood plasma 160,000g pellets reflected the prominent contribution of U1, U5, and U6 small nuclear RNAs’ fragments and at the same time was characterized by a remarkable depletion of small nucleolar RNAs. Besides microRNAs, the variety of fragments of mRNAs and snoRNAs dominated in the set of circulating RNAs differentially expressed in blood fractions of NSCLC patients. Taken together, our data emphasize that not only extracellular microRNAs but also circulating fragments of messenger and small nuclear/nucleolar RNAs represent prominent classes of circulating regulatory ncRNAs as well as promising circulating biomarkers for the development of disease diagnostic approaches.

  7. Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions.

    Science.gov (United States)

    Savelyeva, Anna V; Kuligina, Elena V; Bariakin, Dmitry N; Kozlov, Vadim V; Ryabchikova, Elena I; Richter, Vladimir A; Semenov, Dmitry V

    2017-01-01

    Human peripheral blood contains RNA in cells and in extracellular membrane vesicles, microvesicles and exosomes, as well as in cell-free ribonucleoproteins. Circulating mRNAs and noncoding RNAs, being internalized, possess the ability to modulate vital processes in recipient cells. In this study, with SOLiD sequencing technology, we performed identification, classification, and quantification of RNAs from blood fractions: cells, plasma, plasma vesicles pelleted at 16,000g and 160,000g, and vesicle-depleted plasma supernatant of healthy donors and non-small cell lung cancer (NSCLC) patients. It was determined that 16,000g blood plasma vesicles were enriched with cell-free mitochondria and with a set of mitochondrial RNAs. The variable RNA set of blood plasma 160,000g pellets reflected the prominent contribution of U1, U5, and U6 small nuclear RNAs' fragments and at the same time was characterized by a remarkable depletion of small nucleolar RNAs. Besides microRNAs, the variety of fragments of mRNAs and snoRNAs dominated in the set of circulating RNAs differentially expressed in blood fractions of NSCLC patients. Taken together, our data emphasize that not only extracellular microRNAs but also circulating fragments of messenger and small nuclear/nucleolar RNAs represent prominent classes of circulating regulatory ncRNAs as well as promising circulating biomarkers for the development of disease diagnostic approaches.

  8. Immunoglobulin D multiple myeloma, plasma cell leukemia and chronic myelogenous leukemia in a single patient treated simultaneously with lenalidomide, bortezomib, dexamethasone and imatinib

    Directory of Open Access Journals (Sweden)

    Naveed Ali

    2016-03-01

    Full Text Available Multiple myeloma (MM is a neoplastic lymphoproliferative disorder characterized by uncontrolled monoclonal plasma cell proliferation. Among different isotypes of MM, immunoglobulin D (IgD MM is very rare, representing only 1 to 2% of all isotypes. Chronic myelogenous leukemia (CML is a neoplastic myeloproliferative disorder of pluripotent hematopoietic stem cell, which is characterized by the uncontrolled proliferation of myeloid cells. An 88-year-old male was diagnosed simultaneously with IgD kappa MM and CML. A distinctive feature in this patient was the progression to plasma cell leukemia without any symptomatic myeloma stage. He was treated simultaneously with lenalidomide, bortezomib and imatinib. Synchronous occurrence of these rare hematological malignancies in a single patient is an exceedingly rare event. Multiple hypotheses to explain co-occurrence of CML and MM have been proposed; however, the exact etiological molecular pathophysiology remains elusive.

  9. A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen, E-mail: srrshurology@163.com

    2015-08-14

    The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice.

  10. Miniature Dielectric Barrier Discharge Nonthermal Plasma Induces Apoptosis in Lung Cancer Cells and Inhibits Cell Migration

    Science.gov (United States)

    Eisenmann, Kathryn M.

    2017-01-01

    Traditional cancer treatments like radiotherapy and chemotherapy have drawbacks and are not selective for killing only cancer cells. Nonthermal atmospheric pressure plasmas with dielectric barrier discharge (DBD) can be applied to living cells and tissues and have emerged as novel tools for localized cancer therapy. The purpose of this study was to investigate the different effects caused by miniature DBD (mDBD) plasma to A549 lung cancer cells. In this study, A549 lung cancer cells cultured in 12 well plates were treated with mDBD plasma for specified treatment times to assess the changes in the size of the area of cell detachment, the viability of attached or detached cells, and cell migration. Furthermore, we investigated an innovative mDBD plasma-based therapy for localized treatment of lung cancer cells through apoptotic induction. Our results indicate that plasma treatment for 120 sec causes apoptotic cell death in 35.8% of cells, while mDBD plasma treatment for 60 sec, 30 sec, or 15 sec causes apoptotic cell death in 20.5%, 14.1%, and 6.3% of the cell population, respectively. Additionally, we observed reduced A549 cell migration in response to mDBD plasma treatment. Thus, mDBD plasma system can be a viable platform for localized lung cancer therapy. PMID:28243603

  11. Fine specificity of monoclonal antibodies directed at human T cell receptor variable regions: comparison with oligonucleotide-driven amplification for evaluation of V beta expression.

    Science.gov (United States)

    Diu, A; Romagné, F; Genevée, C; Rocher, C; Bruneau, J M; David, A; Praz, F; Hercend, T

    1993-07-01

    Seven distinct anti-human T cell receptor (TcR) V region monoclonal antibodies (mAb) were generated by immunizing mice with either human T cell lines or transfected murine cells expressing human TcR V beta genes. The specificity of these reagents was determined as follows: T cells recognized by each mAb were purified from the peripheral blood of healthy donors and TcR transcripts expressed in these cells were analyzed using oligonucleotide-driven amplification and cDNA sequencing. Four mAb were found to delineate the V beta 3, V beta 8, V beta 17 and V beta 19 subfamilies, respectively. The remaining reagents recognize subsets within the V beta 2, V beta 5 and V beta 13 subfamilies. Reactivity of the mAb with circulating T cells from 18 unrelated healthy individuals was determined. Limited variability was found from an individual to another. In four donors, mAb staining was compared to oligonucleotide-driven amplification for evaluation of V beta 3, V beta 8, V beta 17 and V beta 19 subfamily expression in the peripheral blood. Although the V gene subfamily-specific oligonucleotides used in this study belong to a carefully controlled series, our results show that this method does not give an accurate estimate of the percentage of peripheral T cells expressing a given TcR beta chain. The present data confirm the necessity to establish a complete set of well-characterized monoclonal reagents to study human T cell responses.

  12. CD27-triggering on primary plasma cell leukaemia cells has anti-apoptotic effects involving mitogen activated protein kinases

    NARCIS (Netherlands)

    Guikema, JEJ; Vellenga, E; Abdulahad, WH; Hovenga, S; Bos, NA

    2004-01-01

    Primary plasma cell leukaemia (PCL) is a rare plasma cell malignancy, which is related to multiple myeloma (MM) and is characterized by a poor prognosis. In a previous study we demonstrated that PCL plasma cells display a high expression of CD27, in contrast to MM plasma cells. The present study was

  13. Monoclonal B-cell lymphocytosis in a hospital-based UK population and a rural Ugandan population: a cross-sectional study.

    Science.gov (United States)

    Rawstron, Andy C; Ssemaganda, Aloysius; de Tute, Ruth; Doughty, Chi; Newton, Darren; Vardi, Anna; Evans, Paul A S; Stamatopoulos, Kostas; Owen, Roger G; Lightfoot, Tracy; Wakeham, Katie; Karabarinde, Alex; Asiki, Gershim; Newton, Robert

    2017-07-01

    Reported incidence of B-cell malignancies shows substantial geographical variation, being more common in the Americas and Europe than in Africa. This variation might reflect differences in diagnostic capability, inherited susceptibility, and infectious exposures. Monoclonal B-cell lymphocytosis (MBL) is a precursor lesion that can be screened for in apparently healthy people, allowing comparison of prevalence across different populations independently of health-care provision. We aimed to compare the prevalence and phenotypic characteristics of MBL in age-and-sex-matched populations from rural Uganda and the UK. In this cross-sectional study, we recruited volunteers aged at least 45 years who were seronegative for HIV-1 from the established Ugandan General Population Cohort and obtained their whole-blood samples. We also obtained blood samples from anonymised waste material of age-and-sex-matched individuals (aged >45 years, with a normal blood count and no history of cancer) in the UK. We used flow cytometry to determine the presence of MBL, defined according to standard diagnostic criteria, in the samples and compared differences in the proportion of cases with chronic lymphocytic leukaemia (CLL)-phenotype MBL and CD5-negative MBL, as well as differences in absolute monoclonal B-cell count between the two cohorts. Between Jan 15 and Dec 18, 2012, we obtained samples from 302 Ugandan volunteers and 302 UK individuals who were matched by age and sex to the Ugandan population. Overall MBL prevalence was higher in the Ugandan participants (42 [14%] individuals) than in the UK cohort (25 [8%]; p=0·038). CLL-phenotype MBL was detected in three (1%) Ugandan participants and 21 (7%) UK participants (p=0·00021); all three Ugandan participants had absolute monoclonal B-cell count below one cell per μL, whereas the 21 UK participants had a median absolute number of circulating neoplastic cells of 4·6 (IQR 2-12) cells per μL. The prevalence of CD5-negative MBL was

  14. Use of a Plackett-Burman statistical design to determine the effect of selected amino acids on monoclonal antibody production in CHO cells.

    Science.gov (United States)

    González-Leal, I J; Carrillo-Cocom, L M; Ramírez-Medrano, A; López-Pacheco, F; Bulnes-Abundis, D; Webb-Vargas, Y; Alvarez, M M

    2011-01-01

    Culture media design is central to the optimization of monoclonal antibody (mAb) production. Although general strategies do not currently exist for optimization of culture media, the combined use of statistical design and analysis of experiments and strategies based on simple material balances can facilitate culture media design. In this study, we evaluate the effect of selected amino acids on the growth rate and monoclonal antibody production of a Chinese hamster ovary DG-44 (CHO-DG44) cell line. These amino acids were selected based on their relative mass fraction in the specific mAb produced in this study, their consumption rate during bioreactor experiments, and also through a literature review. A Plackett-Burman statistical design was conducted to minimize the number of experiments needed to obtain statistically relevant information. The effect of this set of amino acids was evaluated during exponential cell culture (considering viable cell concentration and the specific growth rate as main output variables) and during the high cell-density stage (considering mAb final concentration and specific productivity as relevant output variables). For this particular cell line, leucine (Leu) and arginine (Arg) had the highest negative and positive effects on cell viability, respectively; Leu and threonine (Thr) had the highest negative effect on growth rate, and valine (Val) and Arg demonstrated the highest positive impact on mAb final concentration. Results suggest the pertinence of a two-stage strategy for amino acid supplementation, with a mixture optimized for cell growth and a different amino acid mixture for mAb production at high density.

  15. Biomedical Applications of the Cold Atmospheric Plasma: Cell Responses

    Science.gov (United States)

    Volotskova, Olga

    Current breakthrough research on cold atmospheric plasma (CAP) demonstrates that CAP has great potential in various areas, including medicine and biology, thus providing a new tool for living tissue treatment. Depending on the configuration the cold plasma sources can be used in the following areas: wound healing, skin diseases, hospital hygiene, sterilization, antifungal treatments, dental care, cosmetics targeted cell/tissue removal, and cancer treatments. This dissertation is focused on the studies of biomedical applications of cold atmospheric plasma jet based on helium flow and resultant cell responses to the cold plasma treatment. The studies were carried out on extra-cellular and intra-cellular levels in vitro. The main practical applications are wound healing and alternative to existing cancer therapy methods, areas of great interest and significant challenges. The CAP jet was built in the Micropropulsion and Nanotechnology Laboratory of Dr. Michael Keidar, as a part of multidisciplinary collaboration with the GW Medical School (Dr. M.A. Stepp) concerned with plasma medicine and bioengineering studies. Normal and cancer cells have two fundamental behavioral properties, proliferation and motility, which can be evaluated through cell migration rates and cell cycle progression. Various microscopic, spectroscopic and flow cytometry techniques were used to characterize cell responses to the cold plasma treatment. It was found that CAP effect on the cells is localized within the area of the treatment (of around ˜ 5mm in diameter). The migration rates of the normal skin cells can be reduced up to ˜ 40%. However, depending on the cell type the required treatment time is different, thus differential treatment of various cells presented in tissue is possible. The CAP effect on the migration was explained through the changes of the cell surface proteins/integrins. It was also found that normal and cancer cells respond differently to the CAP treatment under the same

  16. Wheat germ cell-free system-based production of hemagglutinin-neuraminidase protein of human parainfluenza virus type 3: generation and characterization of monoclonal antibody

    Directory of Open Access Journals (Sweden)

    Satoko eMatsunaga

    2014-05-01

    Full Text Available Human parainfluenza virus 3 (HPIV3 commonly causes respiratory disorders in infants and young children. Monoclonal antibodies (MAbs have been produced to several components of HPIV3 and commercially available. However, the utility of these antibodies for several immunological and proteomic assays for understanding the nature of HPIV3 infection remain to be characterized. Herein, we report the development and characterization of monoclonal antibodies against hemagglutinin-neuraminidase (HN of HPIV3. A recombinant full-length HPIV3-HN was successfully synthesized using the wheat-germ cell-free protein production system. After immunization and cell fusion, 36 mouse hybridomas producing MAbs to HPIV3-HN were established. The MAbs obtained were fully characterized using ELISA, immunoblotting and immunofluorescent analyses. Of the MAbs tested, single clone was found to be applicable in both flow cytometry and immunoprecipitation procedures. By utilizing the antibody, we newly identified HPIV3-HN binding host proteins via immunoprecipitation-based mass spectrometry analysis. This study provides the availability of our newly-developed MAbs as a valuable tool for the study of HPIV3 infection as well as the several diagnostic tests of this virus.

  17. Reduction in plasma cell proliferation after initial therapy in newly diagnosed multiple myeloma measures treatment response and predicts improved survival.

    Science.gov (United States)

    Larsen, Jeremy T; Chee, Cheng E; Lust, John A; Greipp, Philip R; Rajkumar, S Vincent

    2011-09-08

    Standard myeloma treatment response criteria are determined principally by changes in the monoclonal protein. Reduction in the size of the proliferative component of malignant plasma cells may be an additional metric of assessing response to therapy. We retrospectively analyzed 176 patients with newly diagnosed myeloma with a measurable plasma cell labeling index (PCLI) at diagnosis and repeat measurement 4 months after initiation of therapy. PCLI response was defined as a ≥ 60% reduction. Baseline PCLI is an independent prognostic factor; therefore, we categorized patients into 3 groups: PCLI ≥ 3% (high), ≥ 1% (intermediate), and compared with 29 months in nonresponders (P = .02). Improved median overall survival with PCLI response occurred in the high initial PCLI group (28 vs 7 months; P = .003) and intermediate group (64 vs 24 months; P = .002). The application of PCLI response and serum M-spike response together provided further prognostic information. On multivariate analysis, the prognostic value of PCLI response was independent of β(2)-microglobulin, elevated creatinine, serum M-spike response, and baseline PCLI. We conclude that a significant reduction in plasma cell proliferation in patients with newly diagnosed myeloma is an important predictor of survival.

  18. CD229 is expressed on the surface of plasma cells carrying an aberrant phenotype and chemotherapy-resistant precursor cells in multiple myeloma.

    Science.gov (United States)

    Yousef, Sara; Kovacsovics-Bankowski, Magdalena; Salama, Mohamed E; Bhardwaj, Neelam; Steinbach, Mary; Langemo, Amanda; Kovacsovics, Tibor; Marvin, James; Binder, Mascha; Panse, Jens; Kröger, Nicolaus; Luetkens, Tim; Atanackovic, Djordje

    2015-01-01

    Multiple Myeloma (MM) is a plasma cell (PC) malignancy, which despite significant therapeutic advances, is still considered incurable. This is due to the persistence of chemotherapy-resistant minimal residual disease in the patients' bone marrow (BM) after an effective induction therapy. Immunotherapies targeting surface molecules expressed on the bulk of tumor cells and the chemotherapy-resistant, myeloma-propagating cells could play a central role in this clinical setting. We recently described surface molecule CD229 as a potential therapeutic target for MM. In our current study we assessed the expression of CD229 on different PC subtypes and on cells with a myeloma-propagating phenotype in a total of 77 patients with PC dyscrasias independently at 2 different cancer centers. We found that CD229 was strongly and homogeneously overexpressed on the PC of patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma, MM, and PC leukemia. CD229 was particularly overexpressed on those PC showing an abnormal phenotype such as expression of CD56. Most importantly, CD229 was also highly expressed on those cells in the patients' BM displaying the phenotype of chemotherapy-resistant and myeloma-propagating cells. In conclusion, our combined findings suggest that immunotherapies targeting CD229 will not only be effective for the bulk of tumor cells but will also help to eradicate chemotherapy-resistant cells remaining in the patients' BM after induction treatment. Hopefully, the design of CD229-specific monoclonal antibodies or chimeric antigen receptor-transduced T cells will help to achieve prolonged remissions or even cures in MM patients.

  19. B cells and plasma cells in coronaries of chronically rejected cardiac transplants.

    Science.gov (United States)

    Wehner, Jennifer R; Fox-Talbot, Karen; Halushka, Marc K; Ellis, Carla; Zachary, Andrea A; Baldwin, William M

    2010-05-15

    BACKGROUND.: Previously, we reported that transcripts of immunoglobulins were increased in coronary arteries dissected from cardiac transplants with arteriopathy, but the prevelance and patterns of B cell and plasma cell infiltration in cardiac allografts has not been documented. METHODS.: In this study, we documented the frequency and distribution of B cells and plasma cells in 16 cardiac transplants with advanced chronic rejection that were explanted during a second transplant procedure. Coronary arteries with pathologically confirmed allograft vasculopathy and controls with native atherosclerosis were immunohistologically stained for markers of T cells, B cells, plasma cells, IgG subclasses, C4d, CD21, and CXCL13. RESULTS.: We found that B cells and plasma cells were prevalent in most of the samples analyzed (14 of 16) and were distributed in three patterns: adventitial nodules, diffuse adventitial infiltrates, and neointimal infiltrates. These cells were found most frequently in nodules, some of which had distinct compartmentalization and granular C4d deposits on follicular dendritic cells (FDCs) that typify tertiary lymphoid nodules. FDCs also stained for CD21 and CXCL13. Diffuse infiltrates of B cells and plasma cells were found in fibrotic areas of the neointima and adventitia. Only a minority of control coronaries with atherosclerosis contained B cells. CONCLUSIONS.: B cells and plasma cell infiltrates are consistent findings in and around coronary arteries with allograft vasculopathy and are significantly more frequent than in coronaries with native atherosclerosis. The presence of C4d on FDCs in tertiary lymphoid nodules suggests active antigen presentation.

  20. Osteoblastlike cell adhesion on titanium surfaces modified by plasma nitriding.

    Science.gov (United States)

    da Silva, Jose Sandro Pereira; Amico, Sandro Campos; Rodrigues, Almir Olegario Neves; Barboza, Carlos Augusto Galvao; Alves, Clodomiro; Croci, Alberto Tesconi

    2011-01-01

    The aim of this study was to evaluate the characteristics of various titanium surfaces modified by cold plasma nitriding in terms of adhesion and proliferation of rat osteoblastlike cells. Samples of grade 2 titanium were subjected to three different surface modification processes: polishing, nitriding by plasma direct current, and nitriding by cathodic cage discharge. To evaluate the effect of the surface treatment on the cellular response, the adhesion and proliferation of osteoblastlike cells (MC3T3) were quantified and the results were analyzed by Kruskal-Wallis and Friedman statistical tests. Cellular morphology was observed by scanning electron microscopy. There was more MC3T3 cell attachment on the rougher surfaces produced by cathodic cage discharge compared with polished samples (P Plasma nitriding improves titanium surface roughness and wettability, leading to osteoblastlike cell adhesion.

  1. CD138-negative clonogenic cells are plasma cells but not B cells in some multiple myeloma patients.

    Science.gov (United States)

    Hosen, N; Matsuoka, Y; Kishida, S; Nakata, J; Mizutani, Y; Hasegawa, K; Mugitani, A; Ichihara, H; Aoyama, Y; Nishida, S; Tsuboi, A; Fujiki, F; Tatsumi, N; Nakajima, H; Hino, M; Kimura, T; Yata, K; Abe, M; Oka, Y; Oji, Y; Kumanogoh, A; Sugiyama, H

    2012-09-01

    Clonogenic multiple myeloma (MM) cells reportedly lacked expression of plasma cell marker CD138. It was also shown that CD19(+) clonotypic B cells can serve as MM progenitor cells in some patients. However, it is unclear whether CD138-negative clonogenic MM plasma cells are identical to clonotypic CD19(+) B cells. We found that in vitro MM colony-forming cells were enriched in CD138(-)CD19(-)CD38(++) plasma cells, while CD19(+) B cells never formed MM colonies in 16 samples examined in this study. We next used the SCID-rab model, which enables engraftment of human MM in vivo. CD138(-)CD19(-)CD38(++) plasma cells engrafted in this model rapidly propagated MM in 3 out of 9 cases, while no engraftment of CD19(+) B cells was detected. In 4 out of 9 cases, CD138(+) plasma cells propagated MM, although more slowly than CD138(-) cells. Finally, we transplanted CD19(+) B cells from 13 MM patients into NOD/SCID IL2Rγc(-/-) mice, but MM did not develop. These results suggest that at least in some MM patients CD138-negative clonogenic cells are plasma cells rather than B cells, and that MM plasma cells including CD138(-) and CD138(+) cells have the potential to propagate MM clones in vivo in the absence of CD19(+) B cells.

  2. Effect of a GABA agonist on the expression and distribution of GABAA receptors in the plasma membrane of cultured cerebellar granule cells: an immunocytochemical study

    DEFF Research Database (Denmark)

    Hansen, G H; Belhage, B; Schousboe, A

    1991-01-01

    The effect of the gamma-aminobutyric acid (GABA) agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 microM) on the localization and density of GABAA receptors in the plasma membrane of rat cerebellar granule cells in primary cultures was studied at the electron microscope (EM) level...... by preembedding immunogold staining using the monoclonal antibody bd-17 directed against the beta-subunit of the GABAA receptor complex. In THIP-treated as well as untreated control cultures, GABAA receptors were found to be evenly distributed in the plasma membrane of cell bodies as well as processes. However...... at the EM level using the preembedding immunogold technique. It is likely that low-affinity GABAA receptors are preferentially located in the cell processes and to a considerable extent in the form of 'hot spots'. However, these 'hot spots' also contain high-affinity receptors....

  3. Prevalence of monoclonal gammopathy of undetermined significance in Thailand.

    Science.gov (United States)

    Watanaboonyongcharoen, Phandee; Nakorn, Thanyaphong Na; Rojnuckarin, Ponlapat; Lawasut, Panisinee; Intragumtornchai, Tanin

    2012-02-01

    Individuals with monoclonal gammopathy of undetermined significance (MGUS) develop multiple myeloma and related malignancies at the rate of 1% per year. Given differences in ethnicity, data on prevalence and risk factors of MGUS in Thai population will be helpful in understanding the pathogenesis of plasma cell disorders and designing an early cancer detection strategy. Subjects of 50 years or older were included. Demographic data and suspected risk factors were collected. Monoclonal proteins were detected using serum protein electrophoresis. Serum was obtained from 3,260 participants; 1,104 males (33.9%) and 2,156 females (66.1%). The median age was 57 years (range 50-93 years). Monoclonal proteins were detectable in 2.3% (95% confidence interval [CI] 1.8-2.8). M spikes were found in gamma- and beta-globulin regions in 50 (1.5%) and 25 (0.8%) subjects, respectively. The prevalence of MGUS in subjects 50-59, 60-69, and 70 years or older was 2.0% (41/1,975), 2.6% (22/851), and 2.8% (12/434), respectively. By multivariate analysis, MGUS was associated with living outside Bangkok (odds ratio 2.25, 95% CI 1.11-4.58). The overall prevalence of MGUS in the Thai population was 2.3%, which was lower than that in Western countries, but comparable to that in Japan.

  4. Combination with a defucosylated anti-HM1.24 monoclonal antibody plus lenalidomide induces marked ADCC against myeloma cells and their progenitors.

    Directory of Open Access Journals (Sweden)

    Takeshi Harada

    Full Text Available The immunomodulatory drug lenalidomide (Len has drawn attention to potentiate antibody-dependent cellular cytotoxicity (ADCC-mediated immunotherapies. We developed the defucosylated version (YB-AHM of humanized monoclonal antibody against HM1.24 (CD317 overexpressed in multiple myeloma (MM cells. In this study, we evaluated ADCC by YB-AHM and Len in combination against MM cells and their progenitors. YB-AHM was able to selectively kill via ADCC MM cells in bone marrow samples from patients with MM with low effector/target ratios, which was further enhanced by treatment with Len. Interestingly, Len also up-regulated HM1.24 expression on MM cells in an effector-dependent manner. HM1.24 was found to be highly expressed in a drug-resistant clonogenic "side population" in MM cells; and this combinatory treatment successfully reduced SP fractions in RPMI 8226 and KMS-11 cells in the presence of effector cells, and suppressed a clonogenic potential of MM cells in colony-forming assays. Collectively, the present study suggests that YB-AHM and Len in combination may become an effective therapeutic strategy in MM, warranting further study to target drug-resistant MM clonogenic cells.

  5. Expression profiles of genes in wild-type DJ-1 and L10P mutant DJ-1 in monoclonal cell strains

    Directory of Open Access Journals (Sweden)

    LIU Zhen-hua

    2013-07-01

    Full Text Available Background DJ-1 gene is a causative gene which contributes to the onset of autosomal recessive early-onset parkinsonism (AREP. Many research suggest that DJ-1 protein may change expression of certain genes through regulate its transcriptional activity, which play a role in the pathogenesis of Parkinson's disease (PD. In our previous study, we found a new mutation of DJ-1 which we named as L10P. DJ-1 gene encodes the first frame 29 bp from the thymine (T→cytosine (C, so that the leucine on the 10th locus of DJ-1 protein was replaced by proline (L10P. To elucidate the effect of the L10P mutation, we identify genes for which expressions are abnormally regulated by L10P mutant DJ-1 protein using DNA microarray analysis. Methods Human embryonic kidney cell 293 (HEK293 monoclonal cell strains which can stably express pCMV-Tag2A-Flag, pCMV-Tag2A-Flag-DJ-1 and pCMV-Tag2A-Flag-DJ-1-L10P were selected by screening, and identified on the basis of DNA, RNA and protein levels to confirm whether the acquired HEK293 monoclonal cell strains can stably express empty vector, wild-type DJ-1 protein and L10P mutant DJ-1 protein. Gene chip technique was used to perform differential gene screening for different groups of HEK293 monoclonal cell strains. Results Compared with the expression in empty vector group, the expression of 14 genes was up-regulated and 28 genes was down-regulated in wild-type group; and the expression of 14 genes was up-regulated and 9 down-regulated in expressing L10P mutant group respectively. Comparison of the expression in wild-type group, expression of 59 genes was up-regulated and 27 genes down-regulated in L10P mutant group. These differential genes all took part in the biological processes including signal transduction, transcriptional regulation, cell cycle, apoptosis, oxidative stress and so on. Conclusion L10P mutant DJ-1 protein may directly or indirectly influence the singal transduction and play a role in the mechanism of PD.

  6. Effects of Nonequilibrium Plasmas on Eukaryotic Cells

    Science.gov (United States)

    2009-05-01

    with cellulosic plates under their cell membrane, the diatom C. hystrix has a siliceous cell wall and organic layer, while A. sanguinea is an...unarmored (naked) dinoflagellate, i.e. without the cellulosic plates (see SEM micrographs below). Some non-motile cells recovered their motility and...formed a somewhat reticulate or porous structure as a cell covering. In the A. sanguinea sample where pH was adjusted to 3.0 (corresponds to the pH

  7. Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay

    OpenAIRE

    Takeda, Hiroyuki; Ogasawara, Tomio; Ozawa, Tatsuhiko; Muraguchi, Atsushi; Jih, Pei-Ju; Morishita, Ryo; Uchigashima, Motokazu; Watanabe, Masahiko; Fujimoto, Toyoshi; Iwasaki, Takahiro; Endo, Yaeta; Sawasaki, Tatsuya

    2015-01-01

    G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-ba...

  8. Distinction between Asymptomatic Monoclonal B-cell Lymphocytosis with Cyclin D1 Overexpression and Mantle Cell Lymphoma: From Molecular Profiling to Flow Cytometry

    Science.gov (United States)

    Espinet, Blanca; Ferrer, Ana; Bellosillo, Beatriz; Nonell, Lara; Salar, Antonio; Fernández-Rodríguez, Concepción; Puigdecanet, Eulàlia; Gimeno, Javier; Garcia-Garcia, Mar; Carmen Vela, Maria; Luño, Elisa; Collado, Rosa; Navarro, José Tomás; de la Banda, Esmeralda; Abrisqueta, Pau; Arenillas, Leonor; Serrano, Cristina; Lloreta, Josep; Miñana, Belén; Cerutti, Andrea; Florensa, Lourdes; Orfao, Alberto; Sanz, Ferran; Solé, Francesc; Dominguez-Sola, David; Serrano, Sergio

    2015-01-01

    Purpose According to current diagnostic criteria, mantle cell lymphoma (MCL) encompasses the usual, aggressive variants and rare, nonnodal cases with monoclonal asymptomatic lymphocytosis, cyclin D1–positive (MALD1). We aimed to understand the biology behind this clinical heterogeneity and to identify markers for adequate identification of MALD1 cases. Experimental Design We compared 17 typical MCL cases with a homogeneous group of 13 untreated MALD1 cases (median follow-up, 71 months). We conducted gene expression profiling with functional analysis in five MCL and five MALD1. Results were validated in 12 MCL and 8 MALD1 additional cases by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in 24 MCL and 13 MALD1 cases by flow cytometry. Classification and regression trees strategy was used to generate an algorithm based on CD38 and CD200 expression by flow cytometry. Results We found 171 differentially expressed genes with enrichment of neoplastic behavior and cell proliferation signatures in MCL. Conversely, MALD1 was enriched in gene sets related to immune activation and inflammatory responses. CD38 and CD200 were differentially expressed between MCL and MALD1 and confirmed by flow cytometry (median CD38, 89% vs. 14%; median CD200, 0% vs. 24%, respectively). Assessment of both proteins allowed classifying 85% (11 of 13) of MALD1 cases whereas 15% remained unclassified. SOX11 expression by qRT-PCR was significantly different between MCL and MALD1 groups but did not improve the classification. Conclusion We show for the first time that MALD1, in contrast to MCL, is characterized by immune activation and driven by inflammatory cues. Assessment of CD38/CD200 by flow cytometry is useful to distinguish most cases of MALD1 from MCL in the clinical setting. MALD1 should be identified and segregated from the current MCL category to avoid overdiagnosis and unnecessary treatment. PMID:24352646

  9. Using simple models to describe the kinetics of growth, glucose consumption, and monoclonal antibody formation in naive and infliximab producer CHO cells.

    Science.gov (United States)

    López-Meza, Julián; Araíz-Hernández, Diana; Carrillo-Cocom, Leydi Maribel; López-Pacheco, Felipe; Rocha-Pizaña, María Del Refugio; Alvarez, Mario Moisés

    2016-08-01

    Despite their practical and commercial relevance, there are few reports on the kinetics of growth and production of Chinese hamster ovary (CHO) cells-the most frequently used host for the industrial production of therapeutic proteins. We characterize the kinetics of cell growth, substrate consumption, and product formation in naive and monoclonal antibody (mAb) producing recombinant CHO cells. Culture experiments were performed in 125 mL shake flasks on commercial culture medium (CD Opti CHO™ Invitrogen, Carlsbad, CA, USA) diluted to different glucose concentrations (1.2-4.8 g/L). The time evolution of cell, glucose, lactic acid concentration and monoclonal antibody concentrations was monitored on a daily basis for mAb-producing cultures and their naive counterparts. The time series were differentiated to calculate the corresponding kinetic rates (rx = d[X]/dt; rs = d[S]/dt; rp = d[mAb]/dt). Results showed that these cell lines could be modeled by Monod-like kinetics if a threshold substrate concentration value of [S]t = 0.58 g/L (for recombinant cells) and [S]t = 0.96 g/L (for naïve cells), below which growth is not observed, was considered. A set of values for μmax, and Ks was determined for naive and recombinant cell cultures cultured at 33 and 37 °C. The yield coefficient (Yx/s) was observed to be a function of substrate concentration, with values in the range of 0.27-1.08 × 10(7) cell/mL and 0.72-2.79 × 10(6) cells/mL for naive and recombinant cultures, respectively. The kinetics of mAb production can be described by a Luedeking-Piret model (d[mAb]/dt = αd[X]/dt + β[X]) with values of α = 7.65 × 10(-7) µg/cell and β = 7.68 × 10(-8) µg/cell/h for cultures conducted in batch-agitated flasks and batch and instrumented bioreactors operated in batch and fed-batch mode.

  10. Universal pooled plasma (Uniplas(®)) does not induce complement-mediated hemolysis of human red blood cells in vitro.

    Science.gov (United States)

    Heger, Andrea; Brandstätter, Hubert; Prager, Bettina; Brainovic, Janja; Cortes, Rhoda; Römisch, Jürgen

    2015-02-01

    Pooling of plasma of different blood groups before large scale manufacturing of Uniplas(®) results in the formation of low levels of soluble immune complexes (CIC). The aim of this study was to investigate the level and removal of CIC during Uniplas(®) manufacturing. In addition, an in vitro hemolysis assay should be developed and investigate if Uniplas(®) does induce complement-mediated hemolysis of human red blood cells (RBC). In-process samples from Uniplas(®) (universal plasma) and Octaplas(LG)(®) (blood group specific plasma) routine manufacturing batches were tested on CIC using commercially available ELISA test kits. In addition, CIC was produced by admixing heat-aggregated immunoglobulins or monoclonal anti-A/anti-B antibodies to plasma and removal of CIC was followed in studies of the Uniplas(®) manufacturing process under down-scale conditions. The extent of RBC lysis was investigated in plasma samples using the in-house hemolysis assay. Levels of CIC in Uniplas(®) are within the normal ranges for plasma and comparable to that found in Octaplas(LG)(®). Down-scale experiments showed that both IgG/IgM-CIC levels are significantly removed on average by 40-50% during Uniplas(®) manufacturing. Uniplas(®) does not induce hemolysis of RBCs in vitro. Hemolysis occurs only after spiking with high titers of anti-A/anti-B antibodies and depends on the antibody specificity (i.e. titer) in the plasma sample. The results of this study confirm the safety of Uniplas(®) regarding transfusion to patients of all ABO blood groups. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Anti-cancer efficacy of nonthermal plasma dissolved in a liquid, liquid plasma in heterogeneous cancer cells

    Science.gov (United States)

    Nguyen, Ngoc Hoan; Park, Hyung Jun; Yang, Sang Sik; Choi, Kyeong Sook; Lee, Jong-Soo

    2016-07-01

    The therapeutic potential of nonthermal plasma for cancer treatment has been reported recently. The heterogeneity of cancer cells need to be addressed to design effective anticancer treatments. Here, we show that treatment with nonthermal atmospheric-pressure plasma dissolved in a liquid (liquid plasma) induces oxidative stress in heterogeneous populations of cancer cells and ultimately kills these cells via apoptosis, regardless of genetic status, e.g., mutations in p53 and other DNA-damage-response genes. We found that liquid plasma markedly increased the concentration of intracellular and mitochondrial reactive oxygen species (ROS), reflecting an influx from the extracellular milieu. Liquid plasma contributed to mitochondrial accumulation of ROS and depolarization of mitochondrial membrane potential with consequent cell death. Healthy normal cells, however, were hardly affected by the liquid-plasma treatment. The antioxidant N-acetylcysteine blocked liquid-plasma-induced cell death. A knockdown of CuZn-superoxide dismutase or Mn-SOD enhanced the plasma-induced cell death, whereas expression of exogenous CuZn-SOD, Mn-SOD, or catalase blocked the cell death. These results suggest that the mitochondrial dysfunction mediated by ROS production is a key contributor to liquid-plasma-induced apoptotic cell death, regardless of genetic variation. Thus, liquid plasma may have clinical applications, e.g., the development of therapeutic strategies and prevention of disease progression despite tumor heterogeneity.

  12. Fully human broadly neutralizing monoclonal antibodies against influenza A viruses generated from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Weibin [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Chen, Aizhong [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Miao, Yi [Shanghai Xuhui Central Hospital, Shanghai 200031 (China); Xia, Shengli [Center for Disease Control and Prevention of Henan Province, Zhengzhou 450016 (China); Ling, Zhiyang; Xu, Ke; Wang, Tongyan [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Xu, Ying; Cui, Jun; Wu, Hongqiang; Hu, Guiyu; Tian, Lin; Wang, Lingling [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Shu, Yuelong [Chinese Center for Disease Control and Prevention, Beijing 102206 (China); Ma, Xiaowei [Hualan Biological Bacterin Company, Xinxiang 453003 (China); Xu, Bianli; Zhang, Jin [Center for Disease Control and Prevention of Henan Province, Zhengzhou 450016 (China); Lin, Xiaojun, E-mail: linxiaojun@hualan.com [Hualan Biological Bacterin Company, Xinxiang 453003 (China); Bian, Chao, E-mail: cbian@sibs.ac.cn [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Sun, Bing, E-mail: bsun@sibs.ac.cn [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)

    2013-01-20

    Whether the 2009 pandemic H1N1 influenza vaccine can induce heterosubtypic cross-protective anti-hemagglutinin (HA) neutralizing antibodies is an important issue. We obtained a panel of fully human monoclonal antibodies from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient. Most of the monoclonal antibodies targeted the HA protein but not the HA1 fragment. Among the analyzed antibodies, seven mAbs exhibited neutralizing activity against several influenza A viruses of different subtypes. The conserved linear epitope targeted by the neutralizing mAbs (FIEGGWTGMVDGWYGYHH) is part of the fusion peptide on HA2. Our work suggests that a heterosubtypic neutralizing antibody response primarily targeting the HA stem region exists in recipients of the 2009 pandemic H1N1 influenza vaccine. The HA stem region contains various conserved neutralizing epitopes with the fusion peptide as an important one. This work may aid in the design of a universal influenza A virus vaccine.

  13. Transplante de células-tronco hematopoéticas em gamopatias monoclonais Hematopoietic stem cell transplantation for monoclonal gammopathies

    Directory of Open Access Journals (Sweden)

    Angelo Maiolino

    2010-05-01

    Full Text Available O transplante de células-tronco hematopoéticas (TCTH é um procedimento de fundamental importância na estratégia terapêutica das gamopatias monoclonais. No mieloma múltiplo, em particular, o TCTH autólogo está indicado como estratégia de primeira linha para pacientes até 70 anos de idade. Nesta capítulo serão discutidas as indicações, estratégias e recomendações envolvendo o TCTH em gamopatias monoclonais, amiloidose e POEMS, frutos da Reunião de Consenso da Sociedade Brasileira de Transplante de Medula Óssea.Hematopoietic stem cell transplantation (HSCT is an important strategy in the treatment of monoclonal gammopathies. For multiple myeloma, in particular, autologous HSCT is indicated as first line therapy for under 70-year-old patients. In this chapter we will discuss indications, strategies and recommendations involving HSCT for monoclonal gammopathies from the Consensus Meeting of the Brazilian Society of Bone Marrow Transplantation.

  14. Plasma cell gingivitis associated with cheilitis: A diagnostic dilemma!

    Directory of Open Access Journals (Sweden)

    Presanthila Janam

    2012-01-01

    Full Text Available Plasma cell gingivitis is a rare condition characterized by diffuse and massive infiltration of plasma cells into the sub-epithelial connective tissue. Clinically, it appears as a diffuse reddening and edematous swelling of the gingiva with a sharp demarcation along the mucogingival border. Though considered as a hypersensitive reaction to an allergen, the etiology of this bizarre condition is still not properly understood. Here, we present an interesting case of plasma cell gingivitis associated with an enlarged and fissured upper lip, which is quite a rarity. The condition was diagnosed based on clinical and histopathologic findings and treated by gingivectomy. The associated cheilitis has dramatically reduced after treatment of the gingival lesion.

  15. Single cell cloning and recombinant monoclonal antibodies generation from RA synovial B cells reveal frequent targeting of citrullinated histones of NETs

    Science.gov (United States)

    Corsiero, Elisa; Bombardieri, Michele; Carlotti, Emanuela; Pratesi, Federico; Robinson, William; Migliorini, Paola; Pitzalis, Costantino

    2016-01-01

    Objectives Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated antigens with generation of anti-citrullinated peptide/proteins antibodies (ACPA). Currently, the nature and source of citrullinated antigens driving the humoral autoimmune response within synovial ectopic lymphoid structures (ELS) is a crucial unknown aspect of RA pathogenesis. Here we characterised the autoreactive B-cell response of lesional B cells isolated from ELS+RA synovium. Methods Single synovial tissue CD19+cells were Fluorescence Activated Cell Sorting (FACS)-sorted and VH/VL Ig genes cloned to generate recombinant monoclonal antibodies (rmAbs) from patients with ELS+/ACPA+RA. Results RA-rmAbs immunoreactivity analysis provided the following key findings: (1) in a chIP-based array containing 300 autoantigens and in a ‘citrullinome’ multiplex assay, a strong reactivity against citrullinated histones H2A/H2B (citH2A/H2B) was observed in ∼40% of RA-rmAbs, followed by cit-fibrinogen and cit-vimentin; (2) anti-citH2A/H2B-reactive RA-rmAbs (but not anti-citH2A/H2B negative) selectively recognised neutrophil extracellular traps (NETs) from peripheral blood and/or RA joint neutrophils; (3) anti-citH2A/citH2B and anti-NET immunobinding was dependent on affinity maturation and was completely abrogated following reversion of hypermutated IgVH/VL genes to germline sequences; (4) ELS+ (not ELS−) RA synovial tissues engrafted into Severe Combined ImmunoDeficiency (SCID) mice released human anti-citH2A/citH2B and anti-NET antibodies in association with the intra-graft expression of CXCL13 and lymphotoxin (LT)-β, two master regulators of ELS. Conclusion We provided novel evidence that B cells differentiated within synovial ELS in the RA joints frequent target deiminated proteins which could be generated during NETosis of RA synovial neutrophils including histones. Thus, NETs could represent a source of citrullinated antigens fuelling the ACPA autoimmune

  16. Intrinsic Constraint on Plasmablast Growth and Extrinsic Limits of Plasma Cell Survival

    OpenAIRE

    2000-01-01

    B cells recruited into splenic antibody responses grow exponentially, either in extrafollicular foci as plasmablasts, or in follicles where they form germinal centers. Both responses yield plasma cells. Although many splenic plasma cells survive

  17. Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells.

    Directory of Open Access Journals (Sweden)

    Dale O Starkie

    Full Text Available Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive. These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking

  18. Prevalence of Monoclonal Gammopathy of Undetermined Significance: A Systematic Review

    Science.gov (United States)

    Wadhera, Rishi K.; Rajkumar, S. Vincent

    2010-01-01

    Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant plasma cell disorder that is associated with a lifelong risk of multiple myeloma. We conducted a systematic review of all studies investigating the prevalence and incidence of MGUS in the online database PubMed. The review was conducted from January 6, 2009, through January 15, 2010. The following MeSH search headings were used: monoclonal gammopathy, benign and prevalence; monoclonal gammopathy, benign and incidence; paraproteinemia and prevalence; and paraproteinemia and incidence. Articles were limited to those written in English and published by January 2009. Fourteen studies that met prespecified criteria were included and systematically assessed to identify the most accurate prevalence estimates of MGUS based on age, sex, and race. On the basis of our systematic review, we estimate that the crude prevalence of MGUS in those older than 50 years is 3.2% in a predominantly white population. Studies in white and Japanese populations demonstrate a clear increase in prevalence with age. The prevalence is also affected by sex: 3.7% and 2.9% in white men and women, respectively; and 2.8% and 1.6% in Japanese men and women, respectively. Additionally, MGUS is significantly more prevalent in black people (5.9%-8.4%) than in white people (3.0%-3.6%). We conclude that MGUS is a common premalignant plasma cell disorder in the general population of those older than 50 years. The prevalence increases with age and is affected by race, sex, family history, immunosuppression, and pesticide exposure. These results are important for counseling, clinical care, and the design of clinical studies in high-risk populations. PMID:20713974

  19. An agonistic monoclonal antibody against DR5 induces ROS production, sustained JNK activation and Endo G release in Jurkat leukemia cells

    Institute of Scientific and Technical Information of China (English)

    Caifeng Chen; Yanxin Liu; Dexian Zheng

    2009-01-01

    We have previously reported that AD5-10, a novel agonistic monoclonal antibody against DRS, possessed a strong cytotoxic activity in various tumor cells, via induction of caspase-dependent and-independent signaling pathways. The present study further demonstrates that reactive oxygen species (ROS) were generated in abundance in Jurkat leukemia cells upon ADS-10 stimulation and that ROS accumulation subsequently evoked sustained activation of c-Jun N-terminal kinase (JNK), loss of mitochondrial membrane potential, and release of endonuclease G (Endo G) from mitochondria into the cytosol. The reducing agent, N-acetylcysteine (NAC), effectively inhibited the sustained activation of JNK, release of Endo G, and cell death in Jurkat cells treated by ADS-10. Moreover, a dominant-nega-tive form of JNK (but not of p38) enhanced NF-KB activation, suppressed caspase-8 recruitment in death-inducing signaling complexes (DISCs), and reduced adverse effects on mitochondria, thereby inhibiting AD5-10-induced cell death in Jurkat leukemia cells. These data provide novel information on the DRS-mediated cell death-signaling path-way and may shed new light on effective strategies for leukemia and solid tumor therapies.

  20. Performance goals for immunoglobulins and serum free light chain measurements in plasma cell dyscrasias can be based on biological variation

    DEFF Research Database (Denmark)

    Hansen, Charlotte Toftmann

    2016-01-01

    , data based on biological variation is a good and reliable method for setting desirable performance standards; this also applies for the measurements of paraprotein and sFLC. The benefits of this approach are several. Among others, it is independent of the clinician, and it provides us with information......Measurements of immunoglobulins and serum free light chains (sFLC) are frequently used in patients with monoclonal plasma cell dyscrasia (PCD). For optimum patient care, well-defined performance standards or goals for the measured concentrations of immunoglobulins and sFLC are required. Generally...... of immunoglobulins and sFLC, studies accounting for number of individuals, samples, and replicates, as well as time length of the studies are needed....

  1. beta-Adrenergic agonist activity of a monoclonal anti-idiotypic antibody.

    Science.gov (United States)

    Guillet, J G; Kaveri, S V; Durieu, O; Delavier, C; Hoebeke, J; Strosberg, A D

    1985-03-01

    Hybridoma cells bearing monoclonal antibody against the beta-adrenergic ligand alprenolol were used as an immunogen to raise monoclonal anti-idiotypic antibodies. Of six anti-idiotypic antibodies, which inhibit ligand binding, three were able to recognize beta-adrenergic receptors. One of them, mAb2B4, an IgM that could be amplified into ascites, binds to the beta-adrenergic catecholamine receptors of intact epidermoid A431 cells and precipitates receptors solubilized from plasma membranes by digitonin. This antibody identifies the beta 2-adrenergic receptor of A431 cells as a single 55-kDa protein and stimulates adenylate cyclase activity. This stimulation is inhibited by the beta-adrenergic antagonist propranolol.

  2. Distinct T helper cell dependence of memory B-cell proliferation versus plasma cell differentiation.

    Science.gov (United States)

    Zabel, Franziska; Fettelschoss, Antonia; Vogel, Monique; Johansen, Pål; Kündig, Thomas M; Bachmann, Martin F

    2017-03-01

    Several memory B-cell subclasses with distinct functions have been described, of which the most effective is the class-switched (CS) memory B-cell population. We have previously shown, using virus-like particles (VLPs), that the proliferative potential of these CS memory B cells is limited and they fail to re-enter germinal centres (GCs). However, VLP-specific memory B cells quickly differentiated into secondary plasma cells (PCs) with the virtue of elevated antibody production compared with primary PCs. Whereas the induction of VLP(+) memory B cells was strongly dependent on T helper cells, we were wondering whether re-stimulation of VLP(+) memory B cells and their differentiation into secondary PCs would also require T helper cells. Global absence of T helper cells led to strongly impaired memory B cell proliferation and PC differentiation. In contrast, lack of interleukin-21 receptor-dependent follicular T helper cells or CD40 ligand signalling strongly affected proliferation of memory B cells, but differentiation into mature secondary PCs exhibiting increased antibody production was essentially normal. This contrasts with primary B-cell responses, where a strong dependence on CD40 ligand but limited importance of interleukin-21 receptor was seen. Hence, T helper cell dependence differs between primary and secondary B-cell responses as well as between memory B-cell proliferation and PC differentiation.

  3. Antibody repertoire diversification through VH gene replacement in mice cloned from an IgA plasma cell.

    Science.gov (United States)

    Kumar, Rashmi; Bach, Martina P; Mainoldi, Federica; Maruya, Mikako; Kishigami, Satoshi; Jumaa, Hassan; Wakayama, Teruhiko; Kanagawa, Osami; Fagarasan, Sidonia; Casola, Stefano

    2015-02-01

    In mammals, VDJ recombination is responsible for the establishment of a highly diversified preimmune antibody repertoire. Acquisition of a functional Ig heavy (H) chain variable (V) gene rearrangement is thought to prevent further recombination at the IgH locus. Here, we describe VHQ52(NT); Vκgr32(NT) Ig monoclonal mice reprogrammed from the nucleus of an intestinal IgA(+) plasma cell. In VHQ52(NT) mice, IgA replaced IgM to drive early B-cell development and peripheral B-cell maturation. In VHQ52(NT) animals, over 20% of mature B cells disrupted the single productive, nonautoimmune IgH rearrangement through VH replacement and exchanged it with a highly diversified pool of IgH specificities. VH replacement occurred in early pro-B cells, was independent of pre-B-cell receptor signaling, and involved predominantly one adjacent VH germ-line gene. VH replacement was also identified in 5% of peripheral B cells of mice inheriting a different productive VH rearrangement expressed in the form of an IgM H chain. In summary, editing of a productive IgH rearrangement through VH replacement can account for up to 20% of the IgH repertoire expressed by mature B cells.

  4. Disturbed B cell subpopulations and increased plasma cells in myasthenia gravis patients.

    Science.gov (United States)

    Kohler, Siegfried; Keil, Thomas Oskar Philipp; Swierzy, Marc; Hoffmann, Sarah; Schaffert, Hanne; Ismail, Mahmoud; Rückert, Jens Carsten; Alexander, Tobias; Hiepe, Falk; Gross, Christian; Thiel, Andreas; Meisel, Andreas

    2013-11-15

    Whether there is a general perturbation of B and plasma cell subsets in myasthenia gravis (MG) has not been investigated so far. Here we performed a detailed flow cytometric analysis of blood and if available thymic tissue in order to detect MG-specific and therapy-induced changes. We observed significant differences in the distribution of B cell subsets in MG patients, yet these were mainly attributable to medical treatment. Furthermore MG is associated with significantly increased frequencies of plasma cells that were especially activated in purely ocular disease manifestation. In contrast to thymoma, B cell subset distribution in hyperplastic thymus could be distinguished from peripheral blood, however both tissues were not significantly enriched with plasma cells. Thus B cell differentiation in general is not defective in MG, but modified by therapy and enhanced frequencies of plasma cells can be detected in MG patients.

  5. Gamopatias monoclonais: critérios diagnósticos e diagnósticos diferenciais Monoclonal gammopathies: diagnosis criteria and differential diagnosis

    Directory of Open Access Journals (Sweden)

    Rosa Malena D. Faria

    2007-03-01

    Full Text Available As gamopatias monoclonais constituem um grupo de desordens caracterizado pela proliferação monoclonal de plasmócitos, que produzem e secretam imunoglobulina ou fragmento de imunoglobulina monoclonal (proteína M . Este artigo propõe uma revisão dos critérios diagnósticos das principais gamopatias monoclonais e diagnósticos diferenciais, uma vez que é comum a sobreposição de muitas características clínicas entre suas variantes. A gamopatia monoclonal de significado indeterminado (MGUS é definida pela presença de proteína M sérica 30% ou plasmocitoma documentado por biópsia. Se a lesão óssea decorre de plasmocitoma solitário ou somente osteoporose, sem fratura, a plasmocitose medular também precisa ser > 30%, para preencher critérios de MM. As gamopatias monoclonais podem estar associadas a diversas doenças, incluindo desordens linfoproliferativas, reumatológicas, neurológicas, dermatológicas e infecciosas. A definição das características clínicas e laboratoriais de cada entidade, maligna ou benigna, facilita o diagnóstico das gamopatias monoclonais e, como conseqüência, seu manejo clínico pelos médicos assistentes.Monoclonal gammopathies are a group of disorders characterized by proliferation of monoclonal plasma cells, which produce and secrete monoclonal immunoglobulin or fragments of monoclonal immunoglobulin (M protein. This paper proposes to review diagnostic criteria of the most important monoclonal gammopathies and their differential diagnosis, because superposition of many clinical characteristics is common between variants. The monoclonal gammopathy of undetermined significance (MGUS is defined by the presence of serum M protein < 3g/dL and/or urinary M protein < 1g/24h, bone marrow plasma cell < 10%, and absence of organ and tissue damage. Asymptomatic multiple myeloma (MM is characterized by the presence of M protein, bone marrow or tissue biopsy plasma cell infiltration, and non-compliance of the

  6. Effect of a GABA agonist on the expression and distribution of GABAA receptors in the plasma membrane of cultured cerebellar granule cells: an immunocytochemical study

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Belhage, B; Schousboe, A

    1991-01-01

    , the density of the GABAA receptors was significantly increased in the THIP-treated cultures as compared to the control cultures and this effect of THIP was particularly pronounced in the processes. GABAA receptors were occasionally observed to form 'hot spots' in process-like structures and again......The effect of the gamma-aminobutyric acid (GABA) agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 microM) on the localization and density of GABAA receptors in the plasma membrane of rat cerebellar granule cells in primary cultures was studied at the electron microscope (EM) level...... by preembedding immunogold staining using the monoclonal antibody bd-17 directed against the beta-subunit of the GABAA receptor complex. In THIP-treated as well as untreated control cultures, GABAA receptors were found to be evenly distributed in the plasma membrane of cell bodies as well as processes. However...

  7. Combination of monoclonal antibodies with DST inhibits accelerated rejection mediated by memory T cells to induce long-lived heart allograft acceptance in mice.

    Science.gov (United States)

    Shao, Wei; Chen, Jibing; Dai, Helong; Peng, Yuanzheng; Wang, Feng; Xia, Junjie; Thorlacius, Henrik; Zhu, Qi; Qi, Zhongquan

    2011-08-30

    Donor-reactive memory T cells mediated accelerated rejection is known as a barrier to the survival of transplanted organs. We investigated the combination of different monoclonal antibodies (mAbs) and donor-specific transfusion (DST) in memory T cells-based adoptive mice model. In the presence of donor-reactive memory T cells, the mean survival time (MST) of grafts in the anti-CD40L/LFA-1/DST group was 49.8d. Adding anti-CD44/CD70 mAbs to anti-CD40L/LFA-1/DST treatment. The MST was more than 100 d (MST>100 d). Compared with anti-CD40L/LFA-1/DST group, anti-CD40L/LFA-1/CD44/CD70/DST group notably reduced the expansion of memory T cells, enhanced the proportion of CD4+Foxp3+ regulatory T cells (Tregs) and suppressed donor-specific responses. Our data suggest that anti-CD40L/LFA-1/CD44/CD70mAbs and DST can synergistically inhibit accelerated rejection mediated by memory T cells to induce long-lived heart allograft acceptance in mice.

  8. A Review of Obinutuzumab (GA101), a Novel Type II Anti-CD20 Monoclonal Antibody, for the Treatment of Patients with B-Cell Malignancies.

    Science.gov (United States)

    Tobinai, Kensei; Klein, Christian; Oya, Naoko; Fingerle-Rowson, Günter

    2017-02-01

    Obinutuzumab (GA101) is a novel, type II, glycoengineered, humanized anti-CD20 monoclonal antibody that has been developed to address the need for new therapeutics with improved efficacy in patients with lymphocytic leukemia and lymphoma of B-cell origin. Obinutuzumab has a distinct mode of action relative to type I anti-CD20 antibodies, such as rituximab, working primarily by inducing direct cell death and antibody-dependent cell-mediated cytotoxicity. Obinutuzumab is under investigation in a wide-ranging program of clinical trials in patients with B-cell malignancies. Efficacy as monotherapy has been reported in patients with relapsed/refractory indolent and aggressive non-Hodgkin lymphoma (NHL) and in chronic lymphocytic leukemia (CLL) of B-cell origin. Improved outcomes have also been noted when obinutuzumab is added to chemotherapy in patients with B-cell NHL, and superiority over rituximab has been reported with combination therapy in patients with CLL. Ongoing research is focusing on developing options for chemotherapy-free treatment and on new combinations of obinutuzumab with novel targeted agents.

  9. Monoclonal antibodies specific for Candida albicans Als3 that immunolabel fungal cells in vitro and in vivo and block adhesion to host surfaces

    Science.gov (United States)

    Coleman, David A.; Oh, Soon-Hwan; Zhao, Xiaomin; Zhao, Hongyuan; Hutchins, Jeff T.; Vernachio, John H.; Patti, Joseph M.; Hoyer, Lois L.

    2009-01-01

    Two monoclonal antibodies (MAbs) were raised against the Candida albicans cell-surface glycoprotein Als3 using the N-terminal domain of the protein as the immunogen. ELISA was used to demonstrate the specificity of the MAbs for the Als3 fragment, but not for the corresponding N-terminal domain fragments from other proteins in the Als family. The anti-Als3 MAbs immunolabeled the surface of germ tubes from a diverse collection of wild-type C. albicans isolates, but did not label yeast cells, an als3Δ/als3Δ deletion mutant strain, nor isolates of other Candida species associated with human disease. Als3 was visualized readily in fresh and formalin-fixed, paraffin-embedded kidney tissue from a murine model of candidiasis. The anti-Als3 MAbs were also useful for immunogold electron microscopy and Western blotting. Both MAbs blocked C. albicans adhesion to vascular endothelial cells and buccal epithelial cells. These versatile MAbs are a valuable addition to the reagents available to study C. albicans cell surface dynamics and interaction of the fungus with host cells. PMID:19427882

  10. High zinc ion supplementation of more than 30 μM can increase monoclonal antibody production in recombinant Chinese hamster ovary DG44 cell culture.

    Science.gov (United States)

    Kim, Bong Gyun; Park, Hong Woo

    2016-03-01

    Effects of high ZnSO4·7H2O supplementation on cell growth and monoclonal antibody (mAb) production in chemically defined suspension cultures of recombinant Chinese hamster ovary (rCHO) DG44 cells were examined. The supplementation of ZnSO4·7H2O up to 120 μM gradually increased specific mAb production rate of rCHO DG44 cells in the early growth phase (0-4 days of culture). The ZnSO4·7H2O concentration for enhancing mAb production without any cytotoxic effects on cell growth was 30-60 μM. In addition of 60 μM ZnSO4·7H2O to in-house protein-free medium and in-house chemically defined medium, mAb production was increased 2.0-fold and 6.5-fold, respectively. Moreover, addition of ZnSO4·7H2O to three kinds of commercial chemically defined media yielded a greater than 1.2-fold enhancement of mAb production. These data indicate that simple supplementation of a relatively high zinc ion concentration to cell culture media without significant changes of rCHO DG44 cell culture process can be useful for achieving high production of mAb.

  11. A novel stem cell associated marker identified by monoclonal antibody HESC5:3 differentiates between neoplastic lesions in follicular thyroid neoplasms.

    Science.gov (United States)

    Heikkilä, Annukka; Fermér, Christian; Hagström, Jaana; Louhimo, Johanna; Mäenpää, Hanna; Siironen, Päivi; Heiskanen, Ilkka; Nilsson, Olle; Arola, Johanna; Haglund, Caj

    2015-07-01

    Follicular thyroid lesions are the bane of cytopathology. Differentiation between adenoma and carcinoma is impossible, and often these neoplasms are indistinguishable even from uninodular goitre. In other cancers as well, a theory of stem cells as the origin of cancer has been discussed in thyroid carcinogenesis. We aimed to examine a novel stem cell associated marker identified by monoclonal antibody HESC5:3 in follicular lesions in an attempt to find a marker for differential diagnosis in thyroid cytopathology. HESC5:3 was raised against and is specific for undifferentiated human embryonic stem cells. The epitope of this novel antibody is to be defined. Immunohistochemical expression of HESC5:3 was examined in clinical material comprised of follicular neoplasms (83 adenomas, 43 carcinomas) and non-neoplastic lesions (41 goitrous, 22 hyperplastic, 23 normal tissue specimens). Staining differed significantly between neoplastic and non-neoplastic lesions. Nuclear staining was increased in non-neoplastic cells, whereas in neoplastic cells expression was mainly cytoplasmic. There was no difference between benign and malignant lesions, suggesting a role in early tumourigenesis. In conclusion, the HESC5:3 epitope may be of benefit as a neoplasia marker in distinguishing between uninodular goitre and neoplasia. Characterization of the epitope would increase the interest in this promising new stem cell associated marker.

  12. Complement-Dependent Lysis of Influenza A Virus-Infected Cells by Broadly Cross-Reactive Human Monoclonal Antibodies ▿

    Science.gov (United States)

    Terajima, Masanori; Cruz, John; Co, Mary Dawn T.; Lee, Jane-Hwei; Kaur, Kaval; Wilson, Patrick C.; Ennis, Francis A.

    2011-01-01

    We characterized human monoclonal antibodies (MAbs) cloned from influenza virus-infected patients and from influenza vaccine recipients by complement-dependent lysis (CDL) assay. Most MAbs active in CDL were neutralizing, but not all neutralizing MAbs can mediate CDL. Two of the three stalk-specific neutralizing MAbs tested were able to mediate CDL and were more cross-reactive to temporally distant H1N1 strains than the conventional hemagglutination-inhibiting and neutralizing MAbs. One of the stalk-specific MAbs was subtype cross-reactive to H1 and H2 hemagglutinins, suggesting a role for stalk-specific antibodies in protection against influenza illness, especially by a novel viral subtype which can cause pandemics. PMID:21994454

  13. Identification of a linear B-cell epitope on the avian leukosis virus P27 protein using monoclonal antibodies.

    Science.gov (United States)

    Li, Xiaofei; Qin, Liting; Zhu, Haibo; Sun, Yingjun; Cui, Xuezhi; Gao, Yadong; Qi, Xiaole; Wang, Yongqiang; Gao, Honglei; Gao, Yulong; Wang, Xiaomei

    2016-10-01

    Avian leukosis virus (ALV) is an avian oncogenic retrovirus that can induce various clinical tumors. The capsid protein P27 is the group-specific antigen of ALV and has many viral antigen sites that are easy to detect. In this study, we produced a monoclonal antibody (mAb), 3A9, that is specific for the P27 protein. A series of partially overlapping peptides were screened to define (181)PPSAR(185) as the minimal linear epitope recognized by mAb 3A9. The identified epitope could be recognized by chicken anti-ALV and mouse anti-ALV P27 sera. The epitope was highly conserved among a number of ALV-A, ALV-B and ALV-J strains. MAb 3A9 might be a valuable tool for the development of new immunodiagnostic approaches for ALV, and the defined linear epitope might help further our understanding of the antigenic structure of the P27 protein.

  14. The effectiveness of an anti-human IL-6 receptor monoclonal antibody combined with chemotherapy to target colon cancer stem-like cells.

    Science.gov (United States)

    Ying, Jin; Tsujii, Masahiko; Kondo, Jumpei; Hayashi, Yoshito; Kato, Motohiko; Akasaka, Tomofumi; Inoue, Takuta; Shiraishi, Eri; Inoue, Tahahiro; Hiyama, Satoshi; Tsujii, Yoshiki; Maekawa, Akira; Kawai, Shoichiro; Fujinaga, Tetsuji; Araki, Maekawa; Shinzaki, Shinichiro; Watabe, Kenji; Nishida, Tsutomu; Iijima, Hideki; Takehara, Tetsuo

    2015-04-01

    Recent studies have demonstrated that cancer stem cells (CSCs) can initiate and sustain tumor growth and exhibit resistance to clinical cytotoxic therapies. Therefore, CSCs represent the main target of anticancer therapy. Interleukin-6 (IL-6) promotes cellular proliferation and drug resistance in colorectal cancer, and its serum levels correlate with patient survival. Therefore, IL-6 and its downstream signaling molecule the signal transducer and activator of transcription-3 (STAT3) represent potential molecular targets. In the present study, we investigated the effects of IL-6 and its downstream signaling components on stem cell biology, particularly the chemoresistance of CSCs, to explore potential molecular targets for cancer therapy. The colon cancer cell line WiDr was cultured in serum-free, non-adherent, and three-dimensional spheroid-forming conditions to enrich the stem cell-like population. Spheroid-forming cells slowly proliferated and expressed high levels of Oct-4, Klf4, Bmi-1, Lgr5, IL-6, and Notch 3 compared with adherent cells. Treatment with an anti-human IL-6 receptor monoclonal antibody reduced spheroid formation, stem cell-related gene expression, and 5-fluorouracil (5-FU) resistance. In addition, IL-6 treatment enhanced the levels of p-STAT3 (Tyr705), the expression of Oct-4, Klf4, Lgr5, and Notch 3, and chemoresistance to 5-FU. siRNA targeting Notch 3 suppressed spheroid formation, Oct-4 and Lgr5 expression, and 5-FU chemoresistance, whereas STAT3 inhibition enhanced Oct-4, Klf4, Lgr5, and Notch 3 expression and 5-FU chemoresistance along with reduced spheroid growth. Taken together, these results indicate that IL-6 functions in dichotomous pathways involving Notch 3 induction and STAT3 activation. The former pathway is involved in cancer stem-like cell biology and enhanced chemoresistance, and the latter pathway leads to accelerated proliferation and reduced chemoresistance. Thus, an anti-human IL-6 receptor monoclonal antibody or Notch 3

  15. Long-lived plasma cells in autoimmunity: lessons from B-cell depleting therapy.

    Science.gov (United States)

    Mahévas, Matthieu; Michel, Marc; Weill, Jean-Claude; Reynaud, Claude-Agnès

    2013-12-27

    A large number of auto-immune diseases are treated with rituximab, an antibody against CD20 that depletes most of the B-cells in the organism. The response to this treatment depends largely on the disease and the type of lymphoid cells involved in the auto-immune process. We recently reported that B-cell depletion in immune thrombocytopenia induced the appearance of pathogenic long-lived plasma cells in the spleen, which were not present before treatment or in non-auto-immune conditions. The spleen of treated patients produced an excess of the cytokine B-cell activating factor, which in in vitro-cultured splenic cells, could increase the longevity of plasma cells. Our results suggested that, paradoxically, the B-cell depletion itself, by altering the splenic milieu, promoted the differentiation of short-lived auto-immune plasma cells into long-lived ones. We describe the cellular and cytokinic components of the splenic plasma cell niche, notably CD4(+) T cells and discuss possible survival factors that could be targeted simultaneously with rituximab-mediated B-cell depletion to interfere with plasma cell persistence.

  16. Controls to validate plasma samples for cell free DNA quantification

    DEFF Research Database (Denmark)

    Pallisgaard, Niels; Spindler, Karen-Lise Garm; Andersen, Rikke Fredslund;

    2015-01-01

    Recent research has focused on the utility of cell free DNA (cfDNA) in serum and plasma for clinical application, especially in oncology. The literature holds promise of cfDNA as a valuable tumour marker to be used for treatment selection, monitoring and follow-up. The results, however, are diver...

  17. Calculated characteristics of an ac plasma display panel cell

    Energy Technology Data Exchange (ETDEWEB)

    Boeuf, J.P.; Pitchford, L.C. [Universite Paul Sabatier, Toulouse (France)

    1996-02-01

    Equipotential contours and contours of constant power deposition into excitation of xenon, calculated from a two-dimensional (2-D) fluid model, are presented at times during the evolution of a single discharge event in an ac plasma display panel cell with a dielectric barrier rib geometry.

  18. Differential expression of SLAMS and other modulatory molecules by human plasma cells during normal maturation.

    Science.gov (United States)

    Rodríguez-Bayona, Beatriz; Ramos-Amaya, Ana; Brieva, José A

    2011-01-30

    Plasma cells (PCs) are specialized in antibody (Ab) production and they are, therefore, responsible for maintaining humoral immune responses. The human PC compartment is heterogeneous. PCs from inductive secondary lymphoid organs and from peripheral blood (PB) show less capability for prolonged survival and Ab production than bone marrow (BM) PCs, a pool consisting of fully mature cells. The HLDA9 workshop has allowed the use of labeled-monoclonal Abs (moAbs) recognizing a variety of recently identified lymphocyte modulatory surface receptors. In this study, flow cytometry analysis has been used to define the presence of these receptors on human PCs obtained from human tonsil (as an example of inductive organ), from PB and from BM. It was found that human PCs commonly expressed SLAMF1 (CD150), SLAMF2 (CD48), SLAMF3 (CD229), SLAMF6 (CD352) and SLAMF7 (CD319), but not SLAMF4 (CD244). In addition, PCs distinctively showed a low level of SLAMF5 (CD84) and a very high level of SLAMF7 expression in comparison with earlier stages of B cell maturation. All PC subsets exhibited a similar pattern of expression of SLAMF receptors suggesting a stage-dependent role for these proteins. In addition, most circulating PCs clearly expressed TNFRSF14 (CD270), BTLA (CD272), B7-1 (CD80) and B7-2 (CD86), and a substantial fraction of them were also positive for TNFRSF18 (CD357), FCRL1 (CD307a) and LAIR-1 (CD305). In contrast, tonsil and BM PCs only exhibited partial expression of TNFRSF14 and B7-2, a pattern of molecular expression similar to that detected on germinal center (GC) B cells. Present results indicate that human PCs exhibit a common pattern of SLAMF proteins, but differ in the rest of the receptors examined; this difference might be associated with their distinctive homing and functional requirements. Copyright © 2010 Elsevier B.V. All rights reserved.

  19. A novel, blocking, Fc-silent anti-CD40 monoclonal antibody prolongs nonhuman primate renal allograft survival in the absence of B cell depletion.

    Science.gov (United States)

    Cordoba, F; Wieczorek, G; Audet, M; Roth, L; Schneider, M A; Kunkler, A; Stuber, N; Erard, M; Ceci, M; Baumgartner, R; Apolloni, R; Cattini, A; Robert, G; Ristig, D; Munz, J; Haeberli, L; Grau, R; Sickert, D; Heusser, C; Espie, P; Bruns, C; Patel, D; Rush, J S

    2015-11-01

    CD40-CD154 pathway blockade prolongs renal allograft survival in nonhuman primates (NHPs). However, antibodies targeting CD154 were associated with an increased incidence of thromboembolic complications. Antibodies targeting CD40 prolong renal allograft survival in NHPs without thromboembolic events but with accompanying B cell depletion, raising the question of the relative contribution of B cell depletion to the efficacy of anti-CD40 blockade. Here, we investigated whether fully silencing Fc effector functions of an anti-CD40 antibody can still promote graft survival. The parent anti-CD40 monoclonal antibody HCD122 prolonged allograft survival in MHC-mismatched cynomolgus monkey renal allograft transplantation (52, 22, and 24 days) with accompanying B cell depletion. Fc-silencing yielded CFZ533, an antibody incapable of B cell depletion but still able to potently inhibit CD40 pathway activation. CFZ533 prolonged allograft survival and function up to a defined protocol endpoint of 98-100 days (100, 100, 100, 98, and 76 days) in the absence of B cell depletion and preservation of good histological graft morphology. CFZ533 was well-tolerated, with no evidence of thromboembolic events or CD40 pathway activation and suppressed a gene signature associated with acute rejection. Thus, use of the Fc-silent anti-CD40 antibody CFZ533 appears to be an attractive approach for preventing solid organ transplant rejection.

  20. Systematization of the Mechanism by Which Plasma Irradiation Causes Cell Growth and Tumor Cell Death

    Science.gov (United States)

    Shimizu, Nobuyuki

    2015-09-01

    New methods and technologies have improved minimally invasive surgical treatment and saved numerous patients. Recently, plasma irradiation has been demonstrated that might be useful in medical field and the plasma irradiation device is expected to become practically applicable. Mild plasma coagulator showed some advantages such as hemostasis and adhesion reduction in experimental animal model, but the mechanism of plasma irradiation remains unclear. Our study group aim to clarify the mechanism of plasma irradiation effects, mainly focusing on oxidative stress using cultured cell lines and small animal model. First, a study using cultured cell lines showed that the culture medium that was activated by plasma irradiation (we called this kind of medium as ``PAM'' -plasma activated medium-) induced tumor cell death. Although this effect was mainly found to be due to hydrogen peroxide, the remaining portion was considered as the specific effect of the plasma irradiation and we are now studying focusing on this effect. Second, we established a mouse intra-peritoneal adhesion model and checked biological reaction that occurred in the adhesion part. Histopathological study showed inflammatory cells infiltration into adhesion part and the expression of PTX3 that might involve tissue repair around adhesion part. We also confirmed that cytokines IL-6 and IL-10 might be useful as a marker of adhesion formation in this model. Applying ``PAM'' or mild plasma irradiation in this model, we examine the effects of plasma on inflamed cells. The samples in these experiments would be applied to targeted proteomics analysis, and we aim to demonstrate the systematization of the cell's reaction by plasma irradiation.

  1. Circulating human B and plasma cells. Age-associated changes in counts and detailed characterization of circulating normal CD138(-) and CD138(+) plasma cells

    NARCIS (Netherlands)

    Caraux, Anouk; Klein, Bernard; Paiva, Bruno; Bret, Caroline; Schmitz, Alexander; Fuhler, Gwenny M.; Bos, Nico A.; Johnsen, Hans E.; Orfao, Alberto; Perez-Andres, Martin

    2010-01-01

    Generation of B and plasma cells involves several organs with a necessary cell trafficking between them. A detailed phenotypic characterization of four circulating B-cell subsets (immature-, naive-, memory- B-lymphocytes and plasma cells) of 106 healthy adults was realized by multiparametric flow cy

  2. The AP-1 transcription factor Fra1 inhibits follicular B cell differentiation into plasma cells.

    Science.gov (United States)

    Grötsch, Bettina; Brachs, Sebastian; Lang, Christiane; Luther, Julia; Derer, Anja; Schlötzer-Schrehardt, Ursula; Bozec, Aline; Fillatreau, Simon; Berberich, Ingolf; Hobeika, Elias; Reth, Michael; Wagner, Erwin F; Schett, Georg; Mielenz, Dirk; David, Jean-Pierre

    2014-10-20

    The cornerstone of humoral immunity is the differentiation of B cells into antibody-secreting plasma cells. This process is tightly controlled by a regulatory gene network centered on the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1). Proliferation of activated B cells is required to foster Blimp1 expression but needs to be terminated to avoid overshooting immune reactions. Activator protein 1 (AP-1) transcription factors become quickly up-regulated upon B cell activation. We demonstrate that Fra1, a Fos member of AP-1, enhances activation-induced cell death upon induction in activated B cells. Moreover, mice with B cell-specific deletion of Fra1 show enhanced plasma cell differentiation and exacerbated antibody responses. In contrast, transgenic overexpression of Fra1 blocks plasma cell differentiation and immunoglobulin production, which cannot be rescued by Bcl2. On the molecular level, Fra1 represses Blimp1 expression and interferes with binding of the activating AP-1 member c-Fos to the Blimp1 promoter. Conversely, overexpression of c-Fos in Fra1 transgenic B cells releases Blimp1 repression. As Fra1 lacks transcriptional transactivation domains, we propose that Fra1 inhibits Blimp1 expression and negatively controls plasma cell differentiation through binding to the Blimp1 promoter. In summary, we demonstrate that Fra1 negatively controls plasma cell differentiation by repressing Blimp1 expression.

  3. T cells and T-cell subsets in mycosis fungoides and parapsoriasis. A study of 18 cases with anti-human T-cell monoclonal antibodies and histochemical techniques.

    Science.gov (United States)

    Buechner, S A; Winkelmann, R K; Banks, P M

    1984-07-01

    Skin lesions from 15 patients with mycosis fungoides (MF) and from three with parapsoriasis were studied immunohistochemically with monoclonal antibodies against T cells (Leu 1) and against T-cell subsets (Leu 2a, Leu 3a). Lymphoid cell reactivity was diverse among these sampled cases. In two cases of parapsoriasis and nine of MF, there was a predominance of helper/inducer (Leu-3a-reactive) cells over suppressor/cytotoxic (Leu-2a-reactive) cells. In one case of parapsoriasis and one (advanced tumor stage) of MF, there was suppressor/cytotoxic cell predominance. One case of MF showed strong reactivity for both T-cell subset markers. Four cases of MF (two plaque-stage and two tumor-stage) featured a predominant cell type in the dermis which was nonreactive for all three antibodies. The intraepidermal lymphoid cellularity was Leu-1-reactive in ten cases of MF and two of parapsoriasis. Among these 12 cases, the intraepidermal cellularity was Leu-2a-reactive in four and Leu-3a-reactive in three. The use of such studies of T-cell subsets on in situ cutaneous lymphoid infiltrates may demonstrate a correlation with cytomorphology, clinical stage, and disease prognosis.

  4. Radiolabeled Monoclonal Antibody and Combination Chemotherapy Before Stem Cell Transplant in Treating Patients With High-Risk Lymphoid Malignancies

    Science.gov (United States)

    2017-01-23

    Recurrent B-Cell Non-Hodgkin Lymphoma; Recurrent Hodgkin Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent T-Cell Non-Hodgkin Lymphoma; Refractory B-Cell Non-Hodgkin Lymphoma; Refractory Hodgkin Lymphoma; Refractory Mantle Cell Lymphoma; Refractory T-Cell Non-Hodgkin Lymphoma

  5. Heterogeneity of monoclonal antibodies.

    Science.gov (United States)

    Liu, Hongcheng; Gaza-Bulseco, Georgeen; Faldu, Dinesh; Chumsae, Chris; Sun, Joanne

    2008-07-01

    Heterogeneity of monoclonal antibodies is common due to the various modifications introduced over the lifespan of the molecules from the point of synthesis to the point of complete clearance from the subjects. The vast number of modifications presents great challenge to the thorough characterization of the molecules. This article reviews the current knowledge of enzymatic and nonenzymatic modifications of monoclonal antibodies including the common ones such as incomplete disulfide bond formation, glycosylation, N-terminal pyroglutamine cyclization, C-terminal lysine processing, deamidation, isomerization, and oxidation, and less common ones such as modification of the N-terminal amino acids by maleuric acid and amidation of the C-terminal amino acid. In addition, noncovalent associations with other molecules, conformational diversity and aggregation of monoclonal antibodies are also discussed. Through a complete understanding of the heterogeneity of monoclonal antibodies, strategies can be employed to better identify the potential modifications and thoroughly characterize the molecules.

  6. Monoclonal antibody "gold rush".

    Science.gov (United States)

    Maggon, Krishan

    2007-01-01

    The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush.

  7. A novel approach to evaluate the pharmacokinetic biocomparability of a monoclonal antibody derived from two different cell lines using simultaneous crossover design.

    Science.gov (United States)

    Han, Chao; McIntosh, Thomas S; Geist, Brian J; Jiao, Trina; Puchalski, Thomas A; Goldberg, Kenneth M; Yang, Tong-Yuan; Pendley, Charles E; Zhou, Honghui; Davis, Hugh M

    2014-01-01

    A parallel study design with a large number of subjects has been a typical path for pharmacokinetic (PK) biocomparability assessment of biotherapeutics with long half-lives and immunogenic propensity, for example, monoclonal antibodies (mAb). A recently published innovative bioanalytical method that can quantify mAb produced from two different cell lines in the same sample opened an avenue to exploring a simultaneous crossover study design for PK biocomparability assessment of biotherapeutics. Siltuximab, a chimeric IgG1 mAb-targeting interleukin-6, was studied as an example. The pharmacokinetic biocomparability of siltuximab derived from mouse myeloma (Sp2/0) cells and Chinese hamster ovary cells was previously assessed and demonstrated in a clinical PK biocomparability study that enrolled more than 140 healthy subjects using a parallel trial design. The biocomparability was successfully shown in six cynomolgus monkeys in a preclinical proof-of-concept study using the new crossover study design supported by the analytical method. The impact of antidrug antibodies on the assessment of biocomparability was minimal. This novel approach opened up a new arena for the evaluation of PK biocomparability of biotherapeutics with unique molecular signatures such as a mAb derived from different cell lines.

  8. Surgical treatment of unicentric plasma cell histological type Castleman's disease

    Directory of Open Access Journals (Sweden)

    Marić Nebojša

    2011-01-01

    Full Text Available Introduction. Castleman’s disease or angiofollicular lymph hyperplasia is a rare disease with two identified clinical forms. Unicentric or localized form is characterized by isolated growth of lymph nodes, most often in mediastinum, and multicentric form is expressed as systemic disease with spread lymphadenopathy, organomegaly and presence of general symptoms of the disease. Histological types are hyalovascular, plasma-cell and transitive (mixed cell. Case report. This case report shows a woman, 59 years old, with unicentric form of plasma-cell type of Castleman’s disease. Unicentric form is usually shown as hyalovascular histological type, extremely rare as plasma-cell type, and transitive (mixed cell type was never described in literature as localized clinical form. The disease was manifested with chest pain, loss of body weight, exhaustion and weakness of legs. Further diagnostic procedures found the presence of enlarged lymph nodes paratracheally right, in a close contact with vena cava superior. The disease was confirmed by histopathological analysis of bioptated mediastinal lymph node after mediastinoscopy. Surgical treatment included extirpation of enlarged lymph nodes. After the regular postoperative condition, a full therapy effect was confirmed. Conclusion. Unicentric form of Castleman’s disease is expressed with enlarged lymph nodes on predilected places, usually in mediastinum. Surgical treatment is best method for the management of the disease and brings a full recovery of patient.

  9. The glycome of normal and malignant plasma cells.

    Directory of Open Access Journals (Sweden)

    Thomas M Moehler

    Full Text Available The glycome, i.e. the cellular repertoire of glycan structures, contributes to important functions such as adhesion and intercellular communication. Enzymes regulating cellular glycosylation processes are related to the pathogenesis of cancer including multiple myeloma. Here we analyze the transcriptional differences in the glycome of normal (n = 10 and two cohorts of 332 and 345 malignant plasma-cell samples, association with known multiple myeloma subentities as defined by presence of chromosomal aberrations, potential therapeutic targets, and its prognostic impact. We found i malignant vs. normal plasma cells to show a characteristic glycome-signature. They can ii be delineated by a lasso-based predictor from normal plasma cells based on this signature. iii Cytogenetic aberrations lead to distinct glycan-gene expression patterns for t(11;14, t(4;14, hyperdiploidy, 1q21-gain and deletion of 13q14. iv A 38-gene glycome-signature significantly delineates patients with adverse survival in two independent cohorts of 545 patients treated with high-dose melphalan and autologous stem cell transplantation. v As single gene, expression of the phosphatidyl-inositol-glycan protein M as part of the targetable glycosyl-phosphatidyl-inositol-anchor-biosynthesis pathway is associated with adverse survival. The prognostically relevant glycome deviation in malignant cells invites novel strategies of therapy for multiple myeloma.

  10. Successful treatment of plasma cell cheilitis with topical tacrolimus: report of two cases.

    Science.gov (United States)

    Hanami, Yuka; Motoki, Yoshikazu; Yamamoto, Toshiyuki

    2011-02-15

    Plasma cell cheilitis is an uncommon chronic inflammatory dermatitis that presents with flat to slightly elevated erosive erythematous plaques. It is histologically characterized by plasma cell infiltrates into the mucosa. Other than the lip, genital areas are often involved, which is called plasma cell balanitis or vulvitis. Plasma cell cheilitis is sometimes resistant to conventional topical corticosteroid therapy. Other choices include oral griseofulvin, topical cyclosporine, and intralesional corticosteroid injection, all of which occasionally fail to produce satisfactory results. Recent reports show that topical calcineurin inhibitors are effective for plasma cell cheilitis, balanitis, and vulvitis. However, there are so far only 2 reports of plasma cell cheilitis successfully treated with topical pimecrolimus and tacrolimus. We present herein two cases of plasma cell cheilitis, in which topical tacrolimus showed beneficial effects, suggesting that this immunomodulatory agent is a promising option for plasma cell cheilitis.

  11. Cold atmospheric plasma for selectively ablating metastatic breast cancer cells.

    Science.gov (United States)

    Wang, Mian; Holmes, Benjamin; Cheng, Xiaoqian; Zhu, Wei; Keidar, Michael; Zhang, Lijie Grace

    2013-01-01

    Traditional breast cancer treatments such as surgery and radiotherapy contain many inherent limitations with regards to incomplete and nonselective tumor ablation. Cold atmospheric plasma (CAP) is an ionized gas where the ion temperature is close to room temperature. It contains electrons, charged particles, radicals, various excited molecules, UV photons and transient electric fields. These various compositional elements have the potential to either enhance and promote cellular activity, or disrupt and destroy them. In particular, based on this unique composition, CAP could offer a minimally-invasive surgical approach allowing for specific cancer cell or tumor tissue removal without influencing healthy cells. Thus, the objective of this research is to investigate a novel CAP-based therapy for selectively bone metastatic breast cancer treatment. For this purpose, human metastatic breast cancer (BrCa) cells and bone marrow derived human mesenchymal stem cells (MSCs) were separately treated with CAP, and behavioral changes were evaluated after 1, 3, and 5 days of culture. With different treatment times, different BrCa and MSC cell responses were observed. Our results showed that BrCa cells were more sensitive to these CAP treatments than MSCs under plasma dose conditions tested. It demonstrated that CAP can selectively ablate metastatic BrCa cells in vitro without damaging healthy MSCs at the metastatic bone site. In addition, our study showed that CAP treatment can significantly inhibit the migration and invasion of BrCa cells. The results suggest the great potential of CAP for breast cancer therapy.

  12. Cold atmospheric plasma for selectively ablating metastatic breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Mian Wang

    Full Text Available Traditional breast cancer treatments such as surgery and radiotherapy contain many inherent limitations with regards to incomplete and nonselective tumor ablation. Cold atmospheric plasma (CAP is an ionized gas where the ion temperature is close to room temperature. It contains electrons, charged particles, radicals, various excited molecules, UV photons and transient electric fields. These various compositional elements have the potential to either enhance and promote cellular activity, or disrupt and destroy them. In particular, based on this unique composition, CAP could offer a minimally-invasive surgical approach allowing for specific cancer cell or tumor tissue removal without influencing healthy cells. Thus, the objective of this research is to investigate a novel CAP-based therapy for selectively bone metastatic breast cancer treatment. For this purpose, human metastatic breast cancer (BrCa cells and bone marrow derived human mesenchymal stem cells (MSCs were separately treated with CAP, and behavioral changes were evaluated after 1, 3, and 5 days of culture. With different treatment times, different BrCa and MSC cell responses were observed. Our results showed that BrCa cells were more sensitive to these CAP treatments than MSCs under plasma dose conditions tested. It demonstrated that CAP can selectively ablate metastatic BrCa cells in vitro without damaging healthy MSCs at the metastatic bone site. In addition, our study showed that CAP treatment can significantly inhibit the migration and invasion of BrCa cells. The results suggest the great potential of CAP for breast cancer therapy.

  13. Inhibition of the receptor tyrosine kinase ROR1 by anti-ROR1 monoclonal antibodies and siRNA induced apoptosis of melanoma cells.

    Directory of Open Access Journals (Sweden)

    Mohammad Hojjat-Farsangi

    Full Text Available The receptor tyrosine kinase (RTK ROR1 is overexpressed and of importance for the survival of various malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL. There is limited information however on ROR1 in melanoma. In the present study we analysed in seven melanoma cell lines ROR1 expression and phosphorylation as well as the effects of anti-ROR1 monoclonal antibodies (mAbs and ROR1 suppressing siRNA on cell survival. ROR1 was overexpressed at the protein level to a varying degree and phosphorylated at tyrosine and serine residues. Three of our four self-produced anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8 induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in complement dependent cytotoxicity (CDC and antibody dependent cellular cytotoxicity (ADCC. The ESTDAB081 and 094 cell lines respectively were resistant to direct apoptosis of the four anti-ROR1 mAbs alone but not in CDC or ADCC. ROR1 siRNA transfection induced downregulation of ROR1 expression both at mRNA and protein levels proceeded by apoptosis of the melanoma cells (ESTDAB049, ESTDAB112, DFW and A375 including ESTDAB081, which was resistant to the direct apoptotic effect of the mAbs. The results indicate that ROR1 may play a role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable target for mAb therapy.

  14. Inhibition of the receptor tyrosine kinase ROR1 by anti-ROR1 monoclonal antibodies and siRNA induced apoptosis of melanoma cells.

    Science.gov (United States)

    Hojjat-Farsangi, Mohammad; Ghaemimanesh, Fatemeh; Daneshmanesh, Amir Hossein; Bayat, Ali-Ahmad; Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Mellstedt, Hakan

    2013-01-01

    The receptor tyrosine kinase (RTK) ROR1 is overexpressed and of importance for the survival of various malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL). There is limited information however on ROR1 in melanoma. In the present study we analysed in seven melanoma cell lines ROR1 expression and phosphorylation as well as the effects of anti-ROR1 monoclonal antibodies (mAbs) and ROR1 suppressing siRNA on cell survival. ROR1 was overexpressed at the protein level to a varying degree and phosphorylated at tyrosine and serine residues. Three of our four self-produced anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8) induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). The ESTDAB081 and 094 cell lines respectively were resistant to direct apoptosis of the four anti-ROR1 mAbs alone but not in CDC or ADCC. ROR1 siRNA transfection induced downregulation of ROR1 expression both at mRNA and protein levels proceeded by apoptosis of the melanoma cells (ESTDAB049, ESTDAB112, DFW and A375) including ESTDAB081, which was resistant to the direct apoptotic effect of the mAbs. The results indicate that ROR1 may play a role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable target for mAb therapy.

  15. Immunotherapy with anti-CD3 monoclonal antibodies and recombinant interleukin 2: stimulation of molecular programs of cytotoxic killer cells and induction of tumor regression.

    Science.gov (United States)

    Nakajima, F; Khanna, A; Xu, G; Lagman, M; Haschemeyer, R; Mouradian, J; Wang, J C; Stenzel, K H; Rubin, A L; Suthanthiran, M

    1994-01-01

    Adoptive cellular immunotherapy, infusions of interleukin 2 (IL-2) in conjunction with in vitro-activated killer cells, has brought new hope to patients with cancer. The broad application of this strategy, however, is constrained by the need for repeated leukapheresis and by the labor-intensive process of in vitro activation of cells. Also, current protocols generally use nonphysiological and toxic concentrations of IL-2. Identification of an in vivo stimulant that renders T cells responsive to physiologic concentrations of IL-2 represents a potential improvement over existing approaches. We have determined whether in vivo administration of monoclonal antibodies (mAbs) directed at the T-cell surface protein CD3 induces T-cell responsiveness to IL-2, stimulates cytolytic molecular programs of natural killer cells and cytotoxic T cells, and induces tumor regression. These hypotheses were explored in a murine hepatic MCA-102 fibrosarcoma model. We report that in vivo administration of anti-CD3 mAbs plus IL-2 results in intrahepatic expression of mRNA-encoding perforin, cytotoxic T-cell-specific serine esterase, and tumor necrosis factor alpha. Anti-CD3 mAbs alone or IL-2 alone failed to induce or induced minimal expression of these molecular mediators of cytotoxicity. The anti-CD3 mAbs plus IL-2 regimen also resulted in a significantly smaller number of hepatic metastases and a significantly longer survival time of tumor-bearing mice, compared to treatment with anti-CD3 mAbs alone or IL-2 alone. Our findings suggest that a regimen of anti-CD3 mAbs plus IL-2 is a more effective antitumor regimen compared with anti-CD3 mAbs alone or IL-2 alone and advance an alternative immunotherapy strategy of potential value for the treatment of cancer in humans. Images PMID:8058730

  16. Influence of electron injection into 27 cm audio plasma cell on the plasma diagnostics

    Energy Technology Data Exchange (ETDEWEB)

    Haleem, N. A.; Ragheb, M. S.; Zakhary, S. G. [Accelerators Department, Nuclear Research Center, AEA, Cairo 13759 (Egypt); El Fiki, S. A.; Nouh, S. A. [Faculty of Science, Ain Shams University, Cairo 11566 (Egypt); El Disoki, T. M. [Faculty of Girls, Ain Shams University, Cairo 11566 (Egypt)

    2013-08-15

    In this article, the plasma is created in a Pyrex tube (L = 27 cm, φ= 4 cm) as a single cell, by a capacitive audio frequency (AF) discharge (f = 10–100 kHz), at a definite pressure of ∼0.2 Torr. A couple of tube linear and deviating arrangements show plasma characteristic conformity. The applied AF plasma and the injection of electrons into two gas mediums Ar and N{sub 2} revealed the increase of electron density at distinct tube regions by one order to attain 10{sup 13}/cm{sup 3}. The electrons temperature and density strengths are in contrast to each other. While their distributions differ along the plasma tube length, they show a decaying sinusoidal shape where their peaks position varies by the gas type. The electrons injection moderates electron temperature and expands their density. The later highest peak holds for the N{sub 2} gas, at electrons injection it changes to hold for the Ar. The sinusoidal decaying density behavior generates electric fields depending on the gas used and independent of tube geometry. The effect of the injected electrons performs a responsive impact on electrons density not attributed to the gas discharge. Analytical tools investigate the interaction of the plasma, the discharge current, and the gas used on the electrodes. It points to the emigration of atoms from each one but for greater majority they behave to a preferred direction. Meanwhile, only in the linear regime, small percentage of atoms still moves in reverse direction. Traces of gas atoms revealed on both electrodes due to sheath regions denote lack of their participation in the discharge current. In addition, atoms travel from one electrode to the other by overcoming the sheaths regions occurring transportation of particles agglomeration from one electrode to the other. The electrons injection has contributed to increase the plasma electron density peaks. These electrons populations have raised the generated electrostatic fields assisting the elemental ions

  17. Selective cytotoxicity of murine monoclonal antibody LAM2 against human small-cell carcinoma in the presence of human complement: possible use for in vitro elimination of tumor cells from bone marrow.

    Science.gov (United States)

    Stahel, R A; Mabry, M; Sabbath, K; Speak, J A; Bernal, S D

    1985-05-15

    LAM2 is a murine IgM monoclonal antibody (MAb) which binds strongly to the cell membrane of human lung small-cell carcinoma (SCC) and squamous-cell carcinoma but not to normal bone-marrow cells. The cytotoxicity of this antibody in the presence of human complement was investigated in vitro by chromium release and clonogenic assays. The optimal treatment conditions included incubation with antibody for 30 min at 37 degrees C followed by 3 additions of human complement 30 min apart. Cell lysis ranged from 94 to 98% in 4 SCC cell lines at antibody dilutions of 1:100: a lower level of lysis (60%) occurred in a lung squamous-cell carcinoma cell line. The cytotoxic effect was strictly complement-dependent. No cytotoxic effect was seen with other human cell lines including lung adenocarcinoma, lung large-cell carcinoma, myeloid leukemia, and lymphoblastic leukemia. No lysis was seen with nucleated marrow cells from healthy volunteers. Normal marrow cells in excess did not inhibit SCC cell lysis. Incubation with antibody and complement resulted in a 100-fold reduction of colony formation of SCC cells, but did not reduce the number of colonies of marrow-cell precursors, including CFU-GEMM, BFU-E, and CFU-C. The selective cytotoxicity of LAM2 antibody to SCC, but not to normal bone-marrow cells, suggests that this antibody may be useful for the in vitro elimination of SCC cells from the bone marrow.

  18. High affinity germinal center B cells are actively selected into the plasma cell compartment.

    Science.gov (United States)

    Phan, Tri Giang; Paus, Didrik; Chan, Tyani D; Turner, Marian L; Nutt, Stephen L; Basten, Antony; Brink, Robert

    2006-10-30

    A hallmark of T cell-dependent immune responses is the progressive increase in the ability of serum antibodies to bind antigen and provide immune protection. Affinity maturation of the antibody response is thought to be connected with the preferential survival of germinal centre (GC) B cells that have acquired increased affinity for antigen via somatic hypermutation of their immunoglobulin genes. However, the mechanisms that drive affinity maturation remain obscure because of the difficulty in tracking the affinity-based selection of GC B cells and their differentiation into plasma cells. We describe a powerful new model that allows these processes to be followed as they occur in vivo. In contrast to evidence from in vitro systems, responding GC B cells do not undergo plasma cell differentiation stochastically. Rather, only GC B cells that have acquired high affinity for the immunizing antigen form plasma cells. Affinity maturation is therefore driven by a tightly controlled mechanism that ensures only antibodies with the greatest possibility of neutralizing foreign antigen are produced. Because the body can sustain only limited numbers of plasma cells, this "quality control" over plasma cell differentiation is likely critical for establishing effective humoral immunity.

  19. Circulating human B and plasma cells. Age-associated changes in counts and detailed characterization of circulating normal CD138- and CD138+ plasma cells. : Blood B-lymphocytes and plasma cells in adults

    OpenAIRE

    2010-01-01

    International audience; Generation of B and plasma cells involves several organs with a necessary cell trafficking between them. A detailed phenotypic characterization of four circulating B-cell subsets (immature-, naïve-, memory- B-lymphocytes and plasma cells) of 106 healthy adults was realized by multiparametric flow cytometry. We show that CD10, CD27 and CD38 is the minimal combination of subsetting markers allowing unequivocal identification of immature (CD10(+)CD27(-)CD38(+), 6+/-6 cell...

  20. Cold atmospheric plasma treatment selectively targets head and neck squamous cell carcinoma cells.

    Science.gov (United States)

    Guerrero-Preston, Rafael; Ogawa, Takenori; Uemura, Mamoru; Shumulinsky, Gary; Valle, Blanca L; Pirini, Francesca; Ravi, Rajani; Sidransky, David; Keidar, Michael; Trink, Barry

    2014-10-01

    The treatment of locoregional recurrence (LRR) of head and neck squamous cell carcinoma (HNSCC) often requires a combination of surgery, radiation therapy and/or chemotherapy. Survival outcomes are poor and the treatment outcomes are morbid. Cold atmospheric plasma (CAP) is an ionized gas produced at room temperature under laboratory conditions. We have previously demonstrated that treatment with a CAP jet device selectively targets cancer cells using in vitro melanoma and in vivo bladder cancer models. In the present study, we wished to examine CAP selectivity in HNSCC in vitro models, and to explore its potential for use as a minimally invasive surgical approach that allows for specific cancer cell or tumor tissue ablation without affecting the surrounding healthy cells and tissues. Four HNSCC cell lines (JHU-022, JHU-028, JHU-029, SCC25) and 2 normal oral cavity epithelial cell lines (OKF6 and NOKsi) were subjected to cold plasma treatment for durations of 10, 30 and 45 sec, and a helium flow of 20 l/min-1 for 10 sec was used as a positive treatment control. We showed that cold plasma selectively diminished HNSCC cell viability in a dose-response manner, as evidenced by MTT assays; the viability of the OKF6 cells was not affected by the cold plasma. The results of colony formation assays also revealed a cell-specific response to cold plasma application. Western blot analysis did not provide evidence that the cleavage of PARP occurred following cold plasma treatment. In conclusion, our results suggest that cold plasma application selectively impairs HNSCC cell lines through non-apoptotic mechanisms, while having a minimal effect on normal oral cavity epithelial cell lines.

  1. Cell lines, Md108 and Md66, from the hemocytes of Malacosoma disstria (Lepidoptera) display aspects of plasma-free innate non-self activities.

    Science.gov (United States)

    Lapointe, Jason F; Dunphy, Gary B; Giannoulis, Paschalis; Mandato, Craig A; Nardi, James B; Gharib, Osama H; Niven, Donald F

    2011-11-01

    The innate non-self response systems of the deciduous tree pest, the forest tent caterpillar, Malacosoma disstria has been documented by us in terms of in vitro and in vivo reactions towards the Gram-positive nonpathogenic bacterium, Bacillus subtilis and Gram-negative pathogenic microbe, Xenorhabdus nematophila and their respective surface antigens, lipopoteichoic acids (LTA) and lipopolysaccharides (LPS). These studies, often conducted in whole and diluted hemolymph, preclude examination of plasma-free cellular (hemocyte) responses. Plasma-free hemocytes as primary cultures are difficult to obtain. The floating cell line Md66 and attached cell line Md108 from M. disstria hemocytes were examined as a model for plasma-free M. disstria hemocyte non-self responses. Herein, it was established that although both lines differed from each other and from the primary hemocyte cultures of M. disstria in growth parameters, cell composition and sizes both cell lines displayed granular cell-like (GL) cells and plasmatocyte-like (PL) cells according to morphological criteria and to some extent antigenic similarities based on labeling with anti-Chrysodeixis includens hemocyte monoclonal antibodies. Hemocyte-specific neuroglian-like protein was detected on cells of both cell lines and in the primary hemocyte cultures albeit with staining patterns differing according to culture and cell types, confluency levels and cell-cell adhesion. Both cell lines bound B. subtilis and X. nematophila, the reaction extent varying with the cell line and its cell types. LPS damaged both cell types in the two cell lines whereas LTA enhanced the adhesion of Md66 GL cells to flask surfaces followed by PL cell adhesion. PL cells of both lines, like the primary cultures, phagocytosed FITC-labeled B. subtilis; only Md108 GL cells phagocytosed B. subtilis. In either case phagocytosis was always less in frequency and intensity than the primary cultures. Proteins released from the cell lines differed in

  2. Lipid signalling dynamics at the β-cell plasma membrane.

    Science.gov (United States)

    Wuttke, Anne

    2015-04-01

    Pancreatic β-cells are clustered in islets of Langerhans and secrete insulin in response to increased concentrations of circulating glucose. Insulin in turn acts on liver, muscle and fat tissue to store energy and normalize the blood glucose level. Inappropriate insulin release may lead to impaired glucose tolerance and diabetes. In addition to glucose, other nutrients, neural stimuli and hormonal stimuli control insulin secretion. Many of these signals are perceived at the plasma membrane, which is also the site where insulin granules undergo exocytosis. Therefore, it is not surprising that membrane lipids play an important role in the regulation of insulin secretion. β-cells release insulin in a pulsatile fashion. Signalling lipids integrate the nutrient and neurohormonal inputs to fine-tune, shape and co-ordinate the pulsatility. An important group of signalling lipids are phosphoinositides and their downstream messengers. This MiniReview will discuss new insights into lipid signalling dynamics in β-cells obtained from live-cell imaging experiments with fluorescent translocation biosensors. The plasma membrane concentration of several phosphoinositides and of their downstream messengers changes rapidly upon nutrient or neurohormonal stimulation. Glucose induces the most complex spatio-temporal patterns, typically involving oscillations of messenger concentrations, which sometimes are locally restricted. The tightly controlled levels of lipid messengers can mediate specific binding of downstream effectors to the plasma membrane, contributing to the appropriate regulation of insulin secretion.

  3. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    Science.gov (United States)

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

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  16. Development of an ErbB4 monoclonal antibody that blocks neuregulin-1-induced ErbB4 activation in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Okazaki, Shogo [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nakatani, Fumi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Masuko, Kazue; Tsuchihashi, Kenji [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Ueda, Shiho; Masuko, Takashi [Cell Biology Laboratory, Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kinki University, Higashiosaka, Osaka 577-8502 Japan (Japan); Saya, Hideyuki [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Nagano, Osamu, E-mail: osmna@sb3.so-net.ne.jp [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan)

    2016-01-29

    The use of monoclonal antibodies (mAbs) for cancer therapy is one of the most important strategies for current cancer treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which regulates cancer cell proliferation, survival, and migration, is a major molecular target for antibody-based therapy. ErbB4/HER4, which contains a ligand-binding extracellular region, is activated by several ligands, including neuregulins (NRGs), heparin-binding EGF-like growth factor, betacellulin and epiregulin. Although there are clinically approved antibodies for ErbB1 and ErbB2, there are no available therapeutic mAbs for ErbB4, and it is not known whether ErbB4 is a useful target for antibody-based cancer therapy. In this study, we developed an anti-ErbB4 mAb (clone P6-1) that suppresses NRG-dependent activation of ErbB4 and examined its effect on breast cancer cell proliferation in the extracellular matrix. - Highlights: • We newly generated four clones of human ErbB4 specific mAb. • ErbB4 mAb clone P6-1 blocks ErbB4 phosphorylation induced by NRG-1. • ErbB4 mAb clone P6-1 suppresses NRG-1-promoted breast cancer cells proliferation on three dimensional culture condition.

  17. Application of a quality by design approach to the cell culture process of monoclonal antibody production, resulting in the establishment of a design space.

    Science.gov (United States)

    Nagashima, Hiroaki; Watari, Akiko; Shinoda, Yasuharu; Okamoto, Hiroshi; Takuma, Shinya

    2013-12-01

    This case study describes the application of Quality by Design elements to the process of culturing Chinese hamster ovary cells in the production of a monoclonal antibody. All steps in the cell culture process and all process parameters in each step were identified by using a cause-and-effect diagram. Prospective risk assessment using failure mode and effects analysis identified the following four potential critical process parameters in the production culture step: initial viable cell density, culture duration, pH, and temperature. These parameters and lot-to-lot variability in raw material were then evaluated by process characterization utilizing a design of experiments approach consisting of a face-centered central composite design integrated with a full factorial design. Process characterization was conducted using a scaled down model that had been qualified by comparison with large-scale production data. Multivariate regression analysis was used to establish statistical prediction models for performance indicators and quality attributes; with these, we constructed contour plots and conducted Monte Carlo simulation to clarify the design space. The statistical analyses, especially for raw materials, identified set point values, which were most robust with respect to the lot-to-lot variability of raw materials while keeping the product quality within the acceptance criteria.

  18. Attachment of an anti-MUC1 monoclonal antibody to 5-FU loaded BSA nanoparticles for active targeting of breast cancer cells.

    Science.gov (United States)

    Kouchakzadeh, Hasan; Shojaosadati, Seyed Abbas; Mohammadnejad, Javad; Paknejad, Malihe; Rasaee, Mohammad Javad

    2012-01-01

    With PR81 as a murine monoclonal antibody (mAb) that was prepared against the human breast cancer, the MUC1 receptor specific targeting is possible. In this study, PR81-conjugated bovine serum albumin (BSA) nanoparticles loaded with anticancer drug 5-fluorouracil (5-FU) were developed. Enzyme linked immunosorbant assay (ELISA) results showed high immunoreactivity of PR81 mAb conjugated to nanoparticles towards MUC1 related peptide or native cancerous MUC1 and almost no cross-reaction to non-specific proteins. In vitro experiments were performed to determine the ability of this new drug delivery system on overcoming MCF-7 breast cancer cells in comparison with four other systems. The results revealed that these cell-type specific drug loaded nanoparticles could achieve more cell death as compared to when the 5-FU was used with no carriers. Stability studies of produced drug delivery system proved high immunoreactivity of conjugated PR81 even after 11 days of storage in room temperature.

  19. Appearance of Human Plasma Cells Following Differentiation of Human B Cells in NOD/SCID Mouse Spleen

    Directory of Open Access Journals (Sweden)

    Kentaro Kikuchi

    2003-01-01

    Full Text Available Relatively little is known for the differentiation and maturation process of human B cells to plasma cells. This is particularly important in reconstitution work involving transfer of autoantibodies. To address this issue, we transplanted human peripheral blood mononuclear cells (PBMC directly into the spleen of irradiated NOD/SCID mice depleted of natural killer cell activity. Within 6 weeks, naïve B cells differentiated into memory B cells and, importantly, the numbers of human CD138+ plasma cells in spleen increased by 100 fold after transplantation. Plasma cell numbers correlated with the detection of human IgM and IgG in serum, indicating that human B cells had differentiated into mature plasma cells in the murine spleen. In addition to CD19+ plasma cells, a distinct CD19- plasma cell population was detected, suggesting that downregulation of CD19 associated with maturation of plasma cells occurred. When purified human B cells were transplanted, those findings were not observed. Our results indicate that differentiation and maturation of human B cells and plasma cells can be investigated by transplantation of human PBMC into the spleen of NOD/SCID mice. The model will be useful for studying the differentiation of human B cells and generation of plasma cells.

  20. Cytokines in Endocrine Dysfunction of Plasma Cell Disorders.

    Science.gov (United States)

    Feigerlová, Eva; Battaglia-Hsu, Shyue-Fang

    2017-01-01

    Monoclonal gammopathies (MG) are classically associated with lytic bone lesions, hypercalcemia, anemia, and renal insufficiency. However, in some cases, symptoms of endocrine dysfunction are more prominent than these classical signs and misdiagnosis can thus be possible. This concerns especially the situation where the presence of M-protein is limited and the serum protein electrophoresis (sPEP) appears normal. To understand the origin of the endocrine symptoms associated with MG, we overview here the current knowledge on the complexity of interactions between cytokines and the endocrine system in MG and discuss the perspectives for both the diagnosis and treatments for this class of diseases. We also illustrate the role of major cytokines and growth factors such as IL-6, IL-1β, TNF-α, and VEGF in the endocrine system, as these tumor-relevant signaling molecules not only help the clonal expansion and invasion of the tumor cells but also influence cellular metabolism through autocrine, paracrine, and endocrine mechanisms. We further discuss the broader impact of these tumor environment-derived molecules and proinflammatory state on systemic hormone signaling. The diagnostic challenges and clinical work-up are illustrated from the point of view of an endocrinologist.

  1. Plasma Cell Gingivits-A Conflict of Diagnosis

    Science.gov (United States)

    Mutyap, Divya Aishwarya; Pantula, Veerandranath Reddy; Akula, Shilpa; Chinthapalli, Bhargavi

    2016-01-01

    Plasma Cell Gingivitis (PCG) is a rare condition of the gingiva which is benign in nature. In this condition there is enlargement of the marginal and attached gingiva. It is not only unaesthetic in appearance but also causes difficulty in speech and mastication. Hence, it creates an area for plaque accumulation because of which there is difficulty in carrying out the routine oral hygiene procedures. The aetiology is unknown and is thought to be a hypersensitive reaction to an allergen. This condition is characterized by massive infiltration of the plasma cells into the sub-epithelial connective tissue layers. Here we are presenting two cases of PCG associated with swollen lips, which is an uncommon condition. These cases were treated by gingivectomy and followed up to six months. PMID:28050510

  2. Hepatic failure caused by plasma cell infiltration in multiple Myeloma

    Institute of Scientific and Technical Information of China (English)

    Fadi E Rahhal; Robert R Schade; Asha Nayak; Teresa A Coleman

    2009-01-01

    Although plasma cell infiltration is not rare in autopsy of patients with multiple myeloma (MM), it is very rarely detected in living patients. This is because MM rarely causes significant liver dysfunction that requires further evaluation. A 49-year-old man presented with acute renal failure and was diagnosed with kappa light chain MM stage ?B. Thalidomide and dexamethasone were initiated. The patient developed a continuous increase in bilirubin that led to severe cholestasis. A liver biopsy revealed plasma cell infiltration. He then rapidly progressed to liver failure and died. Treatment options are limited in MM with significant liver dysfunction.Despite new drug therapies in MM, those patients with rapidly progressive liver failure appear to have a dismal outcome.

  3. Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: KD values, binding pairs, and amino acid sequences.

    Science.gov (United States)

    Peng, Hong; Brimijoin, Stephen; Hrabovska, Anna; Targosova, Katarina; Krejci, Eric; Blake, Thomas A; Johnson, Rudolph C; Masson, Patrick; Lockridge, Oksana

    2015-10-05

    Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at -80 °C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10(-9) M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal.

  4. Transcription factor ABF-1 suppresses plasma cell differentiation but facilitates memory B cell formation.

    Science.gov (United States)

    Chiu, Yi-Kai; Lin, I-Ying; Su, Shin-Tang; Wang, Kuan-Hsiung; Yang, Shii-Yi; Tsai, Dong-Yan; Hsieh, Yi-Ting; Lin, Kuo-I

    2014-09-01

    Ag-primed B cells that result from an immune response can form either memory B cells or Ab-secreting plasma cells; however, the molecular machinery that controls this cellular fate is poorly understood. In this study, we show that activated B cell factor-1 (ABF-1), which encodes a basic helix-loop-helix transcriptional repressor, participates in this regulation. ABF-1 was prevalently expressed in purified memory B cells and induced by T follicular helper cell-mediated signals. ABF-1 expression declined by the direct repression of B lymphocyte-induced maturation protein-1 during differentiation. Ectopic expression of ABF-1 reduced the formation of Ab-secreting cells in an in vitro differentiation system of human memory B cells. Accordingly, knockdown of ABF-1 potentiates the formation of Ab-secreting cells. A transgenic mouse that expresses inducible ABF-1 in a B cell-specific manner was generated to demonstrate that the formation of germinal center and memory B cells was augmented by induced ABF-1 in an immune response, whereas the Ag-specific plasma cell response was dampened. This effect was associated with the ability of ABF-1 to limit cell proliferation. Together, our results demonstrate that ABF-1 facilitates formation of memory B cells but prevents plasma cell differentiation.

  5. Plasma-Sprayed Titanium Patterns for Enhancing Early Cell Responses

    Science.gov (United States)

    Shi, Yunqi; Xie, Youtao; Pan, Houhua; Zheng, Xuebin; Huang, Liping; Ji, Fang; Li, Kai

    2016-06-01

    Titanium coating has been widely used as a biocompatible metal in biomedical applications. However, the early cell responses and long-term fixation of titanium implants are not satisfied. To obviate these defects, in this paper, micro-post arrays with various widths (150-1000 μm) and intervals (100-300 μm) were fabricated on the titanium substrate by template-assisted plasma spraying technology. In vitro cell culture experiments showed that MC3T3-E1 cells exhibited significantly higher osteogenic differentiation as well as slightly improved adhesion and proliferation on the micro-patterned coatings compared with the traditional one. The cell number on the pattern with 1000 µm width reached 130% after 6 days of incubation, and the expressions of osteopontin (OPN) as well as osteocalcin (OC) were doubled. No obvious difference was found in cell adhesion on various size patterns. The present micro-patterned coatings proposed a new modification method for the traditional plasma spraying technology to enhance the early cell responses and convenience for the bone in-growth.

  6. Bach2 represses plasma cell gene regulatory network in B cells to promote antibody class switch.

    Science.gov (United States)

    Muto, Akihiko; Ochiai, Kyoko; Kimura, Yoshitaka; Itoh-Nakadai, Ari; Calame, Kathryn L; Ikebe, Dai; Tashiro, Satoshi; Igarashi, Kazuhiko

    2010-12-01

    Two transcription factors, Pax5 and Blimp-1, form a gene regulatory network (GRN) with a double-negative loop, which defines either B-cell (Pax5 high) or plasma cell (Blimp-1 high) status as a binary switch. However, it is unclear how this B-cell GRN registers class switch DNA recombination (CSR), an event that takes place before the terminal differentiation to plasma cells. In the absence of Bach2 encoding a transcription factor required for CSR, mouse splenic B cells more frequently and rapidly expressed Blimp-1 and differentiated to IgM plasma cells as compared with wild-type cells. Genetic loss of Blimp-1 in Bach2(-/-) B cells was sufficient to restore CSR. These data with mathematical modelling of the GRN indicate that Bach2 achieves a time delay in Blimp-1 induction, which inhibits plasma cell differentiation and promotes CSR (Delay-Driven Diversity model for CSR). Reduction in mature B-cell numbers in Bach2(-/-) mice was not rescued by Blimp-1 ablation, indicating that Bach2 regulates B-cell differentiation and function through Blimp-1-dependent and -independent GRNs.

  7. Nanodomain stabilization dynamics in plasma membranes of biological cells

    Science.gov (United States)

    Das, Tamal; Maiti, Tapas K.; Chakraborty, Suman

    2011-02-01

    We discover that a synergistically amplifying role of stabilizing membrane proteins and continuous lipid recycling can explain the physics governing the stability, polydispersity, and dynamics of lipid raft domains in plasma membranes of biological cells. We establish the conjecture using a generalized order parameter based on theoretical formalism, endorsed by detailed scaling arguments and domain mapping. Quantitative agreements with morphological distributions of raft complexes, as obtained from Förster resonance energy transfer based visualization, support the present theoretical conjecture.

  8. Refraction of $e^-$ beams due to plasma lensing at a plasma-vacuum interface -- applied to beam deflection in a Copper cell with electrical RF-breakdown plasma

    CERN Document Server

    Sahai, Aakash A

    2014-01-01

    We formulate a possible description of the deflection of a relativistic $e^-$ beam in an inhomogeneous copper plasma, encountered by the beam when propagating through a accelerating cell that has undergone a high electric-field RF-breakdown. It is well known that an inhomogeneous plasma forms and may last for up to a few micro-seconds, until recombination in an accelerating structure where a field-emission triggers melting and ionization of RF-cell wall deformity. We present a preliminary model for the beam deflection due to collective plasma response based upon the beam density, plasma density and interaction length.

  9. A monoclonal antibody toolkit for C. elegans.

    Directory of Open Access Journals (Sweden)

    Gayla Hadwiger

    Full Text Available BACKGROUND: Antibodies are critical tools in many avenues of biological research. Though antibodies can be produced in the research laboratory setting, most research labs working with vertebrates avail themselves of the wide array of commercially available reagents. By contrast, few such reagents are available for work with model organisms. METHODOLOGY/PRINCIPAL FINDINGS: We report the production of monoclonal antibodies directed against a wide range of proteins that label specific subcellular and cellular components, and macromolecular complexes. Antibodies were made to synaptobrevin (SNB-1, a component of synaptic vesicles; to Rim (UNC-10, a protein localized to synaptic active zones; to transforming acidic coiled-coil protein (TAC-1, a component of centrosomes; to CENP-C (HCP-4, which in worms labels the entire length of their holocentric chromosomes; to ORC2 (ORC-2, a subunit of the DNA origin replication complex; to the nucleolar phosphoprotein NOPP140 (DAO-5; to the nuclear envelope protein lamin (LMN-1; to EHD1 (RME-1 a marker for recycling endosomes; to caveolin (CAV-1, a marker for caveolae; to the cytochrome P450 (CYP-33E1, a resident of the endoplasmic reticulum; to beta-1,3-glucuronyltransferase (SQV-8 that labels the Golgi; to a chaperonin (HSP-60 targeted to mitochondria; to LAMP (LMP-1, a resident protein of lysosomes; to the alpha subunit of the 20S subcomplex (PAS-7 of the 26S proteasome; to dynamin (DYN-1 and to the alpha-subunit of the adaptor complex 2 (APA-2 as markers for sites of clathrin-mediated endocytosis; to the MAGUK, protein disks large (DLG-1 and cadherin (HMR-1, both of which label adherens junctions; to a cytoskeletal linker of the ezrin-radixin-moesin family (ERM-1, which localized to apical membranes; to an ERBIN family protein (LET-413 which localizes to the basolateral membrane of epithelial cells and to an adhesion molecule (SAX-7 which localizes to the plasma membrane at cell-cell contacts. In addition to

  10. Characterization of a new monoclonal anti-glypican-3 antibody specific to the hepatocellular carcinoma cell line, HepG2.

    Science.gov (United States)

    Vongchan, Preeyanat; Linhardt, Robert J

    2017-03-08

    To characterize the antigen on HepG2 cell that is specifically recognized by a new monoclonal antibody raised against human liver heparan sulfate proteoglycan (HSPG), clone 1E4-1D9. The antigen recognized by mAb 1E4-1D9 was immunoprecipitated and its amino acid sequence was analyzed LC/MS. The transmembrane domain, number of cysteine residues, and glycosylation sites were predicted from these entire sequences. Data from amino acid analysis was aligned with glypican-3 (https://www.ebi.ac.uk/Tools/msa/clustalo/). The competitive reaction of mAb 1E4-1D9 and anti-glypican-3 on HepG2 cells was demonstrated by indirect immunofluorescence and analyzed by flow cytometry. Moreover, co-immunoprecipitation of mAb 1E4-1D9 and anti-glypican-3 was performed in HepG2 cells by Western immunoblotting. The recognition by mAb 1E4-1D9 of a specific epitope on solid tumor and hematopoietic cell lines was studied using indirect immunofluorescence and analyzed by flow cytometry. Monoclonal antibody 1E4-1D9 reacted with an HSPG isolated from human liver and a band of 67 kD was detected under both reducing and non-reducing conditions. The specific antigen pulled down by mAb 1E4-1D9, having a MW of 135 kD, was analyzed. The results showed two sequences of interest, gi30722350 (1478 amino acid) and gi60219551 (1378 amino acid). In both sequences no transmembrane regions were observed. Sequence number gi30722350 was 99.7% showed a match to FYCO1, a molecule involved in induction of autophagy. Sequence number gi60219551 contained 15 cysteines and 11 putative glycosylation sites with 6 predicted N-glycosylation sites. It was also matched with all PDZ domain proteins. Moreover, it showed an 85.7% match to glypican-3. Glypican-3 on HepG2 cells competitively reacted with both phycoerythrin-conjugated anti-glypican-3 and mAb 1E4-1C2 and resulted in an increase of double-stained cell population when higher concentration of mAb 1E4-1D9 was used. Moreover, antigens precipitated from HepG2 cell by anti

  11. Characterization and application of two novel monoclonal antibodies against human OX40: costimulation of T cells and expression on tumor as well as normal gland tissues.

    Science.gov (United States)

    Xie, F; Wang, Q; Chen, Y; Gu, Y; Shi, Q; Ge, Y; Yu, G; Wu, H; Mao, Y; Wang, X; Zhou, Y; Zhang, X

    2006-04-01

    OX40, a membrane-bound molecule of the tumor-necrosis-factor-receptor superfamily, is a critical costimulatory receptor during the immune response. Here, we newly generated two specific mouse antihuman OX40 monoclonal antibodies (mAbs) (2G2 and 1F7), whose specificities are quite different from the available OX40 mAb (ACT35) by competition assay. It was also found that both mAbs could enhance the proliferation, activation and differentiation of T lymphocytes primed by anti-CD3 mAb. These results evidenced that both were functional antihuman OX40 mAbs. Furthermore, stained by 2G2 and 1F7, FCM and immunohistochemistry detected the constitutive expression of OX40 on tumor cell lines from epithelium, breast cancer and glioma tissues. Meanwhile, the non-tumor tissues (thyroid gland, stomach gland) were also found OX40 expression. These results suggested that OX40 is not only expressed in activated T cells, but also in some tumors as well as normal gland tissues. Such expression pattern indicated that OX40 may be a valuable surface antigen in unveiling its expression and function outside the immune system. Briefly, these novel antibodies may contribute to the evaluation of the mechanism of tumor metastasis and eventually shed light on further study of tumor immunotherapy and autoimmune diseases.

  12. Monensin, a small molecule ionophore, can be used to increase high mannose levels on monoclonal antibodies generated by Chinese hamster ovary production cell-lines.

    Science.gov (United States)

    Pande, Sandhya; Rahardjo, Ayu; Livingston, Brittney; Mujacic, Mirna

    2015-07-01

    Asparagine-linked glycosylation of the constant region of monoclonal antibodies (mAbs) plays an important role in their stability and efficacy and is a critical product quality attribute that needs to be consistent between various process changes and production lots. Exact product quality match is also of the utmost importance for the development of biosimilar protein therapeutics. This poses a process development challenge since mAb glycosylation profiles can fluctuate easily with changes in process parameters. Therefore, there is a need to identify methods to modulate glycosylation levels on therapeutic antibodies during a production run in order to maintain consistent product quality profiles between different drug lots. Here, we demonstrate the use of a small molecule ionophore, monensin, to increase high mannose levels on multiple therapeutic human immunoglobulins (IgGs) in both plate-based small scale production models as well as in production bioreactors. This method is simple to implement and readily applicable for multiple production cell lines. Moreover, high mannose levels can be increased without significant negative impact on titer or cell culture performance. As such, monensin gives us a manipulable product quality lever. © 2015 Wiley Periodicals, Inc.

  13. Muscle precursor cells in the developing limbs of two isopods (Crustacea, Peracarida): an immunohistochemical study using a novel monoclonal antibody against myosin heavy chain.

    Science.gov (United States)

    Kreissl, S; Uber, A; Harzsch, S

    2008-05-01

    In the hot debate on arthropod relationships, Crustaceans and the morphology of their appendages play a pivotal role. To gain new insights into how arthropod appendages evolved, developmental biologists recently have begun to examine the expression and function of Drosophila appendage genes in Crustaceans. However, cellular aspects of Crustacean limb development such as myogenesis are poorly understood in Crustaceans so that the interpretative context in which to analyse gene functions is still fragmentary. The goal of the present project was to analyse muscle development in Crustacean appendages, and to that end, monoclonal antibodies against arthropod muscle proteins were generated. One of these antibodies recognises certain isoforms of myosin heavy chain and strongly binds to muscle precursor cells in malacostracan Crustacea. We used this antibody to study myogenesis in two isopods, Porcellio scaber and Idotea balthica (Crustacea, Malacostraca, Peracarida), by immunohistochemistry. In these animals, muscles in the limbs originate from single muscle precursor cells, which subsequently grow to form multinucleated muscle precursors. The pattern of primordial muscles in the thoracic limbs was mapped, and results compared to muscle development in other Crustaceans and in insects.

  14. ING-1(heMAb, a Monoclonal Antibody to Epithelial Cell Adhesion Molecule, Inhibits Tumor Metastases in a Murine Cancer Model

    Directory of Open Access Journals (Sweden)

    Harry H. Ruan

    2003-11-01

    Full Text Available ING-1(heMAb, a human-engineered monoclonal antibody (MAb that specifically targets the epithelial cell adhesion molecule (Ep-CAM, kills adenocarcinoma cells in vitro and inhibits tumor growth in vivo. In the current study, we evaluated the efficacy of ING-1(heMAb in a murine model of cancer metastases. Mice received intravenous dosing of 1 mg/kg ING-1(heMAb, twice a week, starting on day 2 or day 5. A negative control group received 1 mg/kg human immunoglobulin G with the same dose frequency starting on day 2. A positive control group received weekly 100 mg/kg 54lurouracil/leucovorin starting on day 2. ING-1(heMAb/day 2 treatment significantly reduced both the number of visible tumor nodules in body cavities (P < .01 and the number of metastases on lung surfaces (P < .005. The treatment also resulted in a 91% reduction of micrometastases in lung tissues (P <.0001. Delaying ING-1(heMAb treatment until day 5 caused 54% reduction in micrometastases (P <.005. Our results indicate that a number of parameters, including treatment starting day, dose level, and dose frequency, are critical in achieving the optimal efficacy of ING-1(heMAb. We conclude that ING-1(heMAb effectively reduced tumor metastases in a murine cancer model. Immunotherapy with ING-1(heMAb may be beneficial in treating human metastatic diseases.

  15. A human monoclonal IgE antibody that binds to MGL_1304, a major allergen in human sweat, without activation of mast cells and basophils.

    Science.gov (United States)

    Ishii, Kaori; Hiragun, Makiko; Hiragun, Takaaki; Kan, Takanobu; Kawaguchi, Tomoko; Yanase, Yuhki; Tanaka, Akio; Takahagi, Shunsuke; Hide, Michihiro

    MGL_1304, a major allergen in human sweat for patients with atopic dermatitis and/or cholinergic urticaria, is secreted from Malassezia globosa on human skin. The amounts of MGL_1304 and IgE against MGL_1304 are evaluated by the histamine release test using basophils or mast cells sensitized with serum containing IgE against MGL_1304, and enzyme linked sorbent assay (ELISA) using MGL_1304 and anti-MGL_1304 antibodies. Here, we identified a human monoclonal IgE (ABS-IgE) that binds to the high affinity IgE receptor (FcεRI) and MGL_1304 with high affinity (KD = 1.99 nM) but does not release histamine from basophils and mast cells. An ELISA using ABS-IgE as a standard IgE revealed that the amount of IgE against MGL_1304 (1000 U/ml) in the standard sera of patients with AD, employed in our previous report, is 32 ng/ml. A sandwich ELISA using ABS-IgE as a detection antibody showed approximately 10 times lower detection limit for MGL_1304 than ELISA in which MGL_1304 is directly bound to an ELISA plate. Moreover, ABS-IgE prevented histamine release from mast cells and basophils by neutralizing MGL_1304 not only in a free form in solution, but also on FcεRI expressed on the cell surface without cell activation. ABS-IgE may be used both to quantify the amount of MGL_1304 and anti-MGL_1304 IgE, and possibly for the treatment of diseases caused/aggravated by type I allergy to MGL_1304.

  16. Flow cytometry analysis of single-strand DNA damage in neuroblastoma cell lines using the F7-26 monoclonal antibody.

    Science.gov (United States)

    Grigoryan, Rita S; Yang, Bo; Keshelava, Nino; Barnhart, Jerry R; Reynolds, C Patrick

    2007-11-01

    The F7-26 monoclonal antibody (Mab) has been reported to be specific for single-strand DNA damage (ssDNA) and to also identify cells in apoptosis. We carriedout studies to determine if F7-26 binding measured by flow cytometry was able to specifically identify exogenous ssDNA as opposed to DNA damage from apoptosis. Neuroblastoma cells were treated with melphalan (L-PAM), fenretinide, 4-hydroperoxycyclophosphamide (4-HC)+/-pan-caspase inhibitor BOC-d-fmk, topotecan or with 10Gy gamma radiation+/-hydrogen peroxide (H2O2) and fixed immediately postradiation. Cytotoxicity was measured by DIMSCAN digital imaging fluorescence assay. The degree of ssDNA damage was analyzed by flow cytometry using Mab F7-26, with DNA visualized by propidium iodide counterstaining. Flow cytometry was used to measure apoptosis detected by terminal deoxynucleotidyltransferase (TUNEL) assay and reactive oxygen species (ROS) by carboxy-dichlorofluorescein diacetate. Irradiated and immediately fixed neuroblastoma cells showed increased ssDNA, but not apoptosis by TUNEL (TUNEL-negative). 4-HC or L-PAM+/-BOC-d-fmk increased ssDNA (F7-26-positive), but BOC-d-fmk prevented TUNEL staining. Fenretinide increased apoptosis by TUNEL but not ssDNA damage detected with F7-26. Enhanced ssDNA in neuroblastoma cells treated with radiation+H2O2 was associated with increased ROS. Topotecan increased both ssDNA and cytotoxicity in 4-HC-treated cells. These data demonstrate that Mab F7-26 recognized ssDNA due to exogenous DNA damage, rather than apoptosis. This assay should be useful to characterize the mechanism of action of antineoplastic drugs. Copyright (c) 2007 International Society for Analytical Cytology.

  17. A novel monoclonal antibody to human laminin α5 chain strongly inhibits integrin-mediated cell adhesion and migration on laminins 511 and 521.

    Science.gov (United States)

    Wondimu, Zenebech; Omrani, Shahin; Ishikawa, Taichi; Javed, Fawad; Oikawa, Yuko; Virtanen, Ismo; Juronen, Erkki; Ingerpuu, Sulev; Patarroyo, Manuel

    2013-01-01

    Laminins, a large family of αβγ heterotrimeric proteins mainly found in basement membranes, are strong promoters of adhesion and migration of multiple cell types, such as tumor and immune cells, via several integrin receptors. Among laminin α (LMα) chains, α5 displays the widest tissue distribution in adult life and is synthesized by most cell types. Here, we have generated and characterized five novel monoclonal antibodies (mAbs) to the human LMα5 chain to further study the biological relevance of α5 laminins, such as laminins 511 (α5β1γ1) and 521 (α5β2γ1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMα5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited α3β1/α6β1 integrin-mediated adhesion and migration of glioma, melanoma, and carcinoma cells on laminin-511 and, together with mAb 4C7, on laminin-521. Accordingly, mAb 8G9 abolished the interaction of soluble α3β1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMα5 chain but largely hindered by mAb 4E10 to a LMβ1 chain epitope near the globular domain of laminin-511. Thus, mAb 8G9 defines a novel epitope localized at or near the integrin-binding globular domain of the LMα5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.

  18. A novel monoclonal antibody to human laminin α5 chain strongly inhibits integrin-mediated cell adhesion and migration on laminins 511 and 521.

    Directory of Open Access Journals (Sweden)

    Zenebech Wondimu

    Full Text Available Laminins, a large family of αβγ heterotrimeric proteins mainly found in basement membranes, are strong promoters of adhesion and migration of multiple cell types, such as tumor and immune cells, via several integrin receptors. Among laminin α (LMα chains, α5 displays the widest tissue distribution in adult life and is synthesized by most cell types. Here, we have generated and characterized five novel monoclonal antibodies (mAbs to the human LMα5 chain to further study the biological relevance of α5 laminins, such as laminins 511 (α5β1γ1 and 521 (α5β2γ1. As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMα5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited α3β1/α6β1 integrin-mediated adhesion and migration of glioma, melanoma, and carcinoma cells on laminin-511 and, together with mAb 4C7, on laminin-521. Accordingly, mAb 8G9 abolished the interaction of soluble α3β1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMα5 chain but largely hindered by mAb 4E10 to a LMβ1 chain epitope near the globular domain of laminin-511. Thus, mAb 8G9 defines a novel epitope localized at or near the integrin-binding globular domain of the LMα5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.

  19. Evaluation of non-thermal plasma-induced anticancer effects on human colon cancer cells

    Science.gov (United States)

    Choi, Jae-Sun; Kim, Jeongho; Hong, Young-Jun; Bae, Woom-Yee; Choi, Eun Ha; Jeong, Joo-Won; Park, Hun-Kuk

    2017-01-01

    Non-thermal atmospheric-pressure plasma has been introduced in various applications such as sterilization, wound healing, blood coagulation, and other biomedical applications. The most attractive application of non-thermal atmospheric-pressure plasma is in cancer treatment, where the plasma is used to produce reactive oxygen species (ROS) to facilitate cell apoptosis. We investigate the effects of different durations of exposure to dielectric-barrier discharge (DBD) plasma on colon cancer cells using measurement of cell viability and ROS levels, western blot, immunocytochemistry, and Raman spectroscopy. Our results suggest that different kinds of plasma-treated cells can be differentiated from control cells using the Raman data. PMID:28663896

  20. Effect of Wall Charge on Striation in Plasma Display Cells

    Institute of Scientific and Technical Information of China (English)

    HE Feng; OUYANG Jiting; CAO Jing; FENG Shuo; MIAO Jinsong; WANG Jianqi

    2007-01-01

    Different configurations and driving voltages have been employed to investigate the effect of the wall charge on the striations in macroscopic plasma display panel (PDP) cells.The experimental results show that a discharge channel near the dielectric layer is indispensable to striation occurring in the anode area during a discharge,while the pre-accumulated charge on the dielectric layer and the surface state are not important.The origin of the striation is related only to the physical process in the cell.The dielectric layer acts as a charge collector during a PDP discharge.

  1. Fat, Stem Cells, and Platelet-Rich Plasma.

    Science.gov (United States)

    James, Isaac B; Coleman, Sydney R; Rubin, J Peter

    2016-07-01

    The ideal filler for aesthetic surgery is inexpensive and easy to obtain, natural in appearance and texture, immunologically compatible, and long lasting without risk of infection. By most metrics, autologous fat grafts meet these criteria perfectly. Although facial fat grafting is now a commonly accepted surgical procedure, there has been a wave of activity applying stem cells and platelet-rich plasma (PRP) therapies to aesthetic practice. This article addresses technical considerations in the use of autologous fat transfer for facial rejuvenation, and also explores the current evidence for these stem cell and PRP therapies in aesthetic practice.

  2. Multigrid Particle-in-cell Simulations of Plasma Microturbulence

    Energy Technology Data Exchange (ETDEWEB)

    J.L.V. Lewandowski

    2003-06-17

    A new scheme to accurately retain kinetic electron effects in particle-in-cell (PIC) simulations for the case of electrostatic drift waves is presented. The splitting scheme, which is based on exact separation between adiabatic and on adiabatic electron responses, is shown to yield more accurate linear growth rates than the standard df scheme. The linear and nonlinear elliptic problems that arise in the splitting scheme are solved using a multi-grid solver. The multi-grid particle-in-cell approach offers an attractive path, both from the physics and numerical points of view, to simulate kinetic electron dynamics in global toroidal plasmas.

  3. Effects of anti-CXCR4 monoclonal antibody 12G5 on proliferation and apoptosis of human acute myelocytic leukemia cell line HL-60

    Institute of Scientific and Technical Information of China (English)

    WEI Li; KONG Pei-yan; SHI Zhan-zhong; ZENG Dong-feng; CHEN Xing-hua; CHANG Cheng; PENG Xian-gui; ZHANG Yi; LIU Hong

    2007-01-01

    Objective: To investigate the expression of CXCR4 on HL-60 cell line and the proliferation,apoptosis of HL-60 cell line cocultured with bone marrow stromal cells, so as to assess the possibility of 12G5, an anti-CXCR4 monoclonal antibody, in eradicating the minimal residual disease. Methods: The activity of SDF-1 was inhibited by 10 μg/ml 12G5. After treatment with 12G5, the status of adhesion was observed, and the adhesion rates, apoptosis and cell cycles were detected after 24 h of treatment. Cell growth rates were measured by trypan blue exclusion. Cell growth curve was plotted, and the expression of PCNA and apoptosis related protein including PCNA, Bcl-2 and Fas were detected with immunohistochemical technique. Results: (1) There was middling degree expression of CXCR4 on HL-60 membrane. From 0 h to 6 h, as the time of 12G5 incubation along, the expression of CXCR4 decreased gradually. (2)After treatment for 24 h, the adhesion rates in the experiment group and the control were (39.4±7.9)%and (51.4±5.9)%, respectively. (3)After treatment for 24 h, the percentage of HL-60 cells in G0/G1 phase were (55.21±4.9)%, and that in S phase and G2/M phase were (30.40±4.1)% and (14.39±5.2)%, respectively, with the corresponding proportions being (44.67±2.2)%, (45.30±3.7)%, and (10.03±2.6)% in the control. (4) The percentage of apoptotic HL-60 cells was (8.95±1.7)% in the experiment group, compared to (3.97±2.4)% in the control. (5)The survival rates of HL-60 cells decreased markedly at 48 h to 96 h, and the proliferation slowed down at this time duration. (6)The expression of PCNA and Bcl-2 down-regulated significantly, but the Fas protein expression was up-regulated. Conclusion: 12G5 could inhibit the capability of adhesion and proliferation of HL-60 cells and it can induce more cells to enter G0/G1 phase and promote apoptosis. It may be helpful by inhibiting the bioactivity of SDF-1 with 12G5 in the therapy of marrow residual disease.

  4. Endothelin A receptor antagonism enhances inhibitory effects of anti-ganglioside GD2 monoclonal antibody on invasiveness and viability of human osteosarcoma cells.

    Directory of Open Access Journals (Sweden)

    Bo Liu

    Full Text Available Endothelin-1 (ET-1/endothelin A receptor (ETAR signaling is important for osteosarcoma (OS progression. Monoclonal antibodies (mAbs targeting ganglioside GD2 reportedly inhibit tumor cell viability independent of the immune system. A recent study suggests that ganglioside GD2 may play an important role in OS progression. In the present study, we for the first time explored the effects of anti-GD2 mAb alone or in combination with ETAR antagonist on OS cell invasiveness and viability. Human OS cell lines Saos-2, MG-63 and SJSA-1 were treated with control IgG (PK136 mAb, 50 µg/mL, anti-GD2 14G2a mAb (50 µg/mL, selective ETAR antagonist BQ123 (5 µM, or 14G2a (50 µg/mL+BQ123 (5 µM. Cells with knockdown of ETAR (ETAR-shRNA with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K inhibitor BKM120 (50 µM were used as a positive control. Our results showed that BQ123, ETAR-shRNA and 14G2a mAb individually decreased cell invasion and viability, matrix metalloproteinase-2 (MMP-2 expression and activity, PI3k activity, and phosphorylation at serine 473 (ser473 of Akt in OS cells. 14G2a mAb in combination with BQ123 or ETAR-shRNA showed significantly stronger inhibitory effects compared with each individual treatment. In all three cell lines tested, 14G2a mAb in combination with BQ123 showed the strongest inhibitory effects. In conclusion, we provide the first in vitro evidence that anti-ganglioside GD2 14G2a mAb effectively inhibits cell invasiveness, MMP-2 expression and activity, and cell viability in human OS cells. ETAR antagonist BQ123 significantly enhances the inhibitory effects of 14G2a mAb, likely mainly through inhibiting the PI3K/Akt pathway. This study adds novel insights into OS treatment, which will serve as a solid basis for future in vivo studies on the effects of combined treatment of OS with anti-ganglioside GD2 mAbs and ETAR antagonists.

  5. Thermal plasma treatment of cell-phone waste : preliminary result

    Energy Technology Data Exchange (ETDEWEB)

    Ruj, B. [Central Mechanical Engineering Research Inst., Durgapur (India). Thermal Engineering Group; Chang, J.S.; Li, O.L. [McMaster Univ., Hamilton, ON (Canada). Dept. of Engineering Physics; Pietsch, G. [RWTH Aachen Univ., Aachen (Germany)

    2010-07-01

    The cell phone is an indispensable service facilitator, however, the disposal and recycling of cell phones is a major problem. While the potential life span of a mobile phone, excluding batteries, is over 10 years, most of the users upgrade their phones approximately four times during this period. Cell phone waste is significantly more hazardous than many other municipal wastes as it contains thousands of components made of toxic chemicals and metals like lead, cadmium, chromium, mercury, polyvinyl chlorides (PVC), brominated flame retardants, beryllium, antimony and phthalates. Cell phones also use many expensive rare metals. Since cell phones are made up of plastics, metals, ceramics, and trace other substances, primitive recycling or disposal of cell phone waste to landfills and incinerators creates irreversible environmental damage by polluting water and soil, and contaminating air. In order to minimize releases into the environment and threat to human health, the disposal of cell phones needs to be managed in an environmentally friendly way. This paper discussed a safer method of reducing the generation of syngas and hydrocarbons and metal recovery through the treatment of cell phone wastes by a thermal plasma. The presentation discussed the experiment, with particular reference to sample preparation; experimental set-up; and results four samples with different experimental conditions. It was concluded that the plasma treatment of cell phone waste in reduced condition generates gaseous components such as hydrogen, carbon monoxide, and hydrocarbons which are combustible. Therefore, this system is an energy recovery system that contributes to resource conservation and reduction of climate change gases. 5 refs., 2 tabs., 2 figs.

  6. Plasma Cell-Free DNA in Paediatric Lymphomas

    Science.gov (United States)

    Mussolin, Lara; Burnelli, Roberta; Pillon, Marta; Carraro, Elisa; Farruggia, Piero; Todesco, Alessandra; Mascarin, Maurizio; Rosolen, Angelo

    2013-01-01

    Background: Extracellular circulating DNA (cfDNA) can be found in small amounts in plasma of healthy individuals. Increased levels of cfDNA have been reported in patients with cancer of breast, cervix, colon, liver and it was shown that cfDNA can originate from both tumour and non-tumour cells. Objectives: Levels of cfDNA of a large series of children with lymphoma were evaluated and analyzed in relation with clinical characteristics. Methods: plasma cfDNA levels obtained at diagnosis in 201 paediatric lymphoma patients [43 Hodgkin lymphomas (HL), 45 anaplastic large cell lymphomas (ALCL), 88 Burkitt lymphomas (BL), 17 lymphoblastic (LBL), 8 diffuse large B cell lymphoma (DLBCL)] and 15 healthy individuals were determined using a quantitative PCR assay for POLR2 gene and, in addition, for NPM-ALK fusion gene in ALCL patients. Wilcoxon rank sum test was used to compare plasma levels among different patient subgroups and controls and to analyze relationship between levels of cfDNA and clinical characteristics. Results: Levels of cfDNA in lymphoma patients were significantly higher compared with controls (p<0.0001). CfDNA was associated with median age (p=0.01) in HL, and with stage in ALCL (p=0.01). In HL patients high cfDNA levels were correlated with poor prognosis (p=0.03). In ALCL we found that most of the cfDNA (77%) was non-tumor DNA. Conclusion: level of plasma cfDNA might constitute an important non-invasive tool at diagnosis in lymphoma patients' management; in particular in patients with HL, cfDNA seems to be a promising prognostic biomarker. PMID:23678368

  7. [Monoclonal gammopathy of undetermined significance and asymptomatic multiple myelom in the year 2014 ].

    Science.gov (United States)

    Adam, Zdeněk; Krejčí, Marta; Pour, Luděk; Sevčíková, Eva; Křivanová, Andrea; Rehák, Zdeněk; Koukalová, Renata; Cermáková, Zdeňka; Vaníček, Jíří; Sevčíková, Sabina

    2014-10-01

    Presence of monoclonal immunoglobulin in serum or urine is a relatively common event affecting about 3.2 % of people over 50. Isolated increase of only one type of free light chain, either κ or λ, is detected in 0.7-0.8 % of people over 50. Most people with monoclonal immunoglobulin meet the criteria of the so-called "mono-clonal gammopathy of undetermined significance (MGUS)". MGUS is defined by concentration of monoclonal immunoglobulin in serum < 30 g/l, number of plasma cells in the bone marrow < 10 % and the absence of symptoms of multiple myeloma and other lymphoproliferative diseases. A proportion of people with MGUS gradually progresses from asymptomatic into symptomatic myeloma or other malignant lymphoproliferative disease requiring treatment. Therefore, MGUS is considered to be one of the most common premalignant conditions with an average risk of transformation into malignant disease of 1 % per year. Monoclonal gammopathy of IgG and IgA subtype can develop into multiple myeloma. Light chain monoclonal gammopathy can develop not only into light chain multiple myeloma but also into AL-amyloidosis and light chain deposition disease (amorphous deposits of light chains damaging organs). IgM monoclonal gammopathy may develop into Waldenstrom macroglobulinemia or other lymphoproliferative disorder, or into rare IgM subtype of multiple myeloma. Unfortunately, people with MGUS are threatened by more than an increased risk of transformation into multiple myeloma or other severe hematologic disease. Pre-malignant clone of plasma cells in the bone marrow causes changes in the bone marrow that directly affect the person. For people with MGUS, there is an increased incidence of osteoporosis and increased fracture risk when compared to the general population. People with MGUS also have an increased risk of bacterial infections and thromboembolic complications compared with the same age population without MGUS. Clonal plasma cells, which are the basis of MGUS, may in

  8. Selective cytotoxicity of indirect nonequilibrium atmospheric pressure plasma against ovarian clear-cell carcinoma.

    Science.gov (United States)

    Utsumi, Fumi; Kajiyama, Hiroaki; Nakamura, Kae; Tanaka, Hiromasa; Hori, Masaru; Kikkawa, Fumitaka

    2014-01-01

    Ovarian clear cell carcinoma (CCC) is a histological type of epithelial ovarian cancer that is less responsive to chemotherapy and associated with a poorer prognosis than serous and endometrioid carcinoma. Non-thermal atmospheric pressure plasma which produces reactive species has recently led to an explosion of research in plasma medicine. Plasma treatment can be applied to cancer treatment to induce apoptosis and tumor growth arrest. Furthermore, recent studies have shown that a medium exposed to plasma also has an anti-proliferative effect against cancer in the absence of direct exposure to plasma. In this study, we confirmed whether this indirect plasma has an anti-tumor effect against CCC, and investigated whether this efficacy is selective for cancer cells. Non-thermal atmospheric pressure plasma induced apoptosis in CCC cells, while human peritoneal mesothelial cells remained viable. Non-thermal atmospheric pressure plasma exhibits selective cytotoxicity against CCC cells which are resistant to chemotherapy.

  9. Identification of a second T-cell antigen receptor in human and mouse by an anti-peptide. gamma. -chain-specific monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Ioannides, C.G.; Itoh, K.; Fox, F.E.; Pahwa, R.; Good, R.A.; Platsoucas, C.D.

    1987-06-01

    The authors developed a monoclonal antibody (mAb) (9D7) against a synthetic peptide (P13K) selected from the deduced amino acid sequence of the constant region of the lambda chain of the murine T-cell antigen receptor (TCR) (amino acids 118-130). Using this mAb, they identified a putative second TCR expressed on peripheral blood lymphocytes from a patient with severe combined immunodeficiency (SCID) that were propagated in culture with recombinant interleukin 2 (rIL-2) and Con A. This mAb immunoprecipitated two polypeptide chains of 40 and 58 kDa under nonreducing conditions and of 40 and 56 kDa under reducing conditions from /sup 125/I-labeled denatured lysates of T3/sup +/ WT31/sup -/ lymphocytes expanded in culture from a SCID patient. Chemical crosslinking of /sup 125/I-labeled cells followed by immunoprecipitation with anti-Leu-4 mAb under nonreducing or reducing conditions revealed that the 40- and 56-kDa polypeptide chains were associated with the T3 differentiation antigen. These experiments were done with polyclonal cell populations. Cloned T3/sup +/ WT31/sup -/ cell populations are required to determine whether the TCR contains two lambda polypeptide chains. Using the same 9D7 anti-P18K mAb and immunoblotting analysis, they identified a 35 kDa ..gamma..-chain polypeptide under reducing conditions expressed on purified L3T4/sup -/ Lyt2/sup -/ BALB/c mouse thymocytes. This ..gamma..-chain TCR is disulfide linked and has a molecular mass of 80 kDa under nonreducing conditions.

  10. Development of a microfluidic device for cell concentration and blood cell-plasma separation.

    Science.gov (United States)

    Maria, M Sneha; Kumar, B S; Chandra, T S; Sen, A K

    2015-12-01

    This work presents design, fabrication and test of a microfluidic device which employs Fahraeus-Lindqvist and Zweifach-Fung effects for cell concentration and blood cell-plasma separation. The device design comprises a straight main channel with a series of branched channels placed symmetrically on both sides of the main channel. The design implements constrictions before each junction (branching point) in order to direct cells that would have migrated closer to the wall (naturally or after liquid extraction at a junction) towards the centre of the main channel. Theoretical and numerical analysis are performed for design of the microchannel network to ensure that a minimum flow rate ratio (of 2.5:1, main channel-to-side channels) is maintained at each junction and predict flow rate at the plasma outlet. The dimensions and location of the constrictions were determined using numerical simulations. The effect of presence of constrictions before the junctions was demonstrated by comparing the performances of the device with and without constrictions. To demonstrate the performance of the device, initial experiments were performed with polystyrene microbeads (10 and 15 μm size) and droplets. Finally, the device was used for concentration of HL60 cells and separation of plasma and cells in diluted blood samples. The cell concentration and blood-plasma purification efficiency was quantified using Haemocytometer and Fluorescence-Activated Cell Sorter (FACS). A seven-fold cell concentration was obtained with HL60 cells and a purification efficiency of 70 % and plasma recovery of 80 % was observed for diluted (1:20) blood sample. FACS was used to identify cell lysis and the cell viability was checked using Trypan Blue test which showed that more than 99 % cells are alive indicating the suitability of the device for practical use. The proposed device has potential to be used as a sample preparation module in lab on chip based diagnostic platforms.

  11. Circulating human B and plasma cells. Age-associated changes in counts and detailed characterization of circulating normal CD138- and CD138+ plasma cells.

    Science.gov (United States)

    Caraux, Anouk; Klein, Bernard; Paiva, Bruno; Bret, Caroline; Schmitz, Alexander; Fuhler, Gwenny M; Bos, Nico A; Johnsen, Hans E; Orfao, Alberto; Perez-Andres, Martin

    2010-06-01

    Generation of B and plasma cells involves several organs with a necessary cell trafficking between them. A detailed phenotypic characterization of four circulating B-cell subsets (immature-, naïve-, memory- B-lymphocytes and plasma cells) of 106 healthy adults was realized by multiparametric flow cytometry. We show that CD10, CD27 and CD38 is the minimal combination of subsetting markers allowing unequivocal identification of immature (CD10(+)CD27(-)CD38(+), 6+/-6 cells/microL), naïve (CD10(-)CD27(-)CD38(-), 125+/-90 cells/microL), memory B lymphocytes (CD10(-)CD27(+)CD38(-), 58+/-42 cells/microL), and plasma cells (CD10(-)CD27(++)CD38(++), 2.1+/-2.1 cells/microL) within circulating CD19(+) cells. From these four subsets, only memory B lymphocytes and plasma cells decreased with age, both in relative and absolute counts. Circulating plasma cells split into CD138(-) (57+/-12%) and CD138(+) (43+/-12%) cells, the latter displaying a more mature phenotypic profile: absence of surface immunoglobulin, lower CD45 positivity and higher amounts of cytoplasmic immunoglobulin, CD38 and CD27. Unlike B lymphocytes, both populations of plasma cells are KI-67(+) and show weak CXCR4 expression.

  12. Investigating the cell death mechanisms in primary prostate cancer cells using low-temperature plasma treatment

    Science.gov (United States)

    O'Connell, Deborah; Hirst, A. M.; Packer, J. R.; Simms, M. S.; Mann, V. M.; Frame, F. M.; Maitland, N. J.

    2016-09-01

    Atmospheric pressure plasmas have shown considerable promise as a potential cancer therapy. An atmospheric pressure plasma driven with kHz kV excitation, operated with helium and oxygen admixtures is used to investigate the interaction with prostate cancer cells. The cytopathic effect was verified first in two commonly used prostate cancer cell lines (BPH-1 and PC-3 cells) and further extended to examine the effects in paired normal and tumour prostate epithelial cells cultured directly from patient tissues. Through the formation of reactive species in cell culture media, and potentially other plasma components, we observed high levels of DNA damage, together with reduced cell viability and colony-forming ability. We observed differences in response between the prostate cell lines and primary cells, particularly in terms of the mechanism of cell death. The primary cells ultimately undergo necrotic cell death in both the normal and tumour samples, in the complete absence of apoptosis. In addition, we provide the first evidence of an autophagic response in primary cells. This work highlights the importance of studying primary cultures in order to gain a more realistic insight into patient efficacy. EPSRC EP/H003797/1 & EP/K018388/1, Yorkshire Cancer Research: YCR Y257PA.

  13. The effect of tuning cold plasma composition on glioblastoma cell viability.

    Directory of Open Access Journals (Sweden)

    Xiaoqian Cheng

    Full Text Available Previous research in cold atmospheric plasma (CAP and cancer cell interaction has repeatedly proven that the cold plasma induced cell death. It is postulated that the reactive oxygen species (ROS and reactive nitrogen species (RNS play a major role in the CAP cancer therapy. In this paper, we seek to determine a mechanism of CAP therapy on glioblastoma cells (U87 through an understanding of the composition of the plasma, including treatment time, voltage, flow-rate and plasma-gas composition. In order to determine the threshold of plasma treatment on U87, normal human astrocytes (E6/E7 were used as the comparison cell line. Our data showed that the 30 sec plasma treatment caused 3-fold cell death in the U87 cells compared to the E6/E7 cells. All the other compositions of cold plasma were performed based on this result: plasma treatment time was maintained at 30 s per well while other plasma characteristics such as voltage, flow rate of source gas, and composition of source gas were changed one at a time to vary the intensity of the reactive species composition in the plasma jet, which may finally have various effect on cells reflected by cell viability. We defined a term "plasma dosage" to summarize the relationship of all the characteristics and cell viability.

  14. Management of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM).

    Science.gov (United States)

    Kyle, Robert A; Buadi, Francis; Rajkumar, S Vincent

    2011-06-01

    Monoclonal gammopathy of undetermined significance (MGUS) is defined as a serum M protein level of less than 3 g/dL, less than 10% clonal plasma cells in the bone marrow, and the absence of end-organ damage. The prevalence of MGUS is 3.2% in the white population but is approximately twice that high in the black population. MGUS may progress to multiple myeloma, AL amyloidosis, Waldenström macroglobulinemia, or lymphoma. The risk of progression is approximately 1% per year, but the risk continues even after more than 25 years of observation. Risk factors for progression include the size of the serum M protein, the type of serum M protein, the number of plasma cells in the bone marrow, and the serum free light chain ratio. Smoldering (asymptomatic) multiple myeloma (SMM) is characterized by the presence of an M protein level of 3 g/dL or higher and/or 10% or more monoclonal plasma cells in the bone marrow but no evidence of end-organ damage. The overall risk of progression to a malignant condition is 10% per year for the first 5 years, approximately 3% per year for the next 5 years, and 1% to 2% per year for the following 10 years. Patients with both MGUS and SMM must be followed up for their lifetime.

  15. Management of Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM)

    Science.gov (United States)

    KYLE, ROBERT A.; BUADI, FRANC IS; RAJKUMAR, S. VINC ENT

    2014-01-01

    Monoclonal gammopathy of undetermined significance (MGUS) is defined as a serum M protein level of less than 3 g/dL, less than 10% clonal plasma cells in the bone marrow, and the absence of end-organ damage. The prevalence of MGUS is 3.2% in the white population but is approximately twice that high in the black population. MGUS may progress to multiple myeloma, AL amyloidosis, Waldenström macroglobulinemia, or lymphoma. The risk of progression is approximately 1% per year, but the risk continues even after more than 25 years of observation. Risk factors for progression include the size of the serum M protein, the type of serum M protein, the number of plasma cells in the bone marrow, and the serum free light chain ratio. Smoldering (asymptomatic) multiple myeloma (SMM) is characterized by the presence of an M protein level of 3 g/dL or higher and/or 10% or more monoclonal plasma cells in the bone marrow but no evidence of end-organ damage. The overall risk of progression to a malignant condition is 10% per year for the first 5 years, approximately 3% per year for the next 5 years, and 1% to 2% per year for the following 10 years. Patients with both MGUS and SMM must be followed up for their lifetime. PMID:21888255

  16. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

    Sondergeld, P.; van de Donk, N. W. C. J.; Richardson, P. G.;

    2015-01-01

    The development of monoclonal antibodies (mAbs) for the treatment of disease goes back to the vision of Paul Ehrlich in the late 19th century; however, the first successful treatment with a mAb was not until 1982, in a lymphoma patient. In multiple myeloma, mAbs are a very recent and exciting add...

  17. Treatment of severe refractory autoimmune hemolytic anemia in B-cell chronic lymphocytic leukemia with alemtuzumab (humanized CD52 monoclonal antibody).

    Science.gov (United States)

    Karlsson, C; Hansson, L; Celsing, F; Lundin, J

    2007-03-01

    Progressive B-cell chronic lymphocytic leukemia (B-CLL) is often complicated by autoimmune hemolytic anemia (AIHA), which in some cases may be refractory to conventional therapy such as corticosteroids, rituximab and splenectomy. We report here on 5 patients (median age 66 years, range 59-69) with advanced B-CLL, all of whom developed severe transfusion-dependent AIHA resistant to conventional therapy and received subcutaneous (SC) or intravenous (IV) alemtuzumab, a humanized monoclonal antibody that targets the CD52 antigen as salvage treatment for AIHA. Alemtuzumab was well tolerated with only minor 'first dose' reactions. All 5 patients responded with a >or=2.0 g/dl rise in hemoglobin (Hb) concentration, in the absence of further transfusions, after a median time of 5 weeks (range 4-7), and the mean Hb increased from 7.2 g/dl at baseline to 11.9 g/dl at end of treatment. All patients remained stable, without further AIHA episodes, after a median follow-up time of 12 months with a mean Hb of 12.5 g/dl (range 12.2-12.9). For patients with severe, refractory CLL-related AIHA, who have not previously responded to conventional therapy, alemtuzumab is an effective agent.

  18. Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies in Chinese hamster ovary cell culture.

    Science.gov (United States)

    Kishishita, Shohei; Nishikawa, Tomoko; Shinoda, Yasuharu; Nagashima, Hiroaki; Okamoto, Hiroshi; Takuma, Shinya; Aoyagi, Hideki

    2015-06-01

    During the production of therapeutic monoclonal antibodies (mAbs), not only enhancement of mAb productivity but also control of quality attributes is critical. Charge variants, which are among the most important quality attributes, can substantially affect the in vitro and in vivo properties of mAbs. During process development for the production of mAbs in a Chinese hamster ovary cell line, we have observed that an improvement in mAb titer is accompanied by an increase in the content of acidic charge variants. Here, to help maintain comparability among mAbs, we aimed to identify the process parameters that controlled the content of acidic charge variants. First, we used a Plackett-Burman design to identify the effect of selected process parameters on the acidic charge variant content. Eight process parameters were selected by using a failure modes and effects analysis. Among these, temperature shift was identified from the Plackett-Burman design as the factor most influencing the acidic charge variant content. We then investigated in more detail the effects of shift temperature and temperature shift timing on this content. The content decreased with a shift to a lower temperature and with earlier timing of this temperature shift. Our observations suggest that Plackett-Burman designs are advantageous for preliminary screening of bioprocess parameters. We report here for the first time that temperature downshift is beneficial for effective control of the acidic peak variant content.

  19. Evidence for local expansion of IgA plasma cell precursors in human ileum

    NARCIS (Netherlands)

    Yuvaraj, S.; Dijkstra, G.; Burgerhof, J.G.M.; Dammers, P.M.; Stoel, M.; Visser, Annie; Kroese, F.G.M.; Bos, N.A.

    2009-01-01

    IgA plays a crucial role in establishment and maintenance of mucosal homeostasis between host cells and commensal bacteria. To this end, numerous IgA plasma cells are located in the intestinal lamina propria. Whether the (immediate) precursor cells for these plasma cells can expand locally is not

  20. The hormesis effect of plasma-elevated intracellular ROS on HaCaT cells

    Science.gov (United States)

    Szili, Endre J.; Harding, Frances J.; Hong, Sung-Ha; Herrmann, Franziska; Voelcker, Nicolas H.; Short, Robert D.

    2015-12-01

    We have examined the link between ionized-gas plasma delivery of reactive oxygen species (ROS) to immortalized keratinocyte (HaCaT) cells and cell fate, defined in terms of cell viability versus death. Phospholipid vesicles were used as cell mimics to measure the possible intracellular ROS concentration, [ROSi], delivered by various plasma treatments. Cells were exposed to a helium cold atmospheric plasma (CAP) jet for different plasma exposure times (5-60 s) and gas flow rates (50-1000 ml min-1). Based upon the [ROSi] data we argue that plasma-generated ROS in the cell culture medium can readily diffuse into real cells. Plasma exposure that equated to an [ROSi] in the range of 3.81  ×  10-10-9.47  ×  10-8 M, measured at 1 h after the plasma exposure, resulted in increased cell viability at 72 h; whereas a higher [ROSi] at 1 h decreased cell viability after 72 h of culture. This may be because of the manner in which the ROS are delivered by the plasma: HaCaT cells better tolerate a low ROS flux over an extended plasma exposure period of 1 min, compared to a high flux delivered in a few seconds, although the final [ROSi] may be the same. Our results suggest that plasma stimulation of HaCaT cells follows the principle of hormesis.

  1. Physiologically relevant binding affinity quantification of monoclonal antibody PF‐00547659 to mucosal addressin cell adhesion molecule for in vitro in vivo correlation

    Science.gov (United States)

    Wang, Mengmeng; Kussrow, Amanda K; Ocana, Mireia Fernandez; Chabot, Jeffrey R; Lepsy, Christopher S

    2016-01-01

    Background and Purpose A monoclonal antibody (PF‐00547659) against mucosal addressin cell adhesion molecule (MAdCAM), expressed as both soluble (sMAdCAM) and trans‐membrane (mMAdCAM) target forms, showed over 30‐fold difference in antibody‐target KD between in vitro (Biacore) and clinically derived (KD,in‐vivo) values. Back‐scattering interferometry (BSI) was applied to acquire physiologically relevant KD values which were used to establish in vitro and in vivo correlation (IVIVC). Experimental Approach BSI was applied to obtain KD values between PF‐00547659 and recombinant human MAdCAM in buffer or CHO cells and endogenous MAdCAM in human serum or colon tissue. CHO cells and tissue were minimally processed to yield homogenate containing membrane vesicles and soluble proteins. A series of binding affinities in serum with various dilution factors was used to estimate both KD,in‐vivo and target concentrations; MAdCAM concentrations were also measured using LC–MS/MS. Key Results BSI measurements revealed low KD values (higher affinity) for sMAdCAM in buffer and serum, yet a 20‐fold higher KD value (lower affinity) for mMAdCAM in CHO, mMAdCAM and sMAdCAM in tissue. BSI predicted KD,in‐vivo in serum was similar to clinically derived KD,in‐vivo, and the BSI‐estimated serum sMAdCAM concentration also matched the measured concentration by LC–MS/MS. Conclusions and Implications Our results successfully demonstrated that BSI measurements of physiologically relevant KD values can be used to establish IVIVC, for PF‐00547659 to MAdCAM despite the lack of correlation when using Biacore measured KD and accurately estimates endogenous target concentrations. The application of BSI would greatly enhance successful basic pharmacological research and drug development. PMID:27760281

  2. Are neurological complications of monoclonal gammopathy of undetermined significance underestimated?

    Science.gov (United States)

    Steiner, Normann; Schwärzler, Angelika; Göbel, Georg; Löscher, Wolfgang

    2017-01-01

    Objectives Monoclonal gammopathy of undetermined significance (MGUS) is a premalignancy preceding multiple myeloma (MM) or related disorders. Neurological symptoms caused by the monoclonal immunoglobulins or free light-chains are often associated with a high morbidity. We analyzed the prevalence of neuropathy, clinical features and the long-term outcome in 223 patients (pts.) with MGUS. Patients and Methods Between 1/2005 and 3/2015, 223 adult pts. with MGUS were identified in our database. Results In36/223 pts. (16%) a neuropathy was diagnosed (MGUS associated neuropathy, MGUS-N). 20 pts. (55%) had a distal symmetric axonal neuropathy, 10 pts. (28%) had a chronic inflammatory demyelinating polyneuropathy and 6 pts (17%) a distal acquired demyelinating symmetric polyneuropathy. In MGUS-NN (without neuropathy) and in MGUS-N, progression to smoldering MM, MM or Waldenstrom's macroglobulinemia (WM) occurred in 17% of the pts. The Immunoglobulin subtype was predominantly IgG in MGUS-NN and IgM in MGUS-N and ≥5.5% plasma cells in the bone-marrow predicted progression to MM and AL-amyloidosis in MGUS-NN and to WM in MGUS-N (p<0.05). Conclusion Due to the substantial prevalence of neuropathies, MGUS pts. should be monitored carefully and referred to a specialized center if neurological symptoms occur. PMID:27974705

  3. Polyclonal Immunoglobulin G N-Glycosylation in the Pathogenesis of Plasma Cell Disorders.

    Science.gov (United States)

    Mittermayr, Stefan; Lê, Giao N; Clarke, Colin; Millán Martín, Silvia; Larkin, Anne-Marie; O'Gorman, Peter; Bones, Jonathan

    2017-02-03

    The pathological progression from benign monoclonal gammopathy of undetermined significance (MGUS) to smoldering myeloma (SMM) and finally to active myeloma (MM) is poorly understood. Abnormal immunoglobulin G (IgG) glycosylation in myeloma has been reported. Using a glycomic platform composed of hydrophilic interaction UPLC, exoglycosidase digestions, weak anion-exchange chromatography, and mass spectrometry, polyclonal IgG N-glycosylation profiles from 35 patients [MGUS (n = 8), SMM (n = 5), MM (n = 8), complete-response (CR) post-treatment (n = 5), relapse (n = 4), healthy age-matched control (n = 5)] were characterized to map glycan structures in distinct disease phases of multiple myeloma. N-Glycan profiles from MGUS resembled normal control. The abundance of neutral glycans containing terminal galactose was highest in SMM, while agalactosylated glycans and fucosylated glycans were lowest in MM. Three afucosyl-biantennary-digalactosylated-sialylated species (A2G2S1, A2BG2S1, and A2BG2S2) decreased 2.38-, 2.4-, and 4.25-fold, respectively, from benign to active myeloma. Increased light chain sialylation was observed in a longitudinal case of transformation from MGUS to MM. Bisecting N-acetylglucosamine was lowest in the CR group, while highest in relapsed disease. Gene expression levels of FUT 8, ST6GAL1, B4GALT1, RECK, and BACH2 identified from publicly available GEP data supported the glycomic changes seen in MM compared to control. The observed differential glycosylation underlined the heterogeneity of the myeloma spectrum. This study demonstrates the feasibility of mapping glycan modifications on the IgG molecule and provides proof of principle that differential IgG glycosylation patterns can be successfully identified in plasma cell disorders.

  4. Regulation of germinal center responses, memory B cells and plasma cell formation-an update.

    Science.gov (United States)

    Corcoran, Lynn M; Tarlinton, David M

    2016-04-01

    Progress in understanding humoral immunity has been accelerated by the powerful experimental approaches of genetics, genomics and imaging. Excellent reviews of these advances appeared in 2015 in celebration of the 50th anniversary of the discovery of B cell and T cell lineages in the chicken. Here we provide a contemporary model of B cell differentiation, highlighting recent publications illuminating germinal center (GC), memory B cell and antibody-secreting plasma cell biology. The important contributions of CD4T cells to antibody responses have been thoroughly reviewed elsewhere.

  5. Predominant antitumor effects by fully human anti-TRAIL-receptor 2 (DR5) monoclonal antibodies in human glioma cells in vitro and in vivo.

    Science.gov (United States)

    Nagane, Motoo; Shimizu, Saki; Mori, Eiji; Kataoka, Shiro; Shiokawa, Yoshiaki

    2010-07-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2 L) preferentially induces apoptosis in human tumor cells through its cognate death receptors DR4 or DR5, thereby being investigated as a potential agent for cancer therapy. Here, we applied fully human anti-human TRAIL receptor monoclonal antibodies (mAbs) to specifically target one of death receptors for TRAIL in human glioma cells, which could also reduce potential TRAIL-induced toxicity in humans. Twelve human glioma cell lines treated with several fully human anti-human TRAIL receptor mAbs were sensitive to only anti-DR5 mAbs, whereas they were totally insensitive to anti-DR4 mAb. Treatment with anti-DR5 mAbs exerted rapid cytotoxicity and lead to apoptosis induction. The cellular sensitivity was closely associated with cell-surface expression of DR5. Expression of c-FLIP(L), Akt, and Cyclin D1 significantly correlated with sensitivity to anti-DR5 mAbs. Primary cultures of glioma cells were also relatively resistant to anti-DR5 mAbs, exhibiting both lower DR5 and higher c-FLIP(L) expression. Downregulation of c-FLIP(L) expression resulted in the sensitization of human glioma cells to anti-DR5 mAbs, whereas overexpression of c-FLIP(L) conferred resistance to anti-DR5 mAb. Treatment of tumor-burden nude mice with the direct agonist anti-DR5 mAb KMTR2 significantly suppressed growth of subcutaneous glioma xenografts leading to complete regression. Similarly, treatment of nude mice bearing intracerebral glioma xenografts with KMTR2 significantly elongated lifespan without tumor recurrence. These results suggest that DR5 is the predominant TRAIL receptor mediating apoptotic signals in human glioma cells, and sensitivity to anti-DR5 mAbs was determined at least in part by the expression level of c-FLIP(L) and Akt. Specific targeting of death receptor pathway through DR5 using fully human mAbs might provide a novel therapeutic strategy for intractable malignant gliomas.

  6. Identification and characterization of plasma cells in normal human bone marrow by high-resolution flow cytometry

    NARCIS (Netherlands)

    Terstappen, Leon W.M.M.; Johnsen, Steen; Segers-Nolten, Ine M.J.; Loken, Michael R.

    1990-01-01

    The low frequency of plasma cells and the lack of specific cell surface markers has been a major obstacle for a detailed characterization of plasma cells in normal human bone marrow. Multiparameter flow cytometry enabled the identification of plasma cells in normal bone marrow aspirates. The plasma

  7. KTN0158, a Humanized Anti-KIT Monoclonal Antibody, Demonstrates Biologic Activity against both Normal and Malignant Canine Mast Cells.

    Science.gov (United States)

    London, Cheryl A; Gardner, Heather L; Rippy, Sarah; Post, Gerald; La Perle, Krista; Crew, Linda; Lopresti-Morrow, Lori; Garton, Andrew J; McMahon, Gerald; LaVallee, Theresa M; Gedrich, Richard

    2016-11-04

    Purpose: KTN0158 is a novel anti-KIT antibody that potently inhibits wild-type and mutant KIT. This study evaluated the safety, biologic activity, and pharmacokinetic/pharmacodynamics profile of KTN0158 in dogs with spontaneous mast cell tumors (MCT) as a prelude to human clinical applications.Experimental Design: Cell proliferation, KIT phosphorylation, and mast cell degranulation were evaluated in vitro KTN0158 was administered to 4 research dogs to assess clinical effects and cutaneous mast cell numbers. Thirteen dogs with spontaneous MCT were enrolled into a prospective phase I dose-escalating open-label clinical study of KTN0158 evaluating 3 dose levels and 2 schedules and with weekly assessments for response and clinical toxicities.Results: KTN0158 was a potent inhibitor of human and dog KIT activation and blocked mast cell degranulation in vitro In dogs, KTN0158 was well tolerated and reduced cutaneous mast cell numbers in a dose-dependent manner. Clinical benefit of KTN0158 administration in dogs with MCT (n = 5 partial response; n = 7 stable disease) was observed regardless of KIT mutation status, and decreased KIT phosphorylation was demonstrated in tumor samples. Histopathology after study completion demonstrated an absence of neoplastic cells in the primary tumors and/or metastatic lymph nodes from 4 dogs. Reversible hematologic and biochemical adverse events were observed at doses of 10 and 30 mg/kg. The MTD was established as 10 mg/kg.Conclusions: KTN0158 inhibits KIT phosphorylation, demonstrates an acceptable safety profile in dogs, and provides objective responses in canine MCT patients with and without activating KIT mutations, supporting future clinical evaluation of KTN0158 in people. Clin Cancer Res; 1-10. ©2016 AACR.

  8. Cell-Cycle-Dependent Reconfiguration of the DNA Methylome during Terminal Differentiation of Human B Cells into Plasma Cells.

    Science.gov (United States)

    Caron, Gersende; Hussein, Mourad; Kulis, Marta; Delaloy, Céline; Chatonnet, Fabrice; Pignarre, Amandine; Avner, Stéphane; Lemarié, Maud; Mahé, Elise A; Verdaguer-Dot, Núria; Queirós, Ana C; Tarte, Karin; Martín-Subero, José I; Salbert, Gilles; Fest, Thierry

    2015-11-03

    Molecular mechanisms underlying terminal differentiation of B cells into plasma cells are major determinants of adaptive immunity but remain only partially understood. Here we present the transcriptional and epigenomic landscapes of cell subsets arising from activation of human naive B cells and differentiation into plasmablasts. Cell proliferation of activated B cells was linked to a slight decrease in DNA methylation levels, but followed by a committal step in which an S phase-synchronized differentiation switch was associated with an extensive DNA demethylation and local acquisition of 5-hydroxymethylcytosine at enhancers and genes related to plasma cell identity. Downregulation of both TGF-?1/SMAD3 signaling and p53 pathway supported this final step, allowing the emergence of a CD23-negative subpopulation in transition from B cells to plasma cells. Remarkably, hydroxymethylation of PRDM1, a gene essential for plasma cell fate, was coupled to progression in S phase, revealing an intricate connection among cell cycle, DNA (hydroxy)methylation, and cell fate determination.

  9. Cell-Cycle-Dependent Reconfiguration of the DNA Methylome during Terminal Differentiation of Human B Cells into Plasma Cells

    Directory of Open Access Journals (Sweden)

    Gersende Caron

    2015-11-01

    Full Text Available Molecular mechanisms underlying terminal differentiation of B cells into plasma cells are major determinants of adaptive immunity but remain only partially understood. Here we present the transcriptional and epigenomic landscapes of cell subsets arising from activation of human naive B cells and differentiation into plasmablasts. Cell proliferation of activated B cells was linked to a slight decrease in DNA methylation levels, but followed by a committal step in which an S phase-synchronized differentiation switch was associated with an extensive DNA demethylation and local acquisition of 5-hydroxymethylcytosine at enhancers and genes related to plasma cell identity. Downregulation of both TGF-β1/SMAD3 signaling and p53 pathway supported this final step, allowing the emergence of a CD23-negative subpopulation in transition from B cells to plasma cells. Remarkably, hydroxymethylation of PRDM1, a gene essential for plasma cell fate, was coupled to progression in S phase, revealing an intricate connection among cell cycle, DNA (hydroxymethylation, and cell fate determination.

  10. Stromal cells from human long-term marrow cultures, but not cultured marrow fibroblasts, phagocytose horse serum constituents: studies with a monoclonal antibody that reacts with a species-specific epitope common to multiple horse serum proteins.

    Science.gov (United States)

    Charbord, P; Tippens, D; Wight, T S; Gown, A M; Singer, J W

    1987-01-01

    This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These data indicate that marrow stromal cells specifically accumulate horse serum proteins which might partially explain the horse serum requirement for long-term marrow culture maintenance. The data also suggest further similarities between marrow stromal and smooth muscle cells and additional differences between marrow fibroblasts and marrow stromal cells.

  11. Immunological evidence of monoclonal gammopathy in North India: a hospital based study

    Directory of Open Access Journals (Sweden)

    Kalpana Singh

    2010-08-01

    Full Text Available Kalpana Singh1, Bhawna Singh2, Sarika Arora2, Alpana Saxena11Department of Biochemistry, Maulana Azad Medical College and LN Hospital, New Delhi, India; 2Department of Biochemistry, GB Pant Hospital, New Delhi, IndiaBackground: Monoclonal gammopathy of unknown significance (MGUS is a condition in which a paraprotein is found in the blood during standard laboratory tests. It is age-related and characterized by accumulation of bone marrow