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Sample records for antibody mediated cell

  1. Characterization of in vitro antibody-dependent cell-mediated cytotoxicity activity of therapeutic antibodies - impact of effector cells.

    PubMed

    Chung, Shan; Lin, Yuwen L; Reed, Chae; Ng, Carl; Cheng, Zhijie Jey; Malavasi, Fabio; Yang, Jihong; Quarmby, Valerie; Song, An

    2014-05-01

    Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action implicated in the clinical efficacy of several therapeutic antibodies. In vitro ADCC assays employing effector cells capable of inducing lysis of target cells bound by antibodies are routinely performed to support the research and development of therapeutic antibodies. ADCC assays are commonly performed using peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells or engineered cell lines as effector cells. In this study we evaluated the impact of different effector cell types including primary PBMCs, primary NK cells, engineered NK cell lines, and an engineered reporter cell line, on the in vitro ADCC activity of two glycoforms of a humanized IgG1 antibody. The results of this study show the differential effects on both the efficacy and potency of the antibodies by different effector cells and the finding that both the allotype and the expression level of CD16a affect the potency of effector cells in ADCC assays. Our results also show that engineered NK or reporter cell lines provide reduced variability compared to primary effector cells for in vitro ADCC assays.

  2. Antibody mediated transduction of therapeutic proteins into living cells.

    PubMed

    Hansen, James E; Weisbart, Richard H; Nishimura, Robert N

    2005-09-16

    Protein therapy refers to the direct delivery of therapeutic proteins to cells and tissues with the goal of ameliorating or modifying a disease process. Current techniques for delivering proteins across cell membranes include taking advantage of receptor-mediated endocytosis or using protein transduction domains that penetrate directly into cells. The most commonly used protein transduction domains are small cell-penetrating peptides derived from such proteins as the HIV-1 Tat protein. A novel protein transduction domain developed as the single chain fragment (Fv) of a murine anti-DNA autoantibody, mAb 3E10, has recently been developed and used to deliver biologically active proteins to living cells in vitro. This review will provide a brief overview of the development of the Fv fragment and provide a summary of recent studies using Fv to deliver therapeutic peptides and proteins (such as a C-terminal p53 peptide, C-terminal p53 antibody fragment, full-length p53, and micro-dystrophin) to cells.

  3. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

    PubMed

    Wu, Yuling; Li, Jia J; Kim, Hyun Jun; Liu, Xu; Liu, Weiyi; Akhgar, Ahmad; Bowen, Michael A; Spitz, Susan; Jiang, Xu-Rong; Roskos, Lorin K; White, Wendy I

    2015-11-01

    Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function.

  4. What is antibody-mediated pure red cell aplasia (PRCA)?

    PubMed

    Casadevall, Nicole

    2005-05-01

    Antibody (Ab)-mediated pure red cell aplasia (PRCA) is an immunological pathology associated with the production of neutralizing Abs that inhibit the erythropoietic activity of endogenous erythropoietin (EPO) and recombinant erythropoiesis-stimulating agents (ESAs). Although this disorder occurs very rarely, the number of reported cases has increased dramatically in recent years, predominantly in patients with chronic kidney disease (CKD)-associated anaemia receiving subcutaneous (s.c.) injections of one particular formulation of recombinant epoetin-alpha. This disorder is differentiated from classic forms of PRCA that are caused by chemical toxaemia (i.e. erythroblastopenia induced by chemical compounds), lymphoproliferative neoplasms, thymoma, human parvovirus B19 and certain autoimmune disorders. Patients with Ab-mediated PRCA develop resistance to EPO and severe anaemia that follows a period of successful erythropoietic response, and exhibit characteristic decreases in blood haemoglobin (Hb) level and in the number of circulating reticulocytes. However, it is not yet possible to predict which patients will develop PRCA or when in the course of their treatments PRCA may develop. Laboratory confirmation of Ab-mediated PRCA requires bone marrow examination demonstrating few or no erythroid precursors and the presence of serum anti-EPO Abs using a validated assay. These neutralizing anti-EPO Abs recognize the protein core of the EPO molecule; carbohydrate groups on EPO can affect the binding of Abs but are themselves not immunological determinants. Animal models are being developed to increase further our understanding of the immunological mechanisms underlying the onset and progression of Ab-mediated PRCA.

  5. IGE AND IGGA ANTIBODY-MEDIATED RELEASE OF HISTAMINE FROM RAT PERITONEAL CELLS

    PubMed Central

    Bach, Michael K.; Bloch, Kurt J.; Austen, K. Frank

    1971-01-01

    The optimum conditions for antigen-induced release of histamine in the rat IgE and IgGa antibody-mediated systems were studied in vitro. The IgE antibody-mediated reaction could be separated into two steps: preparation of target cells with antibody and challenge with antigen. The optimal conditions for these two steps were distinctly different. Release of histamine by IgGa antibody and antigen could not be separated into two steps, and the optimal conditions for the total reaction were identical to those of the antigen challenge step of the IgE antibody-mediated system. PMID:4100657

  6. Cre-mediated cell ablation contests mast cell contribution in models of antibody- and T cell-mediated autoimmunity.

    PubMed

    Feyerabend, Thorsten B; Weiser, Anne; Tietz, Annette; Stassen, Michael; Harris, Nicola; Kopf, Manfred; Radermacher, Peter; Möller, Peter; Benoist, Christophe; Mathis, Diane; Fehling, Hans Jörg; Rodewald, Hans-Reimer

    2011-11-23

    Immunological functions of mast cells remain poorly understood. Studies in Kit mutant mice suggest key roles for mast cells in certain antibody- and T cell-mediated autoimmune diseases. However, Kit mutations affect multiple cell types of both immune and nonimmune origin. Here, we show that targeted insertion of Cre-recombinase into the mast cell carboxypeptidase A3 locus deleted mast cells in connective and mucosal tissues by a genotoxic Trp53-dependent mechanism. Cre-mediated mast cell eradication (Cre-Master) mice had, with the exception of a lack of mast cells and reduced basophils, a normal immune system. Cre-Master mice were refractory to IgE-mediated anaphylaxis, and this defect was rescued by mast cell reconstitution. This mast cell-deficient strain was fully susceptible to antibody-induced autoimmune arthritis and to experimental autoimmune encephalomyelitis. Differences comparing Kit mutant mast cell deficiency models to selectively mast cell-deficient mice call for a systematic re-evaluation of immunological functions of mast cells beyond allergy. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. Comparison of Antibody-Dependent Cell-Mediated Cytotoxicity and Virus Neutralization by HIV-1 Env-Specific Monoclonal Antibodies

    PubMed Central

    von Bredow, Benjamin; Arias, Juan F.; Heyer, Lisa N.; Moldt, Brian; Le, Khoa; Robinson, James E.; Burton, Dennis R.

    2016-01-01

    ABSTRACT Although antibodies to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been studied extensively for their ability to block viral infectivity, little data are currently available on nonneutralizing functions of these antibodies, such as their ability to eliminate virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 Env-specific antibodies of diverse specificities, including potent broadly neutralizing and nonneutralizing antibodies, were therefore tested for ADCC against cells infected with a lab-adapted HIV-1 isolate (HIV-1NL4-3), a primary HIV-1 isolate (HIV-1JR-FL), and a simian-human immunodeficiency virus (SHIV) adapted for pathogenic infection of rhesus macaques (SHIVAD8-EO). In accordance with the sensitivity of these viruses to neutralization, HIV-1NL4-3-infected cells were considerably more sensitive to ADCC, both in terms of the number of antibodies and magnitude of responses, than cells infected with HIV-1JR-FL or SHIVAD8-EO. ADCC activity generally correlated with antibody binding to Env on the surfaces of virus-infected cells and with viral neutralization; however, neutralization was not always predictive of ADCC, as instances of ADCC in the absence of detectable neutralization, and vice versa, were observed. These results reveal incomplete overlap in the specificities of antibodies that mediate these antiviral activities and provide insights into the relationship between ADCC and neutralization important for the development of antibody-based vaccines and therapies for combating HIV-1 infection. IMPORTANCE This study provides fundamental insights into the relationship between antibody-dependent cell-mediated cytotoxicity (ADCC) and virus neutralization that may help to guide the development of antibody-based vaccines and immunotherapies for the prevention and treatment of HIV-1 infection. PMID:27122574

  8. Comparison of Antibody-Dependent Cell-Mediated Cytotoxicity and Virus Neutralization by HIV-1 Env-Specific Monoclonal Antibodies.

    PubMed

    von Bredow, Benjamin; Arias, Juan F; Heyer, Lisa N; Moldt, Brian; Le, Khoa; Robinson, James E; Zolla-Pazner, Susan; Burton, Dennis R; Evans, David T

    2016-07-01

    Although antibodies to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been studied extensively for their ability to block viral infectivity, little data are currently available on nonneutralizing functions of these antibodies, such as their ability to eliminate virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 Env-specific antibodies of diverse specificities, including potent broadly neutralizing and nonneutralizing antibodies, were therefore tested for ADCC against cells infected with a lab-adapted HIV-1 isolate (HIV-1NL4-3), a primary HIV-1 isolate (HIV-1JR-FL), and a simian-human immunodeficiency virus (SHIV) adapted for pathogenic infection of rhesus macaques (SHIVAD8-EO). In accordance with the sensitivity of these viruses to neutralization, HIV-1NL4-3-infected cells were considerably more sensitive to ADCC, both in terms of the number of antibodies and magnitude of responses, than cells infected with HIV-1JR-FL or SHIVAD8-EO ADCC activity generally correlated with antibody binding to Env on the surfaces of virus-infected cells and with viral neutralization; however, neutralization was not always predictive of ADCC, as instances of ADCC in the absence of detectable neutralization, and vice versa, were observed. These results reveal incomplete overlap in the specificities of antibodies that mediate these antiviral activities and provide insights into the relationship between ADCC and neutralization important for the development of antibody-based vaccines and therapies for combating HIV-1 infection. This study provides fundamental insights into the relationship between antibody-dependent cell-mediated cytotoxicity (ADCC) and virus neutralization that may help to guide the development of antibody-based vaccines and immunotherapies for the prevention and treatment of HIV-1 infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Model for antibody mediated cell aggregation: rosette formation

    SciTech Connect

    Perelson, A.S.

    1984-01-01

    Models are developed for the formation of cellular aggregates, called rosettes, composed of a central lymphocyte and surrounding antibody coated red blood cells. Kinetic and equilibrium models are considered from both the deterministic and stochastic viewpoints. Analytic solutions are given to the system of nonlinear ordinary differential equations that describe the formation of different size rosettes in the cases of both reversible and irreversible red cell adhesion. A comparison of the stochastic model with experimental data indicates that there may exist heterogeneity within the lymphocyte population with regard to the number of chemical bonds required to bind a red cell to a lymphocyte. 24 references, 2 figures.

  10. Reactive oxygen species induced by therapeutic CD20 antibodies inhibit natural killer cell-mediated antibody-dependent cellular cytotoxicity against primary CLL cells.

    PubMed

    Werlenius, Olle; Aurelius, Johan; Hallner, Alexander; Akhiani, Ali A; Simpanen, Maria; Martner, Anna; Andersson, Per-Ola; Hellstrand, Kristoffer; Thorén, Fredrik B

    2016-05-31

    The antibody-dependent cellular cytotoxicity (ADCC) of natural killer (NK) cells is assumed to contribute to the clinical efficacy of monoclonal antibodies (mAbs) in chronic lymphocytic leukemia (CLL) and other hematopoietic malignancies of B cell origin. We sought to determine whether reactive oxygen species (ROS)-producing monocytes regulate the ADCC of NK cells against primary CLL cells using anti-CD20 as the linking antibody. The monoclonal CD20 antibodies rituximab and ofatumumab were found to trigger substantial release of ROS from monocytes. Antibody-exposed monocytes induced NK cell apoptosis and restricted NK cell-mediated ADCC against autologous CLL cells. The presence of inhibitors of ROS formation and scavengers of ROS preserved NK cell viability and restored NK cell-mediated ADCC against primary CLL cells. We propose that limiting the antibody-induced induction of immunosuppressive ROS may improve the anti-leukemic efficacy of anti-CD20 therapy in CLL.

  11. Colostral antibody-mediated and cell-mediated immunity contributes to innate and antigen-specific immunity in piglets

    USDA-ARS?s Scientific Manuscript database

    Immunoglobulins and immune cells are critical components of colostral immunity; however, their transfer to and function in the neonate, especially maternal lymphocytes, is unclear. Cell-mediated and antibody-mediated immunity in sow blood and colostrum and piglet blood before (PS) and after (AS) suc...

  12. HIV-specific antibody immunity mediated through NK cells and monocytes.

    PubMed

    Kramski, Marit; Parsons, Matthew S; Stratov, Ivan; Kent, Stephen J

    2013-07-01

    The partial success of the RV144 trial re-energized the field of HIV vaccine research, which had stalled after vaccines based on neutralizing antibody and cytotoxic T cells had failed to induce protection. A large post-vaccine research effort has focused attention on the role of non-neutralizing antibodies in the protection afforded by the RV144 vaccine. These binding antibodies can initiate immune responses such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) and combine elements of the adaptive and innate immune system in the form of antibodies and effector cells (including NK cells, monocytes and granulocytes). A complex interplay exists between the variable portion of the binding antibody and its HIV antigen target on one hand and the constant region of the antibody and the Fcγ-receptor of the effector cell on the other hand. Technical advances have revolutionized the abilities of scientist to detect the targets of non-neutralizing antibodies, including both envelope and non-envelope epitopes, and their role in forcing escape. Our understanding of the antibody characteristics (including IgG subclasses and Fc glycan profile) is providing valuable insights into their optimal structure and function. We expand on critical research on ADCC effector cells, particularly education of NK cells. We introduce the concept of HIV antibodydependent trogocytosis by monocytes as a potentially important aspect of HIV immunity. In summary, this review highlights recent advances in HIV-specific antibody immunity mediated through NK cells and monocytes.

  13. Cloned transgenic farm animals produce a bispecific antibody for T cell-mediated tumor cell killing

    PubMed Central

    Grosse-Hovest, Ludger; Müller, Sigrid; Minoia, Rosa; Wolf, Eckhard; Zakhartchenko, Valeri; Wenigerkind, Hendrik; Lassnig, Caroline; Besenfelder, Urban; Müller, Mathias; Lytton, Simon D.; Jung, Gundram; Brem, Gottfried

    2004-01-01

    Complex recombinant antibody fragments for modulation of immune function such as tumor cell destruction have emerged at a rapid pace and diverse anticancer strategies are being developed to benefit patients. Despite improvements in molecule design and expression systems, the quantity and stability, e.g., of single-chain antibodies produced in cell culture, is often insufficient for treatment of human disease, and the costs of scale-up, labor, and fermentation facilities are prohibitive. The ability to yield mg/ml levels of recombinant antibodies and the scale-up flexibility make transgenic production in plants and livestock an attractive alternative to mammalian cell culture as a source of large quantities of biotherapeutics. Here, we report on the efficient production of a bispecific single-chain antibody in the serum of transgenic rabbits and a herd of nine cloned, transgenic cattle. The bispecific protein, designated r28M, is directed to a melanoma-associated proteoglycan and the human CD28 molecule on T cells. Purified from the serum of transgenic animals, the protein is stable and fully active in mediating target cell-restricted T cell stimulation and tumor cell killing. PMID:15105446

  14. Chimeric mouse-human IgG1 antibody that can mediate lysis of cancer cells.

    PubMed Central

    Liu, A Y; Robinson, R R; Hellström, K E; Murray, E D; Chang, C P; Hellström, I

    1987-01-01

    A chimeric mouse-human antibody has been created that recognizes an antigen found on the surface of cells from many carcinomas. Immunoglobulin constant (C) domains of the mouse monoclonal antibody L6, C gamma 2a and C kappa, were substituted by the human C gamma 1 and C kappa by recombining cDNA modules encoding variable or C domains. The cDNA constructs were transfected into lymphoid cells for antibody production. The chimeric antibody and mouse L6 antibody bound to carcinoma cells with equal affinity and mediated complement-dependent cytolysis. In the presence of human effector cells, the chimeric antibody gave antibody-dependent cellular cytotoxicity at 100 times lower concentration than that needed for the mouse L6 antibody. The chimeric antibody, but not the mouse L6 antibody, is effective against a melanoma line expressing small amounts of the L6 antigen. The findings point to the usefulness of the chimeric antibody approach for obtaining agents with strong antitumor activity for possible therapeutic use in man. PMID:3106970

  15. Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells

    PubMed Central

    Sapinoro, Ramil; Volcy, Ketna; Shanaka, W.W.; Rodrigo, I.; Schlesinger, Jacob J.; Dewhurst, Stephen

    2008-01-01

    Lambda phage vectors mediate gene transfer in cultured mammalian cells and in live mice, and in vivo phage-mediated gene expression is increased when mice are pre-immunized with bacteriophage lambda. We now show that, like eukaryotic viruses, bacteriophage vectors are subject to Fc receptor-mediated, antibody-dependent enhancement of infection in mammalian cells. Antibody-dependent enhancement of phage gene transfer required FcγRI, but not its associated γ chain, and was not supported by other FcγR family members (FcγRIIA, FcγRIIB and FcγRIII). Studies using chlorpromazine and latrunculin A revealed an important role for clathrin-mediated endocytosis (chlorpromazine) and actin filaments (latrunculin A) in antibody-enhanced phage gene transfer. This was confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized phage particles with early endosome-associated protein-1 (EAA1) . In contrast, microtubule-targeting agents (nocodazole, taxol) increased the efficiency of antibody-enhanced phage gene transfer. These results reveal an unexpected antibody-dependent, FcγRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer. PMID:18191979

  16. Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells.

    PubMed

    Sapinoro, Ramil; Volcy, Ketna; Rodrigo, W W Shanaka I; Schlesinger, Jacob J; Dewhurst, Stephen

    2008-04-10

    Lambda phage vectors mediate gene transfer in cultured mammalian cells and in live mice, and in vivo phage-mediated gene expression is increased when mice are pre-immunized with bacteriophage lambda. We now show that, like eukaryotic viruses, bacteriophage vectors are subject to Fc receptor-mediated, antibody-dependent enhancement of infection in mammalian cells. Antibody-dependent enhancement of phage gene transfer required FcgammaRI, but not its associated gamma-chain, and was not supported by other FcgammaR family members (FcgammaRIIA, FcgammaRIIB, and FcgammaRIII). Studies using chlorpromazine and latrunculin A revealed an important role for clathrin-mediated endocytosis (chlorpromazine) and actin filaments (latrunculin A) in antibody-enhanced phage gene transfer. This was confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized phage particles with early endosome-associated protein-1 (EAA1). In contrast, microtubule-targeting agents (nocodazole, taxol) increased the efficiency of antibody-enhanced phage gene transfer. These results reveal an unexpected antibody-dependent, FcgammaRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer.

  17. Bispecific T cell engager (BiTE®) antibody constructs can mediate bystander tumor cell killing

    PubMed Central

    Ross, Sandra L.; Sherman, Marika; McElroy, Patricia L.; Lofgren, Julie A.; Moody, Gordon; Baeuerle, Patrick A.; Coxon, Angela

    2017-01-01

    For targets that are homogenously expressed, such as CD19 on cells of the B lymphocyte lineage, immunotherapies can be highly effective. Targeting CD19 with blinatumomab, a CD19/CD3 bispecific antibody construct (BiTE®), or with chimeric antigen receptor T cells (CAR-T) has shown great promise for treating certain CD19-positive hematological malignancies. In contrast, solid tumors with heterogeneous expression of the tumor-associated antigen (TAA) may present a challenge for targeted therapies. To prevent escape of TAA-negative cancer cells, immunotherapies with a local bystander effect would be beneficial. As a model to investigate BiTE®-mediated bystander killing in the solid tumor setting, we used epidermal growth factor receptor (EGFR) as a target. We measured lysis of EGFR-negative populations in vitro and in vivo when co-cultured with EGFR-positive cells, human T cells and an EGFR/CD3 BiTE® antibody construct. Bystander EGFR-negative cells were efficiently lysed by BiTE®-activated T cells only when proximal to EGFR-positive cells. Our mechanistic analysis suggests that cytokines released by BiTE®-activated T-cells induced upregulation of ICAM-1 and FAS on EGFR-negative bystander cells, contributing to T cell-induced bystander cell lysis. PMID:28837681

  18. Memory CD4 T Cells Induce Antibody-Mediated Rejection of Renal Allografts.

    PubMed

    Gorbacheva, Victoria; Fan, Ran; Fairchild, Robert L; Baldwin, William M; Valujskikh, Anna

    2016-11-01

    Despite advances in immunosuppression, antibody-mediated rejection is a serious threat to allograft survival. Alloreactive memory helper T cells can induce potent alloantibody responses and often associate with poor graft outcome. Nevertheless, the ability of memory T cells to elicit well characterized manifestations of antibody-mediated rejection has not been tested. We investigated helper functions of memory CD4 T cells in a mouse model of renal transplantation. Whereas the majority of unsensitized C57Bl/6 recipients spontaneously accepted fully MHC-mismatched A/J renal allografts, recipients containing donor-reactive memory CD4 T cells rapidly lost allograft function. Increased serum creatinine levels, high serum titers of donor-specific alloantibody, minimal T cell infiltration, and intense C4d deposition in the grafts of sensitized recipients fulfilled all diagnostic criteria for acute renal antibody-mediated rejection in humans. IFNγ neutralization did not prevent the renal allograft rejection induced by memory helper T cells, and CD8 T cell depletion at the time of transplantation or depletion of both CD4 and CD8 T cells also did not prevent the renal allograft rejection induced by memory helper T cells starting at day 4 after transplantation. However, B cell depletion inhibited alloantibody generation and significantly extended allograft survival, indicating that donor-specific alloantibodies (not T cells) were the critical effector mechanism of renal allograft rejection induced by memory CD4 T cells. Our studies provide direct evidence that recipient T cell sensitization may result in antibody-mediated rejection of renal allografts and introduce a physiologically relevant animal model with which to investigate mechanisms of antibody-mediated rejection and novel therapeutic approaches for its prevention and treatment. Copyright © 2016 by the American Society of Nephrology.

  19. Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells.

    PubMed

    Romain, Gabrielle; Senyukov, Vladimir; Rey-Villamizar, Nicolas; Merouane, Amine; Kelton, William; Liadi, Ivan; Mahendra, Ankit; Charab, Wissam; Georgiou, George; Roysam, Badrinath; Lee, Dean A; Varadarajan, Navin

    2014-11-20

    The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia.

  20. Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells

    PubMed Central

    Romain, Gabrielle; Senyukov, Vladimir; Rey-Villamizar, Nicolas; Merouane, Amine; Kelton, William; Liadi, Ivan; Mahendra, Ankit; Charab, Wissam; Georgiou, George; Roysam, Badrinath; Lee, Dean A.

    2014-01-01

    The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia. PMID:25232058

  1. Phagocytosis of breast cancer cells mediated by anti-MUC-1 monoclonal antibody, DF3, and its bispecific antibody.

    PubMed

    Akewanlop, C; Watanabe, M; Singh, B; Walker, M; Kufe, D W; Hayes, D F

    2001-05-15

    Human epithelial mucin, MUC-1, is commonly expressed in adenocarcinoma including 80% of breast cancers. erbB-2 is overexpressed in approximately 30% of breast cancers. Expression of MUC-1 and erbB-2 may be partially overlapping but discoordinate. Therefore, combined use of antibodies directed against these two antigens might increase the number of patients who benefit from immunotherapy. Monoclonal antibody (MAb) DF3 recognizes the MUC-1 tandem repeat. We investigated phagocytosis and cytolysis of cultured human breast cancer cells by monocyte-derived macrophages mediated by MAb DF3 and its bispecific antibody (BsAb) DF3xH22 with the second epitope directed against the Fc component of phagocytic cells. Purified monocytes from healthy donors were cultured with granulocyte macrophage colony-stimulating factor with or without IFN-gamma. antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) assays were performed with these macrophages and MUC-1-expressing target cells (ZR75-1) in the presence of MAb DF3 and BsAb DF3xH22. ADCP was measured by two-color fluorescence flow cytometry using PKH2 (green fluorescent dye) and R-phytoerythrin (RPE) (red)-conjugated MAb against human CD14 and CD11b and was confirmed by confocal microscopy. ADCC was measured by (51)Cr release assay. Immunohistochemical staining studies of MUC-1 and erbB-2 were performed on 67 primary breast cancer tissues. Expression of MUC-1 and erbB-2 was partially overlapping but discoordinate in 67 consecutive breast cancers. Both MAb DF3 and BsAb DF3xH22 mediated ADCP. However, ADCP mediated by MAb DF3 was greater than that mediated by BsAb DF3xH22. ADCC as detected by (51)Cr release was not seen with either antibody. The addition of IFN-gamma to monocyte-derived macrophage cultures inhibited ADCP compared to granulocyte macrophage colony-stimulating factor alone. Given the partially overlapping but discoordinate expression of MUC-1 and erbB-2 in breast cancer

  2. Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus.

    PubMed

    He, Wenqian; Tan, Gene S; Mullarkey, Caitlin E; Lee, Amanda J; Lam, Mannie Man Wai; Krammer, Florian; Henry, Carole; Wilson, Patrick C; Ashkar, Ali A; Palese, Peter; Miller, Matthew S

    2016-10-18

    The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. The majority of these antibodies bind to the hemagglutinin (HA) head domain and function by blocking the receptor binding site, preventing infection of host cells. Recently, elicitation of broadly neutralizing antibodies which target the conserved HA stalk domain has become a promising "universal" influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza virus-binding antibody mixtures in vivo has never been defined. Here, we demonstrate that interactions among viral glycoprotein-binding antibodies of varying specificities regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We show that the mechanism responsible for this phenotype relies upon competition for binding to HA on the surface of infected cells and virus particles. Nonneutralizing antibodies were poor inducers and did not inhibit antibody-dependent cell-mediated cytotoxicity. Interestingly, anti-neuraminidase antibodies weakly induced antibody-dependent cell-mediated cytotoxicity and enhanced induction in the presence of HA stalk-binding antibodies in an additive manner. Our data demonstrate that antibody specificity plays an important role in the regulation of ADCC, and that cross-talk among antibodies of varying specificities determines the magnitude of Fc receptor-mediated effector functions.

  3. Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus

    PubMed Central

    He, Wenqian; Tan, Gene S.; Mullarkey, Caitlin E.; Lee, Amanda J.; Lam, Mannie Man Wai; Krammer, Florian; Henry, Carole; Wilson, Patrick C.; Ashkar, Ali A.; Palese, Peter; Miller, Matthew S.

    2016-01-01

    The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. The majority of these antibodies bind to the hemagglutinin (HA) head domain and function by blocking the receptor binding site, preventing infection of host cells. Recently, elicitation of broadly neutralizing antibodies which target the conserved HA stalk domain has become a promising “universal” influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza virus-binding antibody mixtures in vivo has never been defined. Here, we demonstrate that interactions among viral glycoprotein-binding antibodies of varying specificities regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We show that the mechanism responsible for this phenotype relies upon competition for binding to HA on the surface of infected cells and virus particles. Nonneutralizing antibodies were poor inducers and did not inhibit antibody-dependent cell-mediated cytotoxicity. Interestingly, anti-neuraminidase antibodies weakly induced antibody-dependent cell-mediated cytotoxicity and enhanced induction in the presence of HA stalk-binding antibodies in an additive manner. Our data demonstrate that antibody specificity plays an important role in the regulation of ADCC, and that cross-talk among antibodies of varying specificities determines the magnitude of Fc receptor-mediated effector functions. PMID:27698132

  4. NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Cancer Immunotherapy

    PubMed Central

    Wang, Wei; Erbe, Amy K.; Hank, Jacquelyn A.; Morris, Zachary S.; Sondel, Paul M.

    2015-01-01

    Natural killer (NK) cells play a major role in cancer immunotherapies that involve tumor-antigen targeting by monoclonal antibodies (mAbs). NK cells express a variety of activating and inhibitory receptors that serve to regulate the function and activity of the cells. In the context of targeting cells, NK cells can be “specifically activated” through certain Fc receptors that are expressed on their cell surface. NK cells can express FcγRIIIA and/or FcγRIIC, which can bind to the Fc portion of immunoglobulins, transmitting activating signals within NK cells. Once activated through Fc receptors by antibodies bound to target cells, NK cells are able to lyse target cells without priming, and secrete cytokines like interferon gamma to recruit adaptive immune cells. This antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells is utilized in the treatment of various cancers overexpressing unique antigens, such as neuroblastoma, breast cancer, B cell lymphoma, and others. NK cells also express a family of receptors called killer immunoglobulin-like receptors (KIRs), which regulate the function and response of NK cells toward target cells through their interaction with their cognate ligands that are expressed on tumor cells. Genetic polymorphisms in KIR and KIR-ligands, as well as FcγRs may influence NK cell responsiveness in conjunction with mAb immunotherapies. This review focuses on current therapeutic mAbs, different strategies to augment the anti-tumor efficacy of ADCC, and genotypic factors that may influence patient responses to antibody-dependent immunotherapies. PMID:26284063

  5. Expanding the antibody-mediated component of plasma cell-rich acute rejection: A case series

    PubMed Central

    Uppin, M. S.; Gudithi, S.; Taduri, G.; Prayaga, A. K.; Raju, S. B.

    2016-01-01

    Renal allograft rejection is mediated by T-cells (T-cell mediated rejection) or by donor-specific antibodies (DSAs) (antibody mediated rejection, ABMR). Plasma cell-rich acute rejection (PCAR) is a unique entity due to its peculiar morphology and poor prognostic behavior. All allograft biopsies done at our center from January 2013 to October 2014 were reviewed, and seven were identified with a diagnosis of PCAR with antibody mediated rejection (ABMR). The allograft biopsies were classified as per the Banff 2007 schema. Immunohistochemistry with C4d, SV 40, CD3, CD20, CD138, kappa and lambda light chain was performed. Total 210 allograft biopsies were performed in the study period of which seven biopsies (3.3%) were diagnosed as PCAR with ABMR. All these were late ABMRs (more than 6 months) with median posttransplant duration of 17 months. The allograft biopsy showed features of PCAR along with glomerulitis, peritubular capillaritis, and positive C4d. DSA was positive in six patients. All the patients were treated with standard therapeutic measures of acute cellular rejection (ACR) and ABMR including steroids, plasma exchange, rituximab and intravenous immunoglobulins. All the patients had persistent graft dysfunction or graft loss on follow-up. PMID:27194831

  6. Hormone Conjugated with Antibody to CD3 Mediates Cytotoxic T Cell Lysis of Human Melanoma Cells

    NASA Astrophysics Data System (ADS)

    Liu, Margaret Ann; Nussbaum, Samuel R.; Eisen, Herman N.

    1988-01-01

    Cytotoxic T lymphocytes can be activated by antibodies to their antigen-specific receptor complex (TCR-CD3) to destroy target cells, regardless of the specificity of the cytotoxic T cells. A novel hormone-antibody conjugate, consisting of an analog of melanocyte-stimulating hormone chemically coupled to a monoclonal antibody to CD3, the invariant component of the T cell receptor complex, was used to target human melanoma cells for destruction by human cytotoxic T lymphocytes that bear no specificity for the tumor cells. As targeting components of such anti-CD3 conjugates, hormones or growth factors are expected to prove more effective than antibodies to tumor-associated antigens in focusing the destructive activity of cytotoxic T cells on tumor target cells.

  7. Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells

    PubMed Central

    2012-01-01

    Introduction Sustained HER2 signaling at the cell surface is an oncogenic mechanism in a significant proportion of breast cancers. While clinically effective therapies targeting HER2 such as mAbs and tyrosine kinase inhibitors exist, tumors overexpressing HER2 eventually progress despite treatment. Thus, abrogation of persistent HER2 expression at the plasma membrane to synergize with current approaches may represent a novel therapeutic strategy. Methods We generated polyclonal anti-HER2 antibodies (HER2-VIA) by vaccinating mice with an adenovirus expressing human HER2, and assessed their signaling effects in vitro and anti-tumor effects in a xenograft model. In addition, we studied the signaling effects of human HER2-specific antibodies induced by vaccinating breast cancer patients with a HER2 protein vaccine. Results HER2-VIA bound HER2 at the plasma membrane, initially activating the downstream kinases extracellular signal-regulated protein kinase 1/2 and Akt, but subsequently inducing receptor internalization in clathrin-coated pits in a HER2 kinase-independent manner, followed by ubiquitination and degradation of HER2 into a 130 kDa fragment phosphorylated at tyrosine residues 1,221/1,222 and 1,248. Following vaccination of breast cancer patients with the HER2 protein vaccine, HER2-specific antibodies were detectable and these antibodies bound to cell surface-expressed HER2 and inhibited HER2 signaling through blocking tyrosine 877 phosphorylation of HER2. In contrast to the murine antibodies, human anti-HER2 antibodies induced by protein vaccination did not mediate receptor internalization and degradation. Conclusion These data provide new insight into HER2 trafficking at the plasma membrane and the changes induced by polyclonal HER2-specific antibodies. The reduction of HER2 membrane expression and HER2 signaling by polyclonal antibodies induced by adenoviral HER2 vaccines supports human clinical trials with this strategy for those breast cancer patients

  8. The Fc and not CD4 Receptor Mediates Antibody Enhancement of HIV Infection in Human Cells

    NASA Astrophysics Data System (ADS)

    Homsy, Jacques; Meyer, Mia; Tateno, Masatoshi; Clarkson, Sarah; Levy, Jay A.

    1989-06-01

    Antibodies that enhance human immunodeficiency virus (HIV) infectivity have been found in the blood of infected individuals and in infected or immunized animals. These findings raise serious concern for the development of a safe vaccine against acquired immunodeficiency syndrome. To address the in vivo relevance and mechanism of this phenomenon, antibody-dependent enhancement of HIV infectivity in peripheral blood macrophages, lymphocytes, and human fibroblastoid cells was studied. Neither Leu3a, a monoclonal antibody directed against the CD4 receptor, nor soluble recombinant CD4 even at high concentrations prevented this enhancement. The addition of monoclonal antibody to the Fc receptor III (anti-FcRIII), but not of antibodies that react with FcRI or FcRII, inhibited HIV type 1 and HIV type 2 enhancement in peripheral blood macrophages. Although enhancement of HIV infection in CD4+ lymphocytes could not be blocked by anti-FcRIII, it was inhibited by the addition of human immunoglobulin G aggregates. The results indicate that the FcRIII receptor on human macrophages and possibly another Fc receptor on human CD4+ lymphocytes mediate antibody-dependent enhancement of HIV infectivity and that this phenomenon proceeds through a mechanism independent of the CD4 protein.

  9. Ocaratuzumab, an Fc-engineered antibody demonstrates enhanced antibody-dependent cell-mediated cytotoxicity in chronic lymphocytic leukemia.

    PubMed

    Cheney, Carolyn M; Stephens, Deborah M; Mo, Xiaokui; Rafiq, Sarwish; Butchar, Jonathan; Flynn, Joseph M; Jones, Jeffrey A; Maddocks, Kami; O'Reilly, Adrienne; Ramachandran, Abhijit; Tridandapani, Susheela; Muthusamy, Natarajan; Byrd, John C

    2014-01-01

    Chronic lymphocytic leukemia (CLL) is common in both developed and developing nations where the need for inexpensive and convenient administration of therapy is apparent. Ocaratuzumab is a novel Fc-engineered humanized IgG1 anti-CD20 monoclonal antibody (mAb) designed for effective antibody-dependent cell-mediated cytotoxicity (ADCC) at very low concentrations that may facilitate sub-cutaneous (vs. intravenous) dosing. Here, we report ocaratuzumab's potency against CLL cells. In vitro assessment of ocaratuzumab's direct cytotoxicity (DC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and ADCC was performed on CLL cells. Ocaratuzumab induced DC, CDC, and ADCP similarly to rituximab or ofatumumab (anti-CD20 mAbs). However, ocaratuzumab showed an advantage in NK cell-mediated ADCC over these antibodies. In allogeneic ADCC, [E:T (effector:target) ratios = 25:1, 12:1, 6:1], ocaratuzumab (10 µg/mL) improved ADCC by ~3-fold compared with rituximab or ofatumumab (P<0.001 all tested E:T ratios). Notably, the superiority of ocaratuzumab-induced ADCC was observed at low concentrations (0.1-10 ug/ml; P<0.03; allogeneic assays). In extended allogeneic ADCC E:T titration, ocaratuzumab (0.1 µg/mL) demonstrated 19.4% more cytotoxicity than rituximab (E:T = 0.38:1; P = 0.0066) and 21.5% more cytotoxicity than ofatumumab (E:T = 1.5:1; P = 0.0015). In autologous ADCC, ocaratuzumab (10 µg/mL) demonstrated ~1.5-fold increase in cytotoxicity compared with rituximab or ofatumumab at all E:T ratios tested (E:Ts = 25:1,12:1,6:1; all P<0.001). Obinutuzumab, a glyco-engineered anti-CD20 mAb, showed no improvement in ADCC activity compared with ocaratuzumab. The enhanced ADCC of ocaratuzumab suggests that it may be effective at low concentrations. If supported by clinical investigation, this feature could potentially allow for subcutaneous dosing at low doses that could expand the potential of administering chemoimmunotherapy in developing

  10. Antibiotics and immunity: effects of antibiotics on natural killer, antibody dependent cell-mediated cytotoxicity and antibody production.

    PubMed

    Ibrahim, M S; Maged, Z A; Haron, A; Khalil, R Y; Attallah, A M

    1987-12-01

    We studied the effects of antibiotics on natural killer (NK), antibody dependent cell-mediated cytotoxicity (ADCC) and immunoglobulin production. When human peripheral blood lymphocytes were incubated overnight with the antibiotic before the assay, nitrofurantoin significantly reduced NK but not ADCC activity. Nitrofurantoin also suppressed both spontaneous and interferon-enhanced NK activities in a dose-dependent fashion. Though it did not affect spontaneous ADCC activity, nitrofurantoin suppressed interferon enhancement of ADCC. Chloramphenicol significantly decreased the number of plaque forming cells in mice. In addition to chloramphenicol, tetracycline, rifampicin, cephalothin, polymyxin B and nitrofurantoin reduced mitogen-induced polycloned immunoglobulin synthesis. Results of this study may have clinical relevance, especially in treating immunocompromised patients.

  11. Inhibition of B-Cell Proliferation and Antibody Production by Mesenchymal Stromal Cells Is Mediated by T Cells

    PubMed Central

    Bernardo, Maria Ester; Scarsella, Marco; Conforti, Antonella; Giorda, Ezio; Biagini, Simone; Cascioli, Simona; Rossi, Francesca; Guzzo, Isabella; Vivarelli, Marina; Dello Strologo, Luca; Emma, Francesco; Locatelli, Franco; Carsetti, Rita

    2015-01-01

    Bone marrow (BM)-derived mesenchymal stromal cells (MSCs), endowed with immunosuppressive and anti-inflammatory properties, represent a promising tool in immunoregulatory and regenerative cell therapy. Clarifying the interactions between MSCs and B-lymphocytes may be crucial for designing innovative MSC-based strategies in conditions in which B cells play a role, including systemic lupus erythematosus (SLE) and rejection of kidney transplantation. In this study, we show that, both in healthy subjects and in patients, in vitro B-cell proliferation, plasma-cell differentiation, and antibody production are inhibited by BM-derived MSCs when peripheral blood lymphocytes are stimulated with CpG, but not when sorted B cells are cultured with MSCs+CpG. Inhibition is restored in CpG+MSC cocultures when sorted T cells are added to sorted B cells, suggesting that this effect is mediated by T cells, with both CD4+ and CD8+ cells playing a role. Moreover, cell–cell contact between MSCs and T cells, but not between MSCs and B cells, is necessary to inhibit B-cell proliferation. Thus, the presence of functional T cells, as well as cell–cell contact between MSCs and T cells, are crucial for B-cell inhibition. This information can be relevant for implementing MSC-based therapeutic immune modulation in patients in whom T-cell function is impaired. PMID:25036865

  12. Mutations That Alter Use of Hepatitis C Virus Cell Entry Factors Mediate Escape From Neutralizing Antibodies

    PubMed Central

    FAUVELLE, CATHERINE; ZAHID, MUHAMMAD NAUMAN; TUREK, MARINE; HEYDMANN, LAURA; CURY, KARINE; HAYER, JULIETTE; COMBET, CHRISTOPHE; COSSET, FRANÇOIS–LOÏC; PIETSCHMANN, THOMAS; HIET, MARIE–SOPHIE; BARTENSCHLAGER, RALF; HABERSETZER, FRANÇOIS; DOFFOËL, MICHEL; KECK, ZHEN–YONG; FOUNG, STEVEN K. H.; ZEISEL, MIRJAM B.; STOLL–KELLER, FRANÇOISE; BAUMERT, THOMAS F.

    2017-01-01

    BACKGROUND & AIMS The development of vaccines and other strategies to prevent hepatitis C virus (HCV) infection is limited by rapid viral evasion. HCV entry is the first step of infection; this process involves several viral and host factors and is targeted by host-neutralizing responses. Although the roles of host factors in HCV entry have been well characterized, their involvement in evasion of immune responses is poorly understood. We used acute infection of liver graft as a model to investigate the molecular mechanisms of viral evasion. METHODS We studied factors that contribute to evasion of host immune responses using patient-derived antibodies, HCV pseudoparticles, and cell culture–derived HCV that express viral envelopes from patients who have undergone liver transplantation. These viruses were used to infect hepatoma cell lines that express different levels of HCV entry factors. RESULTS By using reverse genetic analyses, we identified altered use of host-cell entry factors as a mechanism by which HCV evades host immune responses. Mutations that alter use of the CD81 receptor also allowed the virus to escape neutralizing antibodies. Kinetic studies showed that these mutations affect virus–antibody interactions during postbinding steps of the HCV entry process. Functional studies with a large panel of patient-derived antibodies showed that this mechanism mediates viral escape, leading to persistent infection in general. CONCLUSIONS We identified a mechanism by which HCV evades host immune responses, in which use of cell entry factors evolves with escape from neutralizing antibodies. These findings advance our understanding of the pathogenesis of HCV infection and might be used to develop antiviral strategies and vaccines. PMID:22503792

  13. Mutations that alter use of hepatitis C virus cell entry factors mediate escape from neutralizing antibodies.

    PubMed

    Fofana, Isabel; Fafi-Kremer, Samira; Carolla, Patric; Fauvelle, Catherine; Zahid, Muhammad Nauman; Turek, Marine; Heydmann, Laura; Cury, Karine; Hayer, Juliette; Combet, Christophe; Cosset, François-Loïc; Pietschmann, Thomas; Hiet, Marie-Sophie; Bartenschlager, Ralf; Habersetzer, François; Doffoël, Michel; Keck, Zhen-Yong; Foung, Steven K H; Zeisel, Mirjam B; Stoll-Keller, Françoise; Baumert, Thomas F

    2012-07-01

    The development of vaccines and other strategies to prevent hepatitis C virus (HCV) infection is limited by rapid viral evasion. HCV entry is the first step of infection; this process involves several viral and host factors and is targeted by host-neutralizing responses. Although the roles of host factors in HCV entry have been well characterized, their involvement in evasion of immune responses is poorly understood. We used acute infection of liver graft as a model to investigate the molecular mechanisms of viral evasion. We studied factors that contribute to evasion of host immune responses using patient-derived antibodies, HCV pseudoparticles, and cell culture-derived HCV that express viral envelopes from patients who have undergone liver transplantation. These viruses were used to infect hepatoma cell lines that express different levels of HCV entry factors. By using reverse genetic analyses, we identified altered use of host-cell entry factors as a mechanism by which HCV evades host immune responses. Mutations that alter use of the CD81 receptor also allowed the virus to escape neutralizing antibodies. Kinetic studies showed that these mutations affect virus-antibody interactions during postbinding steps of the HCV entry process. Functional studies with a large panel of patient-derived antibodies showed that this mechanism mediates viral escape, leading to persistent infection in general. We identified a mechanism by which HCV evades host immune responses, in which use of cell entry factors evolves with escape from neutralizing antibodies. These findings advance our understanding of the pathogenesis of HCV infection and might be used to develop antiviral strategies and vaccines. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

  14. Cell-mediated Immunity and Antibodies to Herpesvirus hominis Type 1 in Oral Leukoplakia and Carcinoma

    PubMed Central

    Lehner, T.; Wilton, J. M. A.; Shillitoe, E. J.; Ivanyi, L.

    1973-01-01

    Cell-mediated and humoral immune responses to Herpesvirus hominis type 1 (HVH1) and Candida albicans were studied in patients with leukoplakia, showing a histological spectrum of changes from epithelial keratosis to acanthosis and atypia, and in patients with carcinoma. The results were ranked according to increasing values of stimulation indices of lymphocyte transformation to HVH1, and the corresponding macrophage migration inhibition indices, and complement fixing antibody titres of each patient were correlated. This revealed that most patients with epithelial atypia were clustered to that end of the spectrum which had the highest stimulation and migration indices to HVH1; this relationship was not evident with C. albicans. In patients with keratosis and acanthosis there was a significant lack of correlation between lymphocyte transformation and migration inhibition to both HVH1 and C. albicans. In carcinoma the indices of lymphocyte transformation and migration inhibition to HVH1 and C. albicans were depressed. Furthermore, a significant negative correlation was found between lymphocyte transformation and migration inhibition to HVH1, unlike the positive correlation in control subjects. Complement fixing antibodies to HVH1, HVH2, cytomegalovirus and adenovirus, and fluorescent antibodies to C. albicans failed to show a significant change in titre in any one group of subjects tested. The results suggest a cell-mediated immune defect in leukoplakia, with a dissociation between lymphocyte transformation and macrophage migration inhibition to HVH1 and C. albicans in cases of keratosis or acanthosis. A specific increase in cell-mediated immunity to HVH1 in leukoplakia with epithelial atypia and the sequential changes argue in favour of a possible participation of HVH1 in carcinomatous transformation of some leukoplakias. PMID:4351511

  15. An Fcγ receptor-dependent mechanism drives antibody-mediated target-receptor signaling in cancer cells.

    PubMed

    Wilson, Nicholas S; Yang, Becky; Yang, Annie; Loeser, Stefanie; Marsters, Scot; Lawrence, David; Li, Yun; Pitti, Robert; Totpal, Klara; Yee, Sharon; Ross, Sarajane; Vernes, Jean-Michel; Lu, Yanmei; Adams, Cam; Offringa, Rienk; Kelley, Bob; Hymowitz, Sarah; Daniel, Dylan; Meng, Gloria; Ashkenazi, Avi

    2011-01-18

    Antibodies to cell-surface antigens trigger activatory Fcγ receptor (FcγR)-mediated retrograde signals in leukocytes to control immune effector functions. Here, we uncover an FcγR mechanism that drives antibody-dependent forward signaling in target cells. Agonistic antibodies to death receptor 5 (DR5) induce cancer-cell apoptosis and are in clinical trials; however, their mechanism of action in vivo is not fully defined. Interaction of the DR5-agonistic antibody drozitumab with leukocyte FcγRs promoted DR5-mediated tumor-cell apoptosis. Whereas the anti-CD20 antibody rituximab required activatory FcγRs for tumoricidal function, drozitumab was effective in the context of either activatory or inhibitory FcγRs. A CD40-agonistic antibody required similar FcγR interactions to stimulate nuclear factor-κB activity in B cells. Thus, FcγRs can drive antibody-mediated receptor signaling in target cells.

  16. Key role for CD4 T cells during mixed antibody mediated rejection of renal allografts

    PubMed Central

    Gaughan, A.; Wang, J.; Pelletier, R.P.; Nadasdy, T.; Brodsky, S.; Roy, S.; Lodder, M.; Bobek, D.; Mofatt-Bruce, S.; Fairchild, R.L.; Henry, M.L.; Hadley, G.A.

    2014-01-01

    We utilized mouse models to elucidate the immunologic mechanisms of functional graft loss during mixed antibody mediated rejection of renal allografts (mixed AMR), in which humoral and cellular responses to the graft occur concomitantly. Although the majority of T cells in the graft at the time of rejection were CD8 T cells with only a minor population of CD4 T cells, depletion of CD4 but not CD8 cells prevented acute graft loss during mixed AMR. CD4 depletion eliminated anti-donor alloantibodies and conferred protection from destruction of renal allografts. ELISPOT revealed that CD4 T effectors responded to donor alloantigens by both the direct and indirect pathways of allorecognition. In transfer studies, CD4 T effectors primed to donor alloantigens were highly effective at promoting acute graft dysfunction, and exhibited the attributes of effector T cells. Laser capture microdissection and confirmatory immunostaining studies revealed that CD4 T cells infiltrating the graft produced effector molecules with graft destructive potential. Bioluminescent imaging confirmed that CD4 T effectors traffic to the graft site in immune replete hosts. These data document that host CD4 T cells can promote acute dysfunction of renal allografts by directly mediating graft injury in addition to facilitating anti-donor alloantibody responses. PMID:24410909

  17. Antibody-mediated radiotherapy

    SciTech Connect

    Bloomer, W.D.; Lipsztein, R.; Dalton, J.F.

    1985-05-01

    Antibodies that react with antigens on the surface of tumor cells but not normal cells have great potential for cancer detection and therapy. If radiolabeled without loss of immunologic specificity, such antibodies may be able to deliver cytoxic amounts of radiation. Target- cell specificity and a high extraction coefficient are necessary with any radionuclide in order to minimize normal tissue irradiation. Tumor- cell-retention time and the rate of catabolized radionuclide will also influence ultimate applicability. Among the unanswered questions for choosing a radionuclide is the choice of particle emitter. Although classic beta emitters have been used in a number of clinical situations, they have not had a major impact on disease outcome except in diseases of the thyroid. Unfortunately, Auger emitters such as iodine 125 are cytotoxic only when localized within close proximity to the genome. On the other hand, alpha emitters such as astatine 211 eliminate the need for subcellular sequestration but not cell-specific localization. 34 references.

  18. Phagocytic cells contribute to the antibody-mediated elimination of pulmonary-infected SARS coronavirus.

    PubMed

    Yasui, Fumihiko; Kohara, Michinori; Kitabatake, Masahiro; Nishiwaki, Tetsu; Fujii, Hideki; Tateno, Chise; Yoneda, Misako; Morita, Kouichi; Matsushima, Kouji; Koyasu, Shigeo; Kai, Chieko

    2014-04-01

    While the 2002-2003 outbreak of severe acute respiratory syndrome (SARS) resulted in 774 deaths, patients who were affected with mild pulmonary symptoms successfully recovered. The objective of the present work was to identify, using SARS coronavirus (SARS-CoV) mouse infection models, immune factors responsible for clearing of the virus. The elimination of pulmonary SARS-CoV infection required the activation of B cells by CD4(+) T cells. Furthermore, passive immunization (post-infection) with homologous (murine) anti-SARS-CoV antiserum showed greater elimination efficacy against SARS-CoV than that with heterologous (rabbit) antiserum, despite the use of equivalent titers of neutralizing antibodies. This distinction was mediated by mouse phagocytic cells (monocyte-derived infiltrating macrophages and partially alveolar macrophages, but not neutrophils), as demonstrated both by adoptive transfer from donors and by immunological depletion of selected cell types. These results indicate that the cooperation of anti-SARS-CoV antibodies and phagocytic cells plays an important role in the elimination of SARS-CoV. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy

    PubMed Central

    Roit, Fabio Da; Engelberts, Patrick J.; Taylor, Ronald P.; Breij, Esther C.W.; Gritti, Giuseppe; Rambaldi, Alessandro; Introna, Martino; Parren, Paul W.H.I.; Beurskens, Frank J.; Golay, Josée

    2015-01-01

    The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells, and antibody-dependent cellular cytotoxicity by these cells, as well as phagocytosis by macrophages or neutrophils were inhibited by ibrutinib with a half maximal effective concentration of 0.3–3 μM. Analysis of anti-CD20 mediated activation of natural killer cells isolated from patients on continued oral ibrutinib treatment suggested that repeated drug dosing inhibits these cells in vivo. Finally we show that the phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib similarly inhibited the immune cell-mediated mechanisms induced by anti-CD20 antibodies, although the effects of this drug at 10 μM were weaker than those observed with ibrutinib at the same concentration. We conclude that the design of combined treatment schedules of anti-CD20 antibodies with these kinase inhibitors should consider the multiple negative interactions between these two classes of drugs. PMID:25344523

  20. Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy.

    PubMed

    Da Roit, Fabio; Engelberts, Patrick J; Taylor, Ronald P; Breij, Esther C W; Gritti, Giuseppe; Rambaldi, Alessandro; Introna, Martino; Parren, Paul W H I; Beurskens, Frank J; Golay, Josée

    2015-01-01

    The novel Bruton tyrosine kinase inhibitor ibrutinib and phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib are promising drugs for the treatment of chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma, either alone or in combination with anti-CD20 antibodies. We investigated the possible positive or negative impact of these drugs on all known mechanisms of action of both type I and type II anti-CD20 antibodies. Pretreatment with ibrutinib for 1 hour did not increase direct cell death of cell lines or chronic lymphocytic leukemia samples mediated by anti-CD20 antibodies. Pre-treatment with ibrutinib did not inhibit complement activation or complement-mediated lysis. In contrast, ibrutinib strongly inhibited all cell-mediated mechanisms induced by anti-CD20 antibodies rituximab, ofatumumab or obinutuzumab, either in purified systems or whole blood assays. Activation of natural killer cells, and antibody-dependent cellular cytotoxicity by these cells, as well as phagocytosis by macrophages or neutrophils were inhibited by ibrutinib with a half maximal effective concentration of 0.3-3 μM. Analysis of anti-CD20 mediated activation of natural killer cells isolated from patients on continued oral ibrutinib treatment suggested that repeated drug dosing inhibits these cells in vivo. Finally we show that the phosphatidyl-4-5-biphosphate 3-kinase-δ inhibitor idelalisib similarly inhibited the immune cell-mediated mechanisms induced by anti-CD20 antibodies, although the effects of this drug at 10 μM were weaker than those observed with ibrutinib at the same concentration. We conclude that the design of combined treatment schedules of anti-CD20 antibodies with these kinase inhibitors should consider the multiple negative interactions between these two classes of drugs. Copyright© Ferrata Storti Foundation.

  1. HER2-specific immunoligands engaging NKp30 or NKp80 trigger NK-cell-mediated lysis of tumor cells and enhance antibody-dependent cell-mediated cytotoxicity.

    PubMed

    Peipp, Matthias; Derer, Stefanie; Lohse, Stefan; Staudinger, Matthias; Klausz, Katja; Valerius, Thomas; Gramatzki, Martin; Kellner, Christian

    2015-10-13

    NK cells detect tumors through activating surface receptors, which bind self-antigens that are frequently expressed upon malignant transformation. To increase the recognition of tumor cells, the extracellular domains of ligands of the activating NK cell receptors NKp30, NKp80 and DNAM-1 (i.e. B7-H6, AICL and PVR, respectively) were fused to a single-chain fragment variable (scFv) targeting the human epidermal growth factor receptor 2 (HER2), which is displayed by various solid tumors. The resulting immunoligands, designated B7-H6:HER2-scFv, AICL:HER2-scFv, and PVR:HER2-scFv, respectively, bound HER2 and the addressed NK cell receptor. However, whereas B7-H6:HER2-scFv and AICL:HER2-scFv triggered NK cells to kill HER2-positive breast cancer cells at nanomolar concentrations, PVR:HER2-scFv was not efficacious. Moreover, NK cell cytotoxicity was enhanced synergistically when B7-H6:HER2-scFv or AICL:HER2-scFv were applied in combination with another HER2-specific immunoligand engaging the stimulatory receptor NKG2D. In contrast, no improvements were achieved by combining B7-H6:HER2-scFv with AICL:HER2-scFv. Additionally, B7-H6:HER2-scFv and AICL:HER2-scFv enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) by the therapeutic antibodies trastuzumab and cetuximab synergistically, with B7-H6:HER2-scFv exhibiting a higher efficacy. In summary, antibody-derived proteins engaging NKp30 or NKp80 may represent attractive biologics to further enhance anti-tumor NK cell responses and may provide an innovative approach to sensitize tumor cells for antibody-based immunotherapy.

  2. Heterogeneity of human lymphocyte Fc receptors. II. Relationship to antibody-dependent, cell-mediated cytotoxicity

    PubMed Central

    Gormus, B. J.; Woodson, Mildred; Kaplan, M. E.

    1978-01-01

    Human peripheral blood lymphocytes (PBL) were incubated (stripped) with pronase or papain and compared with unstripped lymphocytes for their ability to mediate antibody-dependent, cell-mediated cytotoxicity (ADCC). Despite marked removal or inactivation of receptors for heat-aggregated IgG (aggG) by proteolytic digestion, and pronounced changes in the percentages of cells rosetting with IgG-sensitized erythrocytes (EA) (decreased by papain, increased by pronase), stripped PBL functioned normally in ADCC. Stripped and unstripped lymphocytes were pre-treated with aggG to determine the role of aggG receptors in ADCC. AggG almost totally abolished ADCC by unstripped PBL, but inhibited ADCC by enzyme-stripped lymphocytes relatively poorly. Neither untreated nor stripped PBL were able to induce cytotoxicity of chicken erythrocyte (CRBC) target cells sensitized with the Fab'2 fragment of anti-CRBC IgG antibody (CRBC-A). Exposure of PBL to EA monolayers composed of CRBC-A or of sheep erythrocytes (SRBC) sensitized with rabbit anti-SRBC IgG antibody (SRBC-A) depleted PBL of cells that rosetted with CRBC-A and with human Rh-positive, type O erythrocytes sensitized with the human anti-Rh serum Ripley (HRBC-A Ripley). Non-adherent cells were incapable of binding aggG and had markedly diminished cytotoxicity in ADCC. Similarly, exposure of PBL to HRBC-A Ripley monolayers resulted in non-adherent cells that were incapable of rosette formation with HRBC-A or CRBC-A, failed to bind aggG, and exhibited significantly diminished ADCC activity. These studies indicated that: (1) cytotoxic effector PBL active in ADCC (K cells) have receptors for aggG and for EA; (2) PBL deficient in functional aggG receptors (enzymatically inactivated or removed) are capable of inducing normal levels of ADCC; (3) aggG and EA receptors appear to be closely associated on native K-cell membranes; (4) there is no clear-cut relationship in a given lymphocyte population between the presence of either aggG or

  3. Interaction with cellular CD4 exposes HIV-1 envelope epitopes targeted by antibody-dependent cell-mediated cytotoxicity.

    PubMed

    Veillette, Maxime; Désormeaux, Anik; Medjahed, Halima; Gharsallah, Nour-Elhouda; Coutu, Mathieu; Baalwa, Joshua; Guan, Yongjun; Lewis, George; Ferrari, Guido; Hahn, Beatrice H; Haynes, Barton F; Robinson, James E; Kaufmann, Daniel E; Bonsignori, Mattia; Sodroski, Joseph; Finzi, Andrés

    2014-03-01

    Anti-HIV-1 envelope glycoprotein (Env) antibodies without broadly neutralizing activity correlated with protection in the RV144 clinical trial, stimulating interest in other protective mechanisms involving antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Env epitopes targeted by many antibodies effective at mediating ADCC are poorly exposed on the unliganded Env trimer. Here we investigated the mechanism of exposure of ADCC epitopes on Env and showed that binding of Env and CD4 within the same HIV-1-infected cell effectively exposes these epitopes. Env capacity to transit to the CD4-bound conformation is required for ADCC epitope exposure. Importantly, cell surface CD4 downregulation by Nef and Vpu accessory proteins and Vpu-mediated BST-2 antagonism modulate exposure of ADCC-mediating epitopes and reduce the susceptibility of infected cells to this effector function in vitro. Significantly, Env conformational changes induced by cell surface CD4 are conserved among Env from HIV-1 and HIV-2/SIVmac lineages. Altogether, our observations describe a highly conserved mechanism required to expose ADCC epitopes that might help explain the evolutionary advantage of downregulation of cell surface CD4 by the HIV-1 Vpu and Nef proteins. HIV-1 envelope epitopes targeted by many antibodies effective at mediating antibody-dependent cell-mediated cytotoxicity (ADCC) are poorly exposed on the unliganded envelope trimer. Here we investigated the mechanism of exposure of these epitopes and found that envelope interaction with the HIV-1 CD4 receptor is required to expose some of these epitopes. Moreover, our results suggest that HIV-1 CD4 downregulation might help avoid the killing of HIV-1-infected cells by this immune mechanism.

  4. HIV-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) -Mediating Antibodies Decline while NK Cell Function Increases during Antiretroviral Therapy (ART).

    PubMed

    Jensen, Sanne Skov; Fomsgaard, Anders; Borggren, Marie; Tingstedt, Jeanette Linnea; Gerstoft, Jan; Kronborg, Gitte; Rasmussen, Line Dahlerup; Pedersen, Court; Karlsson, Ingrid

    2015-01-01

    Understanding alterations in HIV-specific immune responses during antiretroviral therapy (ART), such as antibody-dependent cellular cytotoxicity (ADCC), is important in the development of novel strategies to control HIV-1 infection. This study included 53 HIV-1 positive individuals. We evaluated the ability of effector cells and antibodies to mediate ADCC separately and in combination using the ADCC-PanToxiLux assay. The ability of the peripheral blood mononuclear cells (PBMCs) to mediate ADCC was significantly higher in individuals who had been treated with ART before seroconversion, compared to the individuals initiating ART at a low CD4+ T cell count (<350 cells/μl blood) and the ART-naïve individuals. The frequency of CD16 expressing natural killer (NK) cells correlated with both the duration of ART and Granzyme B (GzB) activity. In contrast, the plasma titer of antibodies mediating ADCC declined during ART. These findings suggest improved cytotoxic function of the NK cells if initiating ART early during infection, while the levels of ADCC mediating antibodies declined during ART.

  5. Antibody penetration into living cells. V. Interference between two fc gamma receptor-mediated functions: antibody penetration and antibody-dependent cellular cytotoxicity.

    PubMed Central

    Llerena, J M; Ruíz-Argüelles, A; Alarcón-Segovia, D; Llorente, L; Díaz-Jouanen, E

    1981-01-01

    The same Fc gamma receptor appears to be shared for two important phenomena: antibody-dependent cellular cytotoxicity (ADCC) and antibody penetration into living cells. ADCC is inhibited through interaction with the Fc gamma receptor during the antibody penetration process, indicating that both mechanisms may modulate each other in vitro. PMID:6972908

  6. Small CD4 Mimetics Prevent HIV-1 Uninfected Bystander CD4 + T Cell Killing Mediated by Antibody-dependent Cell-mediated Cytotoxicity

    PubMed Central

    Richard, Jonathan; Veillette, Maxime; Ding, Shilei; Zoubchenok, Daria; Alsahafi, Nirmin; Coutu, Mathieu; Brassard, Nathalie; Park, Jongwoo; Courter, Joel R.; Melillo, Bruno; Smith, Amos B.; Shaw, George M.; Hahn, Beatrice H.; Sodroski, Joseph; Kaufmann, Daniel E.; Finzi, Andrés

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection causes a progressive depletion of CD4 + T cells. Despite its importance for HIV-1 pathogenesis, the precise mechanisms underlying CD4 + T-cell depletion remain incompletely understood. Here we make the surprising observation that antibody-dependent cell-mediated cytotoxicity (ADCC) mediates the death of uninfected bystander CD4 + T cells in cultures of HIV-1-infected cells. While HIV-1-infected cells are protected from ADCC by the action of the viral Vpu and Nef proteins, uninfected bystander CD4 + T cells bind gp120 shed from productively infected cells and are efficiently recognized by ADCC-mediating antibodies. Thus, gp120 shedding represents a viral mechanism to divert ADCC responses towards uninfected bystander CD4 + T cells. Importantly, CD4-mimetic molecules redirect ADCC responses from uninfected bystander cells to HIV-1-infected cells; therefore, CD4-mimetic compounds might have therapeutic utility in new strategies aimed at specifically eliminating HIV-1-infected cells. PMID:26870823

  7. CD8+ T cells mediate antibody-independent platelet clearance in mice.

    PubMed

    Arthur, Connie M; Patel, Seema R; Sullivan, H Cliff; Winkler, Annie M; Tormey, Chris A; Hendrickson, Jeanne E; Stowell, Sean R

    2016-04-07

    Platelet transfusion provides an important therapeutic intervention in the treatment and prevention of bleeding. However, some patients rapidly clear transfused platelets, preventing the desired therapeutic outcome. Although platelet clearance can occur through a variety of mechanisms, immune-mediated platelet removal often plays a significant role. Numerous studies demonstrate that anti-platelet alloantibodies can induce significant platelet clearance following transfusion. In fact, for nearly 50 years, anti-platelet alloantibodies were considered to be the sole mediator of immune-mediated platelet clearance in platelet-refractory individuals. Although nonimmune mechanisms of platelet clearance can often explain platelet removal in the absence of anti-platelet alloantibodies, many patients experience platelet clearance following transfusion in the absence of a clear mechanism. These results suggest that other processes of antibody-independent platelet clearance may occur. Our studies demonstrate that CD8(+)T cells possess the unique ability to induce platelet clearance in the complete absence of anti-platelet alloantibodies. These results suggest a previously unrecognized form of immune-mediated platelet clearance with significant implications in the appropriate management of platelet-refractory individuals.

  8. Antiparietal cell antibody test

    MedlinePlus

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; Vitamin B12 - anti-gastric ...

  9. Endothelial Cells in Antibody-Mediated Rejection of Kidney Transplantation: Pathogenesis Mechanisms and Therapeutic Implications

    PubMed Central

    Wang, Shuo; Wang, Jina; Yang, Cheng; Xu, Ming

    2017-01-01

    Antibody-mediated rejection (AMR) has been identified as a main obstacle for stable immune tolerance and long survival of kidney allografts. In spite of new insights into the underlying mechanisms of AMR, accurate diagnosis and efficient treatment are still challenges in clinical practice. Endothelium is the first barrier between recipients' immune systems and grafts in vascularized organ transplants. Considering that endothelial cells express a number of antigens that can be attacked by various allo- and autoantibodies, endothelial cells act as main targets for the recipients' humoral immune responses. Importantly, emerging evidence has shown that endothelial cells in transplants could also initiate protective mechanisms in response to immune injuries. A better understanding of the role of endothelial cells during the pathogenesis of AMR might provide novel therapeutic targets. In the present review, we summarize the antigens expressed by endothelial cells and also discuss the activation and accommodation of endothelial cells as well as their clinical implications. Collectively, the progress discussed in this review indicates endothelial cells as promising targets to improve current diagnosis and therapeutic regimens for AMR. PMID:28255564

  10. Endothelial Cells in Antibody-Mediated Rejection of Kidney Transplantation: Pathogenesis Mechanisms and Therapeutic Implications.

    PubMed

    Wang, Shuo; Zhang, Chao; Wang, Jina; Yang, Cheng; Xu, Ming; Rong, Ruiming; Zhu, Tongyu; Zhu, Dong

    2017-01-01

    Antibody-mediated rejection (AMR) has been identified as a main obstacle for stable immune tolerance and long survival of kidney allografts. In spite of new insights into the underlying mechanisms of AMR, accurate diagnosis and efficient treatment are still challenges in clinical practice. Endothelium is the first barrier between recipients' immune systems and grafts in vascularized organ transplants. Considering that endothelial cells express a number of antigens that can be attacked by various allo- and autoantibodies, endothelial cells act as main targets for the recipients' humoral immune responses. Importantly, emerging evidence has shown that endothelial cells in transplants could also initiate protective mechanisms in response to immune injuries. A better understanding of the role of endothelial cells during the pathogenesis of AMR might provide novel therapeutic targets. In the present review, we summarize the antigens expressed by endothelial cells and also discuss the activation and accommodation of endothelial cells as well as their clinical implications. Collectively, the progress discussed in this review indicates endothelial cells as promising targets to improve current diagnosis and therapeutic regimens for AMR.

  11. Colostral antibody-mediated and cell-mediated immunity contributes to innate and antigen-specific immunity in piglets

    PubMed Central

    Bandrick, Meggan; Ariza-Nieto, Claudia; Baidoo, Samuel K.; Molitor, Thomas W.

    2014-01-01

    Immunoglobulins and immune cells are critical components of colostral immunity; however, their transfer to and function in the neonate, especially maternal lymphocytes, is unclear. Cell-mediated and antibody-mediated immunity in sow blood and colostrum and piglet blood before (PS) and after (AS) suckling were assessed to investigate transfer and function of maternal immunity in the piglet. CD4, CD8, and γδ lymphocytes were found in sow blood and colostrum and piglet blood PS and AS; each had a unique T lymphocyte profile. Immunoglobulins were detected in sow blood, colostrum, and in piglet blood AS; the immunoglobulin profile of piglet serum AS mimicked that of sow serum. These results suggest selectivity in lymphocyte concentration into colostrum and subsequent lymphocyte transfer into the neonate, but that immunoglobulin transfer is unimpeded. Assessment of colostral natural killer activity and antigen-specific proliferation revealed that colostral cells are capable of influencing the innate and specific immune response of neonatal pigs. PMID:24252519

  12. The potential role of CD16+ Vγ2Vδ2 T cell-mediated antibody-dependent cell-mediated cytotoxicity in control of HIV type 1 disease.

    PubMed

    He, Xuan; Liang, Hua; Hong, Kunxue; Li, Haishan; Peng, Hong; Zhao, Yangyang; Jia, Manxue; Ruan, Yuhua; Shao, Yiming

    2013-12-01

    Increasing evidence has suggested that HIV infection severely damages the Vγ2Vδ2 (Vδ2) T cells that play an important role in the first-line host response to infectious disease. However, little is known about Vδ2 T cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) in HIV disease. We found that although the CD16(+) Vδ2 T cell subset hardly participated in phosphoantigen responses dominated by the CD16(-) Vδ2 T cell subset, the potency of the ADCC function of Vδ2 T cells was correlated with the frequency of the CD16(+) subset. Thus, two distinct and complementary Vδ2 T cell subsets discriminated by CD16 were characterized to explore the respective impacts of HIV-1 infection on them. HIV-1 disease progression was not only associated with the phosphoantigen responsiveness of the CD16(-) Vδ2 subset, but also with the ability of the CD16(+) Vδ2 subset to kill antibody-coated target cells. Furthermore, both of the two Vδ2 functional subsets could be partially restored in HIV-infected patients with antiretroviral therapy. Notably, in the context of an overall HIV-mediated Vδ2 T cell depletion, despite the decline of phosphoantigen-responsive CD16(-) Vδ2 cells, CD16(+) Vδ2 cell-mediated ADCC was not compromised but exhibited a functional switch with dramatic promotion of degranulation in the early phase of HIV infection and chronic infection with slower disease progression. Our study reveals functional characterizations of the two Vδ2 T cell subsets with different activation pathways during HIV-1 infection and provides a rational direction for activating the CD16(+) Vδ2 T cells capable of mediating ADCC as a means to control HIV-1 disease.

  13. Monoclonal antibody production by receptor-mediated electrically induced cell fusion

    NASA Astrophysics Data System (ADS)

    Lo, Mathew M. S.; Tsong, Tian Yow; Conrad, Mary K.; Strittmatter, Stephen M.; Hester, Lynda D.; Snyder, Solomon H.

    1984-08-01

    Fusion of myeloma cells and B lymphocytes to form hybridomas which produce monoclonal antibodies has been a major advance1-3, but the poor efficiency and randomness of viral or polyethylene glycol fusion techniques generally gives poor yields of specific, high affinity antibodies. High voltage electrical fields with dielectrophoresis to ensure cell alignment can fuse a limited number of cells under direct microscopic examination4-9, but it is not possible to identify B-cells destined to secrete relevant antibodies. However, B-cells express, on their surface, antigen receptor immunoglobulins of the same antigenic specificity as the secreted antibodies. Binding of antigen to surface immunoglobulins stimulates proliferation and differentiation of B-cells into plasma cells. Here we report the use of the selective, high affinity interaction of antigen with surface immunoglobulins on B-cells to facilitate a close adherence to myeloma cells. The antigen, covalently conjugated to avidin, binds to the surface immunoglobulins on B-cells. This B-cell-antigen-avidin complex binds to biotin covalently attached to the surface of myeloma cells. An intense electric field across a bulk cell suspension then produces selective fusion of cells in contact, that is, of myeloma cells with B-cells which make the appropriate antibody. We have used this technique with several antigens, and all resultant hybridomas secrete appropriate antibodies with very high affinity.

  14. Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities

    PubMed Central

    Chung, Shan; Quarmby, Valerie; Gao, Xiaoying; Ying, Yong; Lin, Linda; Reed, Chae; Fong, Chris; Lau, Wendy; Qiu, Zhihua J.; Shen, Amy; Vanderlaan, Martin; Song, An

    2012-01-01

    The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its “regular” fucosylated counterpart and a series of mixtures containing varying proportions of “regular” and afucosylated materials. Compared with the “regular” fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC50 values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells. PMID:22531441

  15. Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities.

    PubMed

    Chung, Shan; Quarmby, Valerie; Gao, Xiaoying; Ying, Yong; Lin, Linda; Reed, Chae; Fong, Chris; Lau, Wendy; Qiu, Zhihua J; Shen, Amy; Vanderlaan, Martin; Song, An

    2012-01-01

    The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its "regular" fucosylated counterpart and a series of mixtures containing varying proportions of "regular" and afucosylated materials. Compared with the "regular" fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC 50 values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.

  16. B cells display an abnormal distribution and an impaired suppressive function in patients with chronic antibody-mediated rejection.

    PubMed

    Nouël, Alexandre; Ségalen, Isabelle; Jamin, Christophe; Doucet, Laurent; Caillard, Sophie; Renaudineau, Yves; Pers, Jacques-Olivier; Le Meur, Yannick; Hillion, Sophie

    2014-03-01

    In kidney transplantation, the composition of the B-cell compartment is increasingly identified as an important determinant for graft outcome. Whereas naive and transitional B cells have been associated with long-term allograft survival and operational tolerance, memory B cells have been linked to graft rejection and graft loss. Chronic antibody-mediated rejection now represents a major complication in transplantation and is a challenge in current therapeutics. Here, we show that patients with chronic antibody-mediated rejection display a unique B-cell phenotype with a reduced ratio of activated to memory B cells associated with an impaired immunosuppressive activity. The regulatory functions of the B cells depended on their maturation status. Thus, phenotypic and functional analyses of the B-cell compartment may be indicated for appropriate follow-up after transplantation and drive therapy in the establishment of transplant tolerance processes.

  17. Comprehensive Cross-Clade Characterization of Antibody-Mediated Recognition, Complement-Mediated Lysis, and Cell-Mediated Cytotoxicity of HIV-1 Envelope-Specific Antibodies toward Eradication of the HIV-1 Reservoir.

    PubMed

    Mujib, Shariq; Liu, Jun; Rahman, A K M Nur-Ur; Schwartz, Jordan A; Bonner, Phil; Yue, Feng Yun; Ostrowski, Mario A

    2017-08-15

    Immunotherapy with passive administration of broadly neutralizing HIV-1 envelope-specific antibodies (bnAbs) in the setting of established infection in vivo has yielded mixed results. The contribution of different antibodies toward the direct elimination of infected cells is poorly understood. In this study, we determined the ability of 12 well-characterized anti-HIV-1 neutralizing antibodies to recognize and eliminate primary CD4 T cells infected with HIV-1 belonging to clades A, B, C, and D, via antibody-dependent complement-mediated lysis (ADCML) and antibody-dependent cell-mediated cytotoxicity (ADCC), in vitro We further tested unique combinations of these antibodies to determine the optimal antibody cocktails to be tested in future clinical trials. We report that antibody binding to infected CD4 T cells is highly variable and correlates with ADCML and ADCC processes. Particularly, antibodies targeting the envelope glycan shield (2G12) and V1/V2 site (PG9, PG16, and PGT145) are best at recognizing HIV-1-infected CD4 T cells. However, only PG9 and PG16 and their combinations with other bnAbs sufficiently induced the elimination of HIV-1-infected CD4 T cells by ADCML, ADCC, or both. Notably, CD4 binding site antibodies VRC01, 3BNC117, and NIH45-46 G54W did not exhibit recognition of infected cells and were unable to induce their killing. Future trials geared toward the development of a cure for HIV/AIDS should incorporate V1/V2 antibodies for maximal clearance of infected cells. With the use of only primary immune cells, we conducted a comprehensive cross-clade physiological analysis to aid the direction of antibodies as therapeutics toward the development of a cure for HIV/AIDS.IMPORTANCE Several antibodies capable of neutralizing the majority of circulating HIV-1 strains have been identified to date and have been shown to prevent infection in animal models. However, the use of combinations of such broadly neutralizing antibodies (bnAbs) for the treatment and

  18. Antibody-mediated rejection in heart transplant recipients: potential efficacy of B-cell depletion and antibody removal.

    PubMed

    Bierl, Charlene; Miller, Barry; Prak, Eline Luning; Gasiewski, Allison; Kearns, Jane; Tsai, Donald; Jessup, Mariell; Kamoun, Malek

    2006-01-01

    We present four patients with late AMR following cardiac transplantation, which was associated with de novo post-transplant anti-HLA class II antibody production. All patients had negative anti-HLA class I and class II antibodies prior to transplantation (as assessed by sensitive Flow PRA bead assays) and had a negative retrospective T- and B-cell flow cytometric cross-match. Upon presentation with late graft rejection due to AMR, all patients were treated with rituximab and serial plasmapheresis with IVIg plus triple-drug immunosuppression therapy. Despite initial responses to therapy, relapses occurred in all of the patients and necessitated prolonged or multiple hospital admissions and second transplants in two cases. Post-transplant serum antibody monitoring did not prove to be predictive of treatment success or failure. Serum anti-HLA antibodies should be monitored after heart transplantation. We recommend an assessment of anti-HLA antibodies following a decline in immunosuppressant drug levels or in the presence of heart failure symptoms. Anti-HLA antibody detection should be performed using very sensitive techniques such as microparticle-based assays.

  19. Antibody-mediated pure red cell aplasia (PRCA): epidemiology, immunogenicity and risks.

    PubMed

    Macdougall, Iain C

    2005-05-01

    Although epoetin-induced antibody (Ab)-mediated pure red cell aplasia (PRCA) was very rare prior to 1998, a large increase in the number of global cases was observed from 1999 to 2002 in patients treated with erythropoiesis-stimulating agents (ESAs) for the anaemia associated with chronic kidney disease (CKD). The number of global cases of this immunological form of PRCA has declined precipitously since 2003 following increased awareness of this disorder and changes in the handling and administration of the formulation of epoetin-alpha (EPO) that was associated with the majority of cases. Current recommendations state that patients should stop treatment immediately following a diagnosis of Ab-mediated PRCA and not resume treatment with the same or another ESA. The feasibility of re-treatment following remission from PRCA or during ongoing immunosuppressive therapy is currently under investigation. The immunological mechanism for developing Ab-mediated PRCA is unknown, but a variety of factors may increase the immunogenicity of epoetin or other ESAs. Product-related factors that have the potential to impact on immunogenicity include sequence variations in proteins, the degree and nature of protein glycosylation, the manufacturing process, handling and storage, and components and properties of the product formulation. Patient-related factors associated with developing Ab-mediated PRCA include skin reactions, immune status and treatment history. Increased awareness among physicians of the factors contributing to the development of PRCA and its distinguishing clinical features has coincided with studies aimed at identifying effective therapies for this disorder. A number of immunosuppressive therapies have been shown recently to reconstitute effectively the erythropoietic response in patients with PRCA. However, therapeutic approaches for this serious immunological reaction to recombinant ESAs remain investigative.

  20. Antibody-mediated pure red cell aplasia (PRCA) treatment and re-treatment: multiple options.

    PubMed

    Rossert, Jérôme; Macdougall, Iain; Casadevall, Nicole

    2005-05-01

    In the vast majority of patients with antibody (Ab)-mediated pure red cell aplasia (PRCA), simple withdrawal of the erythropoiesis-stimulating agent (ESA) does not effectively reverse PRCA. In contrast, immunosuppressive treatments can induce the disappearance of anti-erythropoietin Abs and a reversal of PRCA. Consensus opinion on the optimal therapy has not been established, but individual case reports or case series suggest that kidney transplantation or treatment with corticosteroids plus cyclophosphamide are the most effective therapies. However, treatment with cyclosporine is an interesting alternative, since it appears to be effective in at least two-thirds of patients and with minimal side effects. Due to the key role of ESAs in the management of patients with chronic kidney disease (CKD), some patients have been re-treated with an ESA following resolution of Ab-mediated PRCA. In all reported cases, this treatment increased haemoglobin levels, alleviated the need for transfusions and did not have side effects. However, one should be extremely cautious when deciding to re-treat a patient with ESA, due to the small number of reported cases and the possibility of publication bias.

  1. Bispecific Antibodies that Mediate Killing of Cells Infected with Human Immunodeficiency Virus of Any Strain

    NASA Astrophysics Data System (ADS)

    Berg, Jorg; Lotscher, Erika; Steimer, Kathelyn S.; Capon, Daniel J.; Baenziger, Jurg; Jack, Hans-Martin; Wabl, Matthias

    1991-06-01

    Although AIDS patients lose human immunodeficiency virus (HIV)-specific cytotoxic T cells, their remaining CD8-positive T lymphocytes maintain cytotoxic function. To exploit this fact we have constructed bispecific antibodies that direct cytotoxic T lymphocytes of any specificity to cells that express gp120 of HIV. These bispecific antibodies comprise one heavy/light chain pair from an antibody to CD3, linked to a heavy chain whose variable region has been replaced with sequences from CD4 plus a second light chain. CD3 is part of the antigen receptor on T cells and is responsible for signal transduction. In the presence of these bispecific antibodies, T cells of irrelevant specificity effectively lyse HIV-infected cells in vitro.

  2. Bispecific antibodies that mediate killing of cells infected with human immunodeficiency virus of any strain.

    PubMed Central

    Berg, J; Lötscher, E; Steimer, K S; Capon, D J; Baenziger, J; Jäck, H M; Wabl, M

    1991-01-01

    Although AIDS patients lose human immunodeficiency virus (HIV)-specific cytotoxic T cells, their remaining CD8-positive T lymphocytes maintain cytotoxic function. To exploit this fact we have constructed bispecific antibodies that direct cytotoxic T lymphocytes of any specificity to cells that express gp120 of HIV. These bispecific antibodies comprise one heavy/light chain pair from an antibody to CD3, linked to a heavy chain whose variable region has been replaced with sequences from CD4 plus a second light chain. CD3 is part of the antigen receptor on T cells and is responsible for signal transduction. In the presence of these bispecific antibodies, T cells of irrelevant specificity effectively lyse HIV-infected cells in vitro. Images PMID:1905015

  3. Incidence of erythropoietin antibody-mediated pure red cell aplasia: the Prospective Immunogenicity Surveillance Registry (PRIMS)

    PubMed Central

    Macdougall, Iain C.; Casadevall, Nicole; Locatelli, Francesco; Combe, Christian; London, Gerard M.; Di Paolo, Salvatore; Kribben, Andreas; Fliser, Danilo; Messner, Hans; McNeil, John; Stevens, Paul; Santoro, Antonio; De Francisco, Angel L.M.; Percheson, Paul; Potamianou, Anna; Foucher, Arnaud; Fife, Daniel; Mérit, Véronique; Vercammen, Els

    2015-01-01

    Background Subcutaneous administration of Eprex® (epoetin alfa) in patients with chronic kidney disease (CKD) was contraindicated in the European Union between 2002 and 2006 after increased reports of anti-erythropoietin antibody-mediated pure red cell aplasia (PRCA). The Prospective Immunogenicity Surveillance Registry (PRIMS) was conducted to estimate the incidence of antibody-mediated PRCA with subcutaneous administration of a new coated-stopper syringe presentation of Eprex® and to compare this with the PRCA incidence with subcutaneous NeoRecormon® (epoetin beta) and Aranesp® (darbepoetin alfa). Methods PRIMS was a multicentre, multinational, non-interventional, parallel-group, immunogenicity surveillance registry. Adults with CKD receiving or about to initiate subcutaneous Eprex®, NeoRecormon® or Aranesp® for anaemia were enrolled and followed for up to 3 years. Unexplained loss or lack of effect (LOE), including suspected PRCA, was reported, with antibody testing for confirmation of PRCA. Results Of the 15 333 patients enrolled, 5948 received Eprex® (8377 patient-years) and 9356 received NeoRecormon®/Aranesp® (14 286 patient-years). No treatment data were available for 29 patients. Among 23 patients with LOE, five cases of PRCA were confirmed (Eprex®, n = 3; NeoRecormon®, n = 1; Aranesp®, n = 1). Based on exposed time, PRCA incidence was 35.8/100 000 patient-years (95% CI 7.4–104.7) for Eprex® versus 14.0/100 000 patient-years (95% CI 1.7–50.6) for NeoRecormon®/Aranesp®. The incidence of PRCA with Eprex® was not significantly different versus comparator ESAs (rate ratio: 2.56; 95% CI 0.43–15.31). An analysis based on observed time produced similar findings. Conclusion This large, prospective registry demonstrates that PRCA is rare with subcutaneous administration of either the new coated-stopper syringe presentation of Eprex®, or NeoRecormon® or Aranesp®. PMID:25239637

  4. C-C chemokine receptor-7 mediated endocytosis of antibody cargoes into intact cells.

    PubMed

    Charest-Morin, Xavier; Pépin, Rémy; Gagné-Henley, Angélique; Morissette, Guillaume; Lodge, Robert; Marceau, François

    2013-01-01

    The C-C chemokine receptor-7 (CCR7) is a G protein coupled receptor that has a role in leukocyte homing, but that is also expressed in aggressive tumor cells. Preclinical research supports that CCR7 is a valid target in oncology. In view of the increasing availability of therapeutic monoclonal antibodies that carry cytotoxic cargoes, we studied the feasibility of forcing intact cells to internalize known monoclonal antibodies by exploiting the cycle of endocytosis and recycling triggered by the CCR7 agonist CCL19. Firstly, an anti-CCR7 antibody (CD197; clone 150503) labeled surface recombinant CCR7 expressed in intact HEK 293a cells and the fluorescent antibody was internalized following CCL19 treatment. Secondly, a recombinant myc-tagged CCL19 construction was exploited along the anti-myc monoclonal antibody 4A6. The myc-tagged ligand was produced as a conditioned medium of transfected HEK 293a cells that contained the equivalent of 430 ng/ml of immunoreactive CCL19 (average value, ELISA determination). CCL19-myc, but not authentic CCL19, carried the fluorophore-labeled antibody 4A6 into other recipient cells that expressed recombinant CCR7 (microscopy, cytofluorometry). The immune complexes were apparent in endosomal structures, co-localized well with the small GTPase Rab5 and progressed toward Rab7-positive endosomes. A dominant negative form of Rab5 (GDP-locked) inhibited this endocytosis. Further, endosomes in CCL19-myc- or CCL19-stimulated cells were positive for β-arrestin2, but rarely for β-arrestin1. Following treatment with CCL19-myc and the 4A6 antibody, the melanoma cell line A375 that expresses endogenous CCR7 was specifically stained using a secondary peroxidase-conjugated antibody. Agonist-stimulated CCR7 can transport antibody-based cargoes, with possible therapeutic applications in oncology.

  5. LGR5 expressing cells of hair follicle as potential targets for antibody mediated anti-cancer laser therapy

    NASA Astrophysics Data System (ADS)

    Popov, Boris V.

    2013-02-01

    Near infrared laser immunotherapy becomes now a new promising research field to cure the patients with cancers. One of the critical limitation in medical application of this treatment is availability of the specific markers for delivery of laser-sensitive nanoparticles. When coupled to antibodies to the cancer stem cells markers these nanoparticles may be delivered to the cancer tissue and mediate the laser induced thermolysis of the cancer stem cells that initiate and drive growth of cancer. This paper addresses the Lgr5 cell surface marker mediating the Wnt/β-catenin signal transduction as a potential target for anti-cancer laser immunotherapy of skin cancers.

  6. Acute T cell-mediated rejection accompanied by C4d-negative acute antibody-mediated rejection and cell debris in tubulus: A case report.

    PubMed

    Takamura, Tsuyoshi; Yamamoto, Izumi; Nakada, Yasuyuki; Katsumata, Haruki; Yamakawa, Takafumi; Furuya, Maiko; Mafune, Aki; Kobayashi, Akimitsu; Tanno, Yudo; Miki, Jun; Ohkido, Ichiro; Tsuboi, Nobuo; Yamamoto, Hiroyasu; Yokoo, Takashi

    2015-07-01

    Herein, we report a complicated case of acute T-cell-mediated rejection (ACR) accompanied by C4d-negative acute antibody-mediated rejection (AMR) and cell debris in tubulus. A 32 year-old male was admitted for an episode biopsy with a serum creatinine (S-Cr) level of 1.83 mg/dL and pyuria (20-29 white blood cells per high power field) 49 days following kidney transplantation. Histological features included three distinct entities, mainly, in one of the three specimens: 1) focal aggressive tubulointerstitial inflammatory cell infiltration with moderate tubulitis, 2) inflammatory cell infiltration in peritubular capillaries (including neutrophils) and glomerular capillaries, and 3) cell debris consisting mainly of neutrophils in tubulus. Laboratory examination revealed evidence of non-human leukocyte antigen donor-specific antibodies. However, urinary culture and gram staining were negative. Considering both the histological and laboratory findings, the patient was diagnosed with ACR accompanied by C4d-negative AMR and suspicion of a urinary tract infection (UTI). The patient was treated for three consecutive days with steroid pulse therapy. The patient's S-Cr level decreased to ~1.5 mg/dL following treatment and did not increase thereafter. A second biopsy 133 days following kidney transplantation showed an excellent response to treatment and revealed no evidence of rejection. This case report demonstrates the difficulty in the diagnosis of, and therapy for, the complicated pathological findings of ACR, AMR and suspicion of a UTI.

  7. Excess reactive oxygen species production mediates monoclonal antibody-induced human embryonic stem cell death via oncosis.

    PubMed

    Zheng, Ji Yun; Tan, Heng Liang; Matsudaira, Paul Thomas; Choo, Andre

    2017-03-01

    Antibody-mediated cell killing has significantly facilitated the elimination of undesired cells in therapeutic applications. Besides the well-known Fc-dependent mechanisms, pathways of antibody-induced apoptosis were also extensively studied. However, with fewer studies reporting the ability of antibodies to evoke an alternative form of programmed cell death, oncosis, the molecular mechanism of antibody-mediated oncosis remains underinvestigated. In this study, a monoclonal antibody (mAb), TAG-A1 (A1), was generated to selectively kill residual undifferentiated human embryonic stem cells (hESC) so as to prevent teratoma formation upon transplantation of hESC-derived products. We revealed that A1 induces hESC death via oncosis. Aided with high-resolution scanning electron microscopy (SEM), we uncovered nanoscale morphological changes in A1-induced hESC oncosis, as well as A1 distribution on hESC surface. A1 induces hESC oncosis via binding-initiated signaling cascade, most likely by ligating receptors on surface microvilli. The ability to evoke excess reactive oxygen species (ROS) production via the Nox2 isoform of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is critical in the cell death pathway. Excess ROS production occurs downstream of microvilli degradation and homotypic adhesion, but upstream of actin reorganization, plasma membrane damage and mitochondrial membrane permeabilization. To our knowledge, this is the first mechanistic model of mAb-induced oncosis on hESC revealing a previously unrecognized role for NAPDH oxidase-derived ROS in mediating oncotic hESC death. These findings in the cell death pathway may potentially be exploited to improve the efficiency of A1 in eliminating undifferentiated hESC and to provide insights into the study of other mAb-induced cell death.

  8. Disappearance of T Cell-Mediated Rejection Despite Continued Antibody-Mediated Rejection in Late Kidney Transplant Recipients.

    PubMed

    Halloran, Philip F; Chang, Jessica; Famulski, Konrad; Hidalgo, Luis G; Salazar, Israel D R; Merino Lopez, Maribel; Matas, Arthur; Picton, Michael; de Freitas, Declan; Bromberg, Jonathan; Serón, Daniel; Sellarés, Joana; Einecke, Gunilla; Reeve, Jeff

    2015-07-01

    The prevalent renal transplant population presents an opportunity to observe the adaptive changes in the alloimmune response over time, but such studies have been limited by uncertainties in the conventional biopsy diagnosis of T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR). To circumvent these limitations, we used microarrays and conventional methods to investigate rejection in 703 unselected biopsies taken 3 days to 35 years post-transplant from North American and European centers. Using conventional methods, we diagnosed rejection in 205 biopsy specimens (28%): 67 pure TCMR, 110 pure ABMR, and 28 mixed (89 designated borderline). Using microarrays, we diagnosed rejection in 228 biopsy specimens (32%): 76 pure TCMR, 124 pure ABMR, and 28 mixed (no borderline). Molecular assessment confirmed most conventional diagnoses (agreement was 90% for TCMR and 83% for ABMR) but revealed some errors, particularly in mixed rejection, and improved prediction of failure. ABMR was strongly associated with increased graft loss, but TCMR was not. ABMR became common in biopsy specimens obtained >1 year post-transplant and continued to appear in all subsequent intervals. TCMR was common early but progressively disappeared over time. In 108 biopsy specimens obtained 10.2-35 years post-transplant, TCMR defined by molecular and conventional features was never observed. We conclude that the main cause of kidney transplant failure is ABMR, which can present even decades after transplantation. In contrast, TCMR disappears by 10 years post-transplant, implying that a state of partial adaptive tolerance emerges over time in the kidney transplant population.

  9. Impact of Signal Peptides on Furin-2A Mediated Monoclonal Antibody Secretion in CHO Cells.

    PubMed

    Lin, Jian'er; Neo, Shu Hui; Ho, Steven C L; Yeo, Jessna H M; Wang, Tianhua; Zhang, Wei; Bi, Xuezhi; Chao, Sheng-Hao; Yang, Yuansheng

    2017-09-01

    Studies had shown the benefits of using furin-2A peptides for high monoclonal antibody (mAb) expression in mammalian cells. How signal peptides affect furin-2A mediated mAb secretion has yet to be investigated. The impact of signal peptides on mAb secretion in furin-2A based tricistronic vectors in CHO cells is evaluated. In each tricistronic vector, heavy chain (HC) is arranged as the first cistron and followed by a furin recognition sequence, a 2A peptide, light chain (LC), an internal ribosome entry site (IRES), and dihydrofolate reductase (DHFR). Signal peptides for HC and LC are either removed or changed in different vectors. The vectors with signal peptides on both HC and LC genes gIve the highest mAb secretion levels. Changing to signal peptides with different strengths on either HC or LC do not change the mAb secretion level. IgG is still secreted when the signal peptide on the LC gene is removed but at a lower level compared to the vectors containing signal peptides on both HC and LC genes. Removing the HC signal peptide results in almost no IgG secretion regardless of whether the downstream LC carries any signal peptide. Removing the furin cleavage site does not affect mAb secretion levels while removing the 2A sequence results in low mAb secretion. The results present here will be beneficial for designing furin-2A based vectors for expressing mAb in mammalian cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Antibody-mediated inhibition of Nogo-A signaling promotes neurite growth in PC-12 cells

    PubMed Central

    Yazdi, Iman K; Taghipour, Nima; Hmaidan, Sarah; Palomba, Roberto; Scaria, Shilpa; Munoz, Alvaro; Boone, Timothy B; Tasciotti, Ennio

    2016-01-01

    The use of a monoclonal antibody to block the neurite outgrowth inhibitor Nogo-A has been of great interest for promoting axonal recovery as a treatment for spinal cord injury. While several cellular and non-cellular assays have been developed to quantify the bioactive effects of Nogo-A signaling, demand still exists for the development of a reliable approach to characterize the effectiveness of the anti-Nogo-A antibody. In this study, we developed and validated a novel cell-based approach to facilitate the biological quantification of a Nogo-A antibody using PC-12 cells as an in vitro neuronal cell model. Changes in the mRNA levels of the neuronal differentiation markers, growth-associated protein 43 and neurofilament light-polypeptide, suggest that activation of the Nogo-A pathway suppresses axonal growth and dendrite formation in the tested cell line. We found that application of anti-Nogo-A monoclonal antibody can significantly enhance the neuronal maturity of PC-12 cells by blocking the Nogo-A inhibitory effects, providing enhanced effects on neural maturity at the molecular level. No adverse effects were observed on cell viability. PMID:27027860

  11. High-multiplicity HIV-1 infection and neutralizing antibody evasion mediated by the macrophage-T cell virological synapse.

    PubMed

    Duncan, Christopher J A; Williams, James P; Schiffner, Torben; Gärtner, Kathleen; Ochsenbauer, Christina; Kappes, John; Russell, Rebecca A; Frater, John; Sattentau, Quentin J

    2014-02-01

    Macrophage infection is considered to play an important role in HIV-1 pathogenesis and persistence. Using a primary cell-based coculture model, we show that monocyte-derived macrophages (MDM) efficiently transmit a high-multiplicity HIV-1 infection to autologous CD4(+) T cells through a viral envelope glycoprotein (Env) receptor- and actin-dependent virological synapse (VS), facilitated by interactions between ICAM-1 and LFA-1. Virological synapse (VS)-mediated transmission by MDM results in high levels of T cell HIV-1 integration and is 1 to 2 orders of magnitude more efficient than cell-free infection. This mode of cell-to-cell transmission is broadly susceptible to the activity of CD4 binding site (CD4bs) and glycan or glycopeptide epitope-specific broadly neutralizing monoclonal antibodies (bNMAbs) but shows resistance to bNMAbs targeting the Env gp41 subunit membrane-proximal external region (MPER). These data define for the first time the structure and function of the macrophage-to-T cell VS and have important implications for bNMAb activity in HIV-1 prophylaxis and therapy. IMPORTANCE The ability of HIV-1 to move directly between contacting immune cells allows efficient viral dissemination with the potential to evade antibody attack. Here, we show that HIV-1 spreads from infected macrophages to T cells via a structure called a virological synapse that maintains extended contact between the two cell types, allowing transfer of multiple infectious events to the T cell. This process allows the virus to avoid neutralization by a class of antibody targeting the gp41 subunit of the envelope glycoproteins. These results have implications for viral spread in vivo and the specificities of neutralizing antibody elicited by antibody-based vaccines.

  12. Antibody-dependent cell-mediated cytotoxicity of mononuclear cells against Langerhans islets of Wistar rats in normal man and in patients at diabetes risk.

    PubMed

    Köhler, E; Knospe, S; Woltanski, G; Maciejewski, R; Salzsieder, C; Rjasanowski, I; Strese, J; Michaelis, D

    1984-01-01

    Antibody-dependent cell-mediated cytotoxicity against pancreatic islets was investigated in 13 newly diagnosed insulin-dependent diabetics, in 38 patients at high risk for the disease and in 20 age-matched healthy controls. For this purpose 51Cr-labeled neonatal rat pancreatic islets incubated with the specific anti-rat islet cell antiserum 339 or with serum of the lymphocyte donors were used as targets. The antibody-mediated cytotoxic activity of mononuclear cells was evaluated from the specific chromium release after 6 h exposure of pretreated islets to the effector cells. The specific cytotoxic effect of mononuclear cells from healthy controls on pancreatic islets pretreated with serum is weak. ADCC mediated by the specific antirat islet cell antiserum is significantly increased in 54% of newly diagnosed diabetics as well as in 32% of patients at high risk for insulin-dependent diabetes mellitus. 46% of the newly diagnosed diabetics were also ADCC-positive when their own serum was used for the pretreatment of islets, regardless of whether they were islet cell antibody- or islet cell surface antibody positive or not. Subsets of mononuclear cells (non E-rosette-forming cells, high affinity E-rosette-forming cells, low affinity E-rosette-forming cells) were prepared and their cytotoxic potential was analysed in patients with positive ADCC test results.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. CD1d-mediated interaction between activated T cells and B cells is essential to B-cell proliferation and anti-alpha-Gal antibody production.

    PubMed

    Liu, S; Kandeva, T; Tchervenkov, J

    2009-01-01

    Antibody-mediated rejection is central to ABO-incompatible transplantation as well as to xenotransplantation. The carbohydrate structure of xenoantigen alpha-Gal is highly analogous to the human blood group antigens. Both require memory B-cell activation for antibody production. We hypothesized that B cells, reactive to the alpha-Gal xenoantigen, required the presence of fully activated T cells to survive and proliferate in vitro. This hypothesis was contrary to the traditional theory that the response of B cells to carbohydrate antigens is T cell independent (Wong and Arsequell: Immunobiology of Carbohydrates. New York: Kluwer; 2003). When we compared the capacity of B cells to proliferate, we observed that activated T cells were necessary for B-cell proliferation. However, this proliferation was independent of the presence of antigen. A relevant question was also to investigate the role of the specific class of T cells: the CD1d-restricted iNKT (iNKT) cells in the activation of alpha-Gal-reactive B cells. The iNKT cells are reactive to glycolipids and capable of producing both Th1 and Th2 cytokine responses. We therefore wanted to determine the role of the iNKT cells as mediators of a Th2-type response when B cells were exposed to a glycolipid antigen extracted from pig red blood cells, which express blockade of the alpha-Gal epitope. We observed that the interaction between B cells and iNKT cells prevents B-cell proliferation and anti-alpha-Gal antibody production.

  14. Adlayer-mediated antibody immobilization to stainless steel for potential application to endothelial progenitor cell capture.

    PubMed

    Benvenuto, Pasquale; Neves, Miguel A D; Blaszykowski, Christophe; Romaschin, Alexander; Chung, Timothy; Kim, Sa Rang; Thompson, Michael

    2015-05-19

    This work describes the straightforward surface modification of 316L stainless steel with BTS, S-(11-trichlorosilylundecanyl)-benzenethiosulfonate, a thiol-reactive trichlorosilane cross-linker molecule designed to form intermediary coatings with subsequent biofunctionalization capability. The strategy is more specifically exemplified with the immobilization of intact antibodies and their Fab' fragments. Both surface derivatization steps are thoroughly characterized by means of X-ray photoelectron spectroscopy. The antigen binding capability of both types of biofunctionalized surfaces is subsequently assessed by fluorescence microscopy. It was determined that BTS adlayers achieve robust immobilization of both intact and fragmented antibodies, while preserving antigen binding activity. Another key finding was the observation that the Fab' fragment immobilization strategy would constitute a preferential option over that involving intact antibodies in the context of in vivo capture of endothelial progenitor cells in stent applications.

  15. X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity

    PubMed Central

    Evans, M K; Sauer, S J; Nath, S; Robinson, T J; Morse, M A; Devi, G R

    2016-01-01

    Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy. PMID:26821068

  16. Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase.

    PubMed

    López-Alemany, Roser; Longstaff, Colin; Hawley, Stephen; Mirshahi, Massoud; Fábregas, Pere; Jardí, Merce; Merton, Elizabeth; Miles, Lindsey A; Félez, Jordi

    2003-04-01

    Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells.

  17. A flow cytometry-based assay for the evaluation of antibody-dependent cell-mediated cytotoxicity (ADCC) in cancer cells.

    PubMed

    Salinas-Jazmín, Nohemí; Hisaki-Itaya, Emiliano; Velasco-Velázquez, Marco A

    2014-01-01

    The development of new therapeutic monoclonal antibodies (mAbs) for cancer therapy will rise in the following years. The evaluation of biological activity of mAbs is required during drug development and during drug production as quality control test. Antibody-dependent cell-mediated cytotoxicity (ADCC) is a desirable activity of anticancer mAbs. Here, we describe a flow cytometry-based method to quantify ADCC that combines the staining of cancer cells, effector cells, and dead cells, with specific dyes. This method is inexpensive, has low background, and avoids the use of radioisotopes.

  18. Natural killer cells play a critical role in mediating inflammation and graft failure during antibody-mediated rejection of kidney allografts

    PubMed Central

    Kohei, Naoki; Tanaka, Toshiaki; Tanabe, Kazunari; Masumori, Naoya; Dvorina, Nina; Valujskikh, Anna; Baldwin, William M.; Fairchild, Robert L.

    2016-01-01

    While the incidence of antibody-mediated kidney graft rejection has increased, the key cellular and molecular participants underlying this graft injury remain unclear. Rejection of kidney allografts in mice lacking the chemokine receptor CCR5 is dependent on production of donor-specific antibody. Here we determine if cells expressing cytotoxic function contributed to antibody-mediated kidney allograft rejection in these recipients. Wild type C57BL/6, B6.CCR5−/− and B6.CD8−/−/CCR5−/− mice were transplanted with complete MHC mismatched A/J kidney grafts and intra-graft inflammatory components were followed to rejection. B6.CCR5−/− and B6.CD8−/−/CCR5−/− recipients rejected kidney allografts by day 35 whereas 65% of allografts in wild type recipients survived past day 80 post-transplant. Rejected allografts in wild-type C57BL/6, B6.CCR5−/− and B6.CD8−/−/CCR5−/− recipients expressed high levels of VCAM-1 and MMP7 mRNA that was associated with high serum titers of donor-specific antibody. High levels of perforin and granzyme B mRNA expression peaked on day 6 post-transplant in allografts in all recipients, but were absent in isografts. Depletion of natural killer cells in B6.CD8−/−/CCR5−/− recipients reduced this expression to background levels and promoted the long-term survival of 40% of the kidney allografts. Thus, natural killer cells have a role in increased inflammation during antibody-mediated kidney allograft injury and in rejection of the grafts. PMID:27165816

  19. Natural killer cells play a critical role in mediating inflammation and graft failure during antibody-mediated rejection of kidney allografts.

    PubMed

    Kohei, Naoki; Tanaka, Toshiaki; Tanabe, Kazunari; Masumori, Naoya; Dvorina, Nina; Valujskikh, Anna; Baldwin, William M; Fairchild, Robert L

    2016-06-01

    While the incidence of antibody-mediated kidney graft rejection has increased, the key cellular and molecular participants underlying this graft injury remain unclear. Rejection of kidney allografts in mice lacking the chemokine receptor CCR5 is dependent on production of donor-specific antibody. Here we determine if cells expressing cytotoxic function contributed to antibody-mediated kidney allograft rejection in these recipients. Wild-type C57BL/6, B6.CCR5(-/-), and B6.CD8(-/-)/CCR5(-/-) mice were transplanted with complete MHC-mismatched A/J kidney grafts, and intragraft inflammatory components were followed to rejection. B6.CCR5(-/-) and B6.CD8(-/-)/CCR5(-/-) recipients rejected kidney allografts by day 35, whereas 65% of allografts in wild-type recipients survived past day 80 post-transplant. Rejected allografts in wild-type C57BL/6, B6.CCR5(-/-), and B6.CD8(-/-)/CCR5(-/-) recipients expressed high levels of VCAM-1 and MMP7 mRNA that was associated with high serum titers of donor-specific antibody. High levels of perforin and granzyme B mRNA expression peaked on day 6 post-transplant in allografts in all recipients, but were absent in isografts. Depletion of natural killer cells in B6.CD8(-/-)/CCR5(-/-) recipients reduced this expression to background levels and promoted the long-term survival of 40% of the kidney allografts. Thus, natural killer cells have a role in increased inflammation during antibody-mediated kidney allograft injury and in rejection of the grafts.

  20. Control of Toll-like receptor-mediated T cell-independent type 1 antibody responses by the inducible nuclear protein IκB-ζ.

    PubMed

    Hanihara-Tatsuzawa, Fumito; Miura, Hanae; Kobayashi, Shuhei; Isagawa, Takayuki; Okuma, Atsushi; Manabe, Ichiro; MaruYama, Takashi

    2014-11-07

    Antibody responses have been classified as being either T cell-dependent or T cell-independent (TI). TI antibody responses are further classified as being either type 1 (TI-1) or type 2 (TI-2), depending on their requirement for B cell-mediated antigen receptor signaling. Although the mechanistic basis of antibody responses has been studied extensively, it remains unclear whether different antibody responses share similarities in their transcriptional regulation. Here, we show that mice deficient in IκB-ζ, specifically in their B cells, have impaired TI-1 antibody responses but normal T cell-dependent and TI-2 antibody responses. The absence of IκB-ζ in B cells also impaired proliferation triggered by Toll-like receptor (TLR) activation, plasma cell differentiation, and class switch recombination (CSR). Mechanistically, IκB-ζ-deficient B cells could not induce TLR-mediated induction of activation-induced cytidine deaminase (AID), a class-switch DNA recombinase. Retroviral transduction of AID in IκB-ζ-deficient B cells restored CSR activity. Furthermore, acetylation of histone H3 in the vicinity of the transcription start site of the gene that encodes AID was reduced in IκB-ζ-deficient B cells relative to IκB-ζ-expressing B cells. These results indicate that IκB-ζ regulates TLR-mediated CSR by inducing AID. Moreover, IκB-ζ defines differences in the transcriptional regulation of different antibody responses.

  1. Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies

    PubMed Central

    Yee, Michael; Konopka, Krystyna; Balzarini, Jan; Düzgüneş, Nejat

    2011-01-01

    Background: Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy. Results: Cell-cell fusion was inhibited completely by the carbohydrate binding proteins (CBPs), Hippeastrum hybrid (Amaryllis) agglutinin (HHA), and Galanthus nivalis (Snowdrop) agglutinin (GNA), and by the peptide, T-20, at relatively low concentrations. Anti-gp120 and anti-gp41 antibodies, at concentrations much higher than those required for neutralization, were not particularly effective in inhibiting fusion. Monoclonal antibodies b12, m14 IgG and 2G12 had moderate inhibitory activity; the IC50 of 2G12 was about 80 µg/ml. Antibodies 4E10 and 2F5 had no inhibitory activity at the concentrations tested. Conclusions: These observations raise concerns about the ability of neutralizing antibodies to inhibit the spread of viral genetic material from infected cells to uninfected cells via cell-cell fusion. The interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion reactions, and may explain the differential effects of antibodies in these two systems. The fluorescence assay described here may be useful in high throughput screening of potential HIV fusion inhibitors. PMID:21660189

  2. Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies.

    PubMed

    Yee, Michael; Konopka, Krystyna; Balzarini, Jan; Düzgüneş, Nejat

    2011-01-01

    Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy. Cell-cell fusion was inhibited completely by the carbohydrate binding proteins (CBPs), Hippeastrum hybrid (Amaryllis) agglutinin (HHA), and Galanthus nivalis (Snowdrop) agglutinin (GNA), and by the peptide, T-20, at relatively low concentrations. Anti-gp120 and anti-gp41 antibodies, at concentrations much higher than those required for neutralization, were not particularly effective in inhibiting fusion. Monoclonal antibodies b12, m14 IgG and 2G12 had moderate inhibitory activity; the IC(50) of 2G12 was about 80 µg/ml. Antibodies 4E10 and 2F5 had no inhibitory activity at the concentrations tested. These observations raise concerns about the ability of neutralizing antibodies to inhibit the spread of viral genetic material from infected cells to uninfected cells via cell-cell fusion. The interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion reactions, and may explain the differential effects of antibodies in these two systems. The fluorescence assay described here may be useful in high throughput screening of potential HIV fusion inhibitors.

  3. TCR-like antibody drug conjugates mediate killing of tumor cells with low peptide/HLA targets.

    PubMed

    Lowe, Devin B; Bivens, Camille K; Mobley, Alexis S; Herrera, Christian E; McCormick, Amanda L; Wichner, Timea; Sabnani, Manoj K; Wood, Laurence M; Weidanz, Jon A

    The currently marketed antibody-drug conjugates (ADC) destabilize microtubule assembly in cancer cells and initiate apoptosis in patients. However, few tumor antigens (TA) are expressed at high densities on cancer lesions, potentially minimizing the therapeutic index of current ADC regimens. The peptide/human leukocyte antigen (HLA) complex can be specifically targeted by therapeutic antibodies (designated T cell receptor [TCR]-like antibodies) and adequately distinguish malignant cells, but has not been the focus of ADC development. We analyzed the killing potential of TCR-like ADCs when cross-linked to the DNA alkylating compound duocarmycin. Our data comprise proof-of-principle results that TCR-like ADCs mediate potent tumor cytotoxicity, particularly under common scenarios of low TA/HLA density, and support their continued development alongside agents that disrupt DNA replication. Additionally, TCR-like antibody ligand binding appears to play an important role in ADC functionality and should be addressed during therapy development to avoid binding patterns that negate ADC killing efficacy.

  4. Women with Cervicovaginal Antibody-Dependent Cell-Mediated Cytotoxicity Have Lower Genital HIV-1 RNA Loads

    PubMed Central

    Nag, Pratip; Kim, Jenney; Sapiega, Vytautas; Landay, Alan L.; Bremer, James W.; Mestecky, Jiri; Reichelderfer, Patricia; Kovacs, Andrea; Cohn, Jonathan; Weiser, Barbara; Baum, Linda L.

    2011-01-01

    Antibodies that mediate human immunodeficiency virus (HIV)-specific antibody-dependent cell-mediated cytotoxicity (ADCC) are present in the cervical fluid of many HIV-positive women; however, the role that these antibodies play in host defense against HIV is not known. To understand the contribution of ADCC in cervical secretions as a protective mechanism against HIV, we evaluated ADCC titers in paired serum and cervical-lavage (CVL) samples from >300 HIV-1–positive women who participated in the multicenter Division of AIDS Treatment Research Initiative Study 009. The present study demonstrates that women with CVL ADCC activity had lower genital viral loads than did women with serum ADCC activity only. Women with CVL ADCC activity were likely to have HIV-1 gp120–specific immunoglobulin (Ig) G, but not IgA, in their cervical fluid. This finding suggests that specific IgG in cervical fluid can mediate ADCC activity that inversely correlates with genital viral load. PMID:15529262

  5. Antibody- and cell-mediated immune responses to a synthetic oligosaccharide conjugate vaccine after booster immunization.

    PubMed

    Safari, Dodi; Dekker, Huberta A Th; de Jong, Ben; Rijkers, Ger T; Kamerling, Johannis P; Snippe, Harm

    2011-09-02

    Memory formation to CRM-neoglycoconjugate, a synthetic branched tetrasaccharide of Streptococcus pneumoniae type 14 polysaccharide (Pn14PS) that is conjugated to a CRM197 protein, was investigated using mice models. Mice were first immunized with the CRM-neoglycoconjugate and then boosted with either the same neoglycoconjugate or a native Pn14PS in order to investigate the effect of booster immunization. Boosting with the CRM-neoglycoconjugate resulted in increased levels of interleukin 5 (IL-5) in the serum on Day 1, followed by the appearance of high levels of specific anti-Pn14PS IgG antibodies on Day 7. Boosting with native Pn14PS resulted in neither IL-5 induction nor the generation of anti-Pn14PS IgG antibodies. In vitro (re)stimulation of spleen cells after booster injection with the neoglycoconjugate revealed the presence of IL-4 and IL-5. This was not seen in spleen cells obtained from mice boosted with the polysaccharide. When stimulated with heat-inactivated bacteria, however, the polysaccharide-boosted mice did have higher levels of IFN-γ and lower levels of IL-17 than both the CRM-neoglycoconjugate-boosted mice and the mock-immunized mice. In conclusion, neoglycoconjugate boosting is responsible for the activation of memory cells and the establishment of sustained immunity. Not only is a booster with native polysaccharide ineffective in inducing opsonic antibodies, but it also interferes with several immunoregulatory mechanisms.

  6. Generation of a human antibody that inhibits TSPAN8-mediated invasion of metastatic colorectal cancer cells.

    PubMed

    Kim, Taek-Keun; Park, Chang Sik; Jeoung, Mee Hyun; Lee, Woo Ran; Go, Nam Kyung; Choi, Jong Rip; Lee, Tae Sup; Shim, Hyunbo; Lee, Sukmook

    2015-12-25

    Tetraspanin 8 (TSPAN8) is a tumor-associated antigen implicated in tumor progression and metastasis. However, the validation of TSPAN8 as a potential therapeutic target in metastatic colorectal cancer (mCRC) has not yet been studied. In this study, through several in vitro methodologies, we identified a large extracellular loop of TSPAN8 (TSPAN8-LEL) as a key domain for regulating mCRC invasion. Using phage display technology, we developed a novel anti-TSPAN8-LEL human antibody with subnanomolar affinity that specifically recognizes amino acids 140-205 of TSPAN8-LEL in a conformation-dependent manner. Finally, we demonstrated that the antibody specifically reduces invasion in the HCT116 and LoVo mCRC cell lines more potently than in the HCT-8 and SW480 non-mCRC cell lines. Our data suggest that TSPAN8-LEL may play an important role in mCRC cell invasion, and that the antibody we have developed could be a useful tool for inhibiting the invasion of TSPAN8-expressing mCRCs. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Non-neutralizing Antibodies Targeting the V1V2 Domain of HIV Exhibit Strong Antibody-Dependent Cell-mediated Cytotoxic Activity.

    PubMed

    Mayr, Luzia M; Decoville, Thomas; Schmidt, Sylvie; Laumond, Géraldine; Klingler, Jéromine; Ducloy, Camille; Bahram, Seiamak; Zolla-Pazner, Susan; Moog, Christiane

    2017-10-04

    The development of an effective vaccine against HIV-1 has proven to be challenging. Broadly neutralizing antibodies (bNAbs), whilst exhibiting neutralization breadth and potency, are elicited only in a small subset of infected individuals and have yet to be induced by vaccination. Case-control studies of RV144 identified an inverse correlation of HIV-1 infection risk with antibodies (Abs) to the V1V2 region of gp120 with high antibody-dependent cellular cytotoxicity (ADCC) activity. The neutralizing activity of Abs was not found to contribute to this protective outcome. Using primary effector and target cells and primary virus isolates, we studied the ADCC profile of different monoclonal Abs targeting the V1V2 loop of gp120 that had low or no neutralizing activity. We compared their ADCC activity to some bNAbs targeting different regions of gp120. We found that mAbs targeting the V1V2 domain induce up to 60% NK cell mediated lysis of HIV-1 infected PBMCs in a physiologically relevant ADCC model, highlighting the interest in inducing such Abs in future HIV vaccine trials. Our data also suggest that in addition to neutralization, lysis of infected cells by Abs can effectively participate in HIV protection, as suggested by the RV144 immune correlate analysis.

  8. Evidence of T-cell mediated neuronal injury in stiff-person syndrome with anti-amphiphysin antibodies.

    PubMed

    Poh, Mervyn Q W; Simon, Neil G; Buckland, Michael E; Salisbury, Elizabeth; Watson, Shaun

    2014-02-15

    Paraneoplastic stiff-person syndrome (SPS) has been associated with antibodies against amphiphysin. Current evidence supports a pathogenic role for anti-amphiphysin antibodies. A 74-year-old female was diagnosed with amphiphysin-associated paraneoplastic stiff-person syndrome and associated encephalomyelitis. She had initial response to IVIG, however her symptoms worsened after two months and were resistant to further treatment. Subsequently the patient died and a post-mortem was performed. Neuropathology revealed perivascular and parenchymal lymphocytic infiltrates, with neuronophagia mediated by CD8+ T cells and microglia in brainstem, spinal cord, and mesial temporal lobe structures. These findings suggest a pathogenic role of cytotoxic CD8+ T-cells, with potential implication for therapy of future patients.

  9. Antibody-Dependent Cell-Mediated Cytotoxicity to Hemagglutinin of Influenza A Viruses After Influenza Vaccination in Humans

    PubMed Central

    Zhong, Weimin; Liu, Feng; Wilson, Jason R.; Holiday, Crystal; Li, Zhu-Nan; Bai, Yaohui; Tzeng, Wen-Pin; Stevens, James; York, Ian A.; Levine, Min Z.

    2016-01-01

    Background. Detection of neutralizing antibodies (nAbs) to influenza A virus hemagglutinin (HA) antigens by conventional serological assays is currently the main immune correlate of protection for influenza vaccines However, current prepandemic avian influenza vaccines are poorly immunogenic in inducing nAbs despite considerable protection conferred. Recent studies show that Ab-dependent cell-mediated cytotoxicity (ADCC) to HA antigens are readily detectable in the sera of healthy individuals and patients with influenza infection. Methods. Virus neutralization and ADCC activities of serum samples from individuals who received either seasonal or a stock-piled H5N1 avian influenza vaccine were evaluated by hemagglutination inhibition assay, microneutralization assay, and an improved ADCC natural killer (NK) cell activation assay. Results. Immunization with inactivated seasonal influenza vaccine led to strong expansion of both nAbs and ADCC-mediating antibodies (adccAbs) to H3 antigen of the vaccine virus in 24 postvaccination human sera. In sharp contrast, 18 individuals vaccinated with the adjuvanted H5N1 avian influenza vaccine mounted H5-specific antibodies with strong ADCC activities despite moderate virus neutralization capacity. Strength of HA-specific ADCC activities is largely associated with the titers of HA-binding antibodies and not with the fine antigenic specificity of anti-HA nAbs. Conclusions. Detection of both nAbs and adccAbs may better reflect protective capacity of HA-specific antibodies induced by avian influenza vaccines. PMID:27419174

  10. Characterization of lymphokines mediating B cell growth and differentiation from monoclonal anti-CD3 antibody-stimulated T cells.

    PubMed

    Sherris, D; Stohl, W; Mayer, L

    1989-04-01

    Addition of anti-CD3 mAb 147 (IgG1), 446 (IgG1), or 454 (IgG2a) to cultures of T plus non-T cells can result in both B cell growth and differentiation. To determine whether lymphokines mediating these activities were similar to those described from conventional mitogen-induced T cell activation, normal peripheral blood T cells were stimulated with anti-CD3 mAb for 48 h. The supernatants were assayed for factors inducing B cell growth or differentiation (BCDF). A marked increase in Ig secretion was observed when either EBV-transformed B cell lines or normal B cells, pre-activated with Staphylococcus aureus Cowan I strain, were cultured in the presence of mAb 446 (anti-CD3) stimulated T cell supernatant whereas no significant increase in Ig secretion was noted with either mAb 454- or 147-induced T cell supernatant despite equivalent T cell proliferative responses to these antibodies. In contrast, IL-2 secretion was detectable in T cell supernatants from T cells stimulated with either mAb 454 or 147 but not 446. Factors promoting B cell proliferation were detected in all antibody-stimulated T cell supernatants but, contrary to BCDF, appear to act only on non-activated B cells. To determine whether these effector activities were due to distinct lymphokines, supernatants were pooled and concentrated by ammonium sulfate precipitation. Superose 12 permeation chromatography revealed BCDF activity with an apparent Mr of approximately 30,000 Da. The growth factor activity eluted over a wider and higher molecular weight range which overlapped the differentiation factor activity. Fractions containing BCDF activity were pooled, dialyzed, applied to a Mono Q anion-exchange column, and eluted with a linear NaCl gradient. The growth factor activity came off in a single-peak while BCDF was found divided into two major areas. The growth factor eluted at an ionic strength between the two BCDF activities. BCDF has an apparent isoelectric point (pI) of 6, in contrast to the reported p

  11. The CD25-binding antibody Daclizumab High-Yield Process has a distinct glycosylation pattern and reduced antibody-dependent cell-mediated cytotoxicity in comparison to Zenapax®.

    PubMed

    Ganguly, Bishu; Balasa, Balaji; Efros, Lyubov; Hinton, Paul R; Hartman, Stephen; Thakur, Archana; Xiong, Joanna M; Schmidt, Brian; Robinson, Randy R; Sornasse, Thierry; Vexler, Vladimir; Sheridan, James P

    2016-10-01

    The CD25-binding antibody daclizumab high-yield process (DAC HYP) is an interleukin (IL)-2 signal modulating antibody that shares primary amino acid sequence and CD25 binding affinity with Zenapax®, a distinct form of daclizumab, which was approved for the prevention of acute organ rejection in patients receiving renal transplants as part of an immunosuppressive regimen that includes cyclosporine and corticosteroids. Comparison of the physicochemical properties of the two antibody forms revealed the glycosylation profile of DAC HYP differs from Zenapax in both glycan distribution and the types of oligosaccharides, most notably high-mannose, galactosylated and galactose-α-1,3-galactose (α-Gal) oligosaccharides, resulting in a DAC HYP antibody material that is structurally distinct from Zenapax. Although neither antibody elicited complement-dependent cytotoxicity in vitro, DAC HYP antibody had significantly reduced levels of antibody-dependent cell-mediated cytotoxicity (ADCC). The ADCC activity required natural killer (NK) cells, but not monocytes, suggesting the effects were mediated through binding to Fc-gamma RIII (CD16). Incubation of each antibody with peripheral blood mononuclear cells also caused the down-modulation of CD16 expression on NK cells and the CD16 down-modulation was greater for Zenapax in comparison to that observed for DAC HYP. The substantive glycosylation differences between the two antibody forms and corresponding greater Fc-mediated effector activities by Zenapax, including cell killing activity, manifest as a difference in the biological function and pharmacology between DAC HYP and Zenapax.

  12. The Fc-region of a new class of intact bispecific antibody mediates activation of accessory cells and NK cells and induces direct phagocytosis of tumour cells

    PubMed Central

    Zeidler, R; Mysliwietz, J; Csánady, M; Walz, A; Ziegler, I; Schmitt, B; Wollenberg, B; Lindhofer, H

    2000-01-01

    Bispecific antibodies (bsAb) are considered as promising tools for the elimination of disseminated tumour cells in a minimal residual disease situation. The bsAb-mediated recruitment of an immune effector cell in close vicinity of a tumour cell is thought to induce an antitumoural immune response. However, classical bispecific molecules activate only a single class of immune effector cell that may not yield optimal immune responses. We therefore constructed an intact bispecific antibody, BiUII (anti-CD3 × anti-EpCAM), that not only recognizes tumour cells and T lymphocytes with its two binding arms, but also binds and activates Fcγ-receptor positive accessory cells through its Fc-region. We have demonstrated recently that activated accessory cells contribute to the bsAb-induced antitumoural activity. We now analyse this stimulation in more detail and demonstrate here the BiUll-induced upregulation of activation markers like CD83 and CD95 on accessory cells and the induction of neopterin and biopterin synthesis. Experiments with pure cell subpopulations revealed binding of BiUll to CD64+ accessory cells and CD16+ NK cells, but not to CD32+ B lymphocytes. We provide further evidence for the importance of the Fc-region in that this bispecific molecule stimulates Fcγ-R-positive accessory cells to eliminate tumour cells in vitro by direct phagocytosis. © 2000 Cancer Research Campaign PMID:10901380

  13. Antibody-mediated FOXP3 protein therapy induces apoptosis in cancer cells in vitro and inhibits metastasis in vivo.

    PubMed

    Heinze, Emil; Baldwin, Scott; Chan, Grace; Hansen, James; Song, Jason; Clements, Douglas; Aragon, Robert; Nishimura, Robert; Reeves, Mark; Weisbart, Richard

    2009-07-01

    In addition to its immune suppressive function in T-regulatory cells, the nuclear transcription factor, FOXP3, has been identified as a tumor suppressor. To evaluate the clinical efficacy of monoclonal antibody (mAb) 3E10 Fv antibody-mediated FOXP3 protein therapy of cancer, the Fv-FOXP3 fusion protein produced in Pichia pastoris was tested on breast, ovarian, and colon cancer cells in vitro, and with colon cancer cells in vivo in a mouse model of colon cancer metastasis to liver. Treatment with Fv-FOXP3 resulted in dose-dependent cell death of cancer cells in vitro. Apoptosis was established as a mechanism of cell death by demonstrating increased production of the p17 activated fragment of caspase-3 by cancer cells in response to Fv-FOXP3 and inhibition of cell killing by the caspase inhibitor, Z-VAD-FMK. Fv-FOXP3 treatment resulted in clinically significant reduction in tumor burden in a syngeneic model of colon cancer metastasis to liver in Balb/c mice. These results represent the first demonstration of effective full-length FOXP3 protein therapy and emphasize the clinical potential of mAb 3E10 as an intracellular and intranuclear delivery vehicle of FOXP3 for prevention and treatment of cancer metastasis.

  14. Differential timing of antibody-mediated phagocytosis and cell-free killing of invasive African Salmonella allows immune evasion.

    PubMed

    Siggins, Matthew K; O'Shaughnessy, Colette M; Pravin, John; Cunningham, Adam F; Henderson, Ian R; Drayson, Mark T; MacLennan, Calman A

    2014-04-01

    Nontyphoidal Salmonellae commonly cause fatal bacteraemia in African children lacking anti-Salmonella antibodies. These are facultative intracellular bacteria capable of cell-free and intracellular survival within macrophages. To better understand the relationship between extracellular and intracellular infection in blood and general mechanisms of Ab-related protection against Salmonella, we used human blood and sera to measure kinetics of Ab and complement deposition, serum-mediated bactericidal killing and phagocytosis of invasive African Salmonella enterica serovar Typhimurium D23580. Binding of antibodies peaked by 30 s, but C3 deposition lagged behind, peaking after 2-4 min. C5b-9 deposition was undetectable until between 2 and 6 min and peaked after 10 min, after which time an increase in serum-mediated killing occurred. In contrast, intracellular, opsonized Salmonellae were readily detectable within 5 min. By 10 min, around half of monocytes and most neutrophils contained bacteria. The same kinetics of serum-mediated killing and phagocytosis were observed with S. enterica Typhimurium laboratory strain SL1344, and the S. enterica Enteritidis African invasive isolate D24954 and laboratory strain PT4. The differential kinetics between cell-free killing and phagocytosis of invasive nontyphoidal Salmonella allows these bacteria to escape the blood and establish intracellular infection before they are killed by the membrane attack complex.

  15. One step antibody-mediated isolation and patterning of multiple cell types in microfluidic devices

    PubMed Central

    Bavli, Danny; Ezra, Elishai; Kitsberg, Daniel; Murthy, Shashi K.; Nahmias, Yaakov

    2016-01-01

    Cell-cell interactions play a key role in regeneration, differentiation, and basic tissue function taking place under physiological shear forces. However, current solutions to mimic such interactions by micro-patterning cells within microfluidic devices have low resolution, high fabrication complexity, and are limited to one or two cell types. Here, we present a microfluidic platform capable of laminar patterning of any biotin-labeled peptide using streptavidin-based surface chemistry. The design permits the generation of arbitrary cell patterns from heterogeneous mixtures in microfluidic devices. We demonstrate the robust co-patterning of α-CD24, α-ASGPR-1, and α-Tie2 antibodies for rapid isolation and co-patterning of mixtures of hepatocytes and endothelial cells. In addition to one-step isolation and patterning, our design permits step-wise patterning of multiple cell types and empty spaces to create complex cellular geometries in vitro. In conclusion, we developed a microfluidic device that permits the generation of perfusable tissue-like patterns in microfluidic devices by directly injecting complex cell mixtures such as differentiated stem cells or tissue digests with minimal sample preparation. PMID:27051469

  16. Fc-mediated activity of EGFR x c-Met bispecific antibody JNJ-61186372 enhanced killing of lung cancer cells.

    PubMed

    Grugan, Katharine D; Dorn, Keri; Jarantow, Stephen W; Bushey, Barbara S; Pardinas, Jose R; Laquerre, Sylvie; Moores, Sheri L; Chiu, Mark L

    2017-01-01

    Epidermal growth factor receptor (EGFR) mutant non-small cell lung cancers acquire resistance to EGFR tyrosine kinase inhibitors through multiple mechanisms including c-Met receptor pathway activation. We generated a bispecific antibody targeting EGFR and c-Met (JNJ-61186372) demonstrating anti-tumor activity in wild-type and mutant EGFR settings with c-Met pathway activation. JNJ-61186372 was engineered with low fucosylation (<10 %), resulting in enhanced antibody-dependent cell-mediated cytotoxicity and FcγRIIIa binding. In vitro and in vivo studies with the single-arm EGFR or c-Met versions of JNJ-61186372 identified that the Fc-activity of JNJ-61186372 is mediated by binding of the anti-EGFR arm and required for inhibition of EGFR-driven tumor cells. In a tumor model driven by both EGFR and c-Met, treatment with Fc-silent JNJ-61186372 or with c-Met single-arm antibody reduced tumor growth inhibition compared to treatment with JNJ-61186372, suggesting that the Fc function of JNJ-61186372 is essential for maximal tumor inhibition. Moreover in this same model, downregulation of both EGFR and c-Met receptors was observed upon treatment with Fc-competent JNJ-61186372, suggesting that the Fc interactions are necessary for down-modulation of the receptors in vivo and for efficacy. These Fc-mediated activities, in combination with inhibition of both the EGFR and c-Met signaling pathways, highlight the multiple mechanisms by which JNJ-61186372 combats therapeutic resistance in EGFR mutant patients.

  17. Fc-mediated activity of EGFR x c-Met bispecific antibody JNJ-61186372 enhanced killing of lung cancer cells

    PubMed Central

    Grugan, Katharine D.; Dorn, Keri; Bushey, Barbara S.; Pardinas, Jose R.; Laquerre, Sylvie; Moores, Sheri L.; Chiu, Mark L.

    2017-01-01

    ABSTRACT Epidermal growth factor receptor (EGFR) mutant non-small cell lung cancers acquire resistance to EGFR tyrosine kinase inhibitors through multiple mechanisms including c-Met receptor pathway activation. We generated a bispecific antibody targeting EGFR and c-Met (JNJ-61186372) demonstrating anti-tumor activity in wild-type and mutant EGFR settings with c-Met pathway activation. JNJ-61186372 was engineered with low fucosylation (<10 %), resulting in enhanced antibody-dependent cell-mediated cytotoxicity and FcγRIIIa binding. In vitro and in vivo studies with the single-arm EGFR or c-Met versions of JNJ-61186372 identified that the Fc-activity of JNJ-61186372 is mediated by binding of the anti-EGFR arm and required for inhibition of EGFR-driven tumor cells. In a tumor model driven by both EGFR and c-Met, treatment with Fc-silent JNJ-61186372 or with c-Met single-arm antibody reduced tumor growth inhibition compared to treatment with JNJ-61186372, suggesting that the Fc function of JNJ-61186372 is essential for maximal tumor inhibition. Moreover in this same model, downregulation of both EGFR and c-Met receptors was observed upon treatment with Fc-competent JNJ-61186372, suggesting that the Fc interactions are necessary for down-modulation of the receptors in vivo and for efficacy. These Fc-mediated activities, in combination with inhibition of both the EGFR and c-Met signaling pathways, highlight the multiple mechanisms by which JNJ-61186372 combats therapeutic resistance in EGFR mutant patients. PMID:27786612

  18. Complement and Antibody-mediated Enhancement of Red Blood Cell Invasion and Growth of Malaria Parasites.

    PubMed

    Biryukov, Sergei; Angov, Evelina; Landmesser, Mary E; Spring, Michele D; Ockenhouse, Christian F; Stoute, José A

    2016-07-01

    Plasmodium falciparum malaria is a deadly pathogen. The invasion of red blood cells (RBCs) by merozoites is a target for vaccine development. Although anti-merozoite antibodies can block invasion in vitro, there is no efficacy in vivo. To explain this discrepancy we hypothesized that complement activation could enhance RBC invasion by binding to the complement receptor 1 (CR1). Here we show that a monoclonal antibody directed against the merozoite and human polyclonal IgG from merozoite vaccine recipients enhanced RBC invasion in a complement-dependent manner and that soluble CR1 inhibited this enhancement. Sialic acid-independent strains, that presumably are able to bind to CR1 via a native ligand, showed less complement-dependent enhancement of RBC invasion than sialic acid-dependent strains that do not utilize native CR1 ligands. Confocal fluorescent microscopy revealed that complement-dependent invasion resulted in aggregation of CR1 at the RBC surface in contact with the merozoite. Finally, total anti-P. berghei IgG enhanced parasite growth and C3 deficiency decreased parasite growth in mice. These results demonstrate, contrary to current views, that complement activation in conjunction with antibodies can paradoxically aid parasites invade RBCs and should be considered in future design and testing of merozoite vaccines. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Antibody-mediated pure red cell aplasia (PRCA) on switching from darbepoetin alfa to epoetin beta: what are the implications?

    PubMed Central

    Assunção, José; Vinhas, José

    2008-01-01

    We report the development of antibody-mediated pure red cell aplasia (PRCA) in a 63-year-old man with end-stage renal disease following a switch from darbepoetin alfa to epoetin beta. Haemoglobin levels began to decrease 6 months after the switch. Increasing the epoetin beta dose produced no response and regular blood transfusions were required; PRCA was confirmed and epoetin beta was discontinued. The patient responded positively to immunosuppression; after 2 months on prednisone and cyclophosphamide, haemoglobin levels stabilized and no further transfusions were required. This case highlights the difficulty in establishing a cause-effect relationship where more than one erythropoiesis-stimulating agent is involved. PMID:25983889

  20. Antibody-mediated pure red cell aplasia (PRCA) on switching from darbepoetin alfa to epoetin beta: what are the implications?

    PubMed

    Assunção, José; Vinhas, José

    2008-08-01

    We report the development of antibody-mediated pure red cell aplasia (PRCA) in a 63-year-old man with end-stage renal disease following a switch from darbepoetin alfa to epoetin beta. Haemoglobin levels began to decrease 6 months after the switch. Increasing the epoetin beta dose produced no response and regular blood transfusions were required; PRCA was confirmed and epoetin beta was discontinued. The patient responded positively to immunosuppression; after 2 months on prednisone and cyclophosphamide, haemoglobin levels stabilized and no further transfusions were required. This case highlights the difficulty in establishing a cause-effect relationship where more than one erythropoiesis-stimulating agent is involved.

  1. Prospective Evaluation of Cetuximab-Mediated Antibody-Dependent Cell Cytotoxicity in Metastatic Colorectal Cancer Patients Predicts Treatment Efficacy.

    PubMed

    Trotta, Anna Maria; Ottaiano, Alessandro; Romano, Carmela; Nasti, Guglielmo; Nappi, Anna; De Divitiis, Chiara; Napolitano, Maria; Zanotta, Serena; Casaretti, Rossana; D'Alterio, Crescenzo; Avallone, Antonio; Califano, Daniela; Iaffaioli, Rosario Vincenzo; Scala, Stefania

    2016-04-01

    Cetuximab is a monoclonal antibody to the EGFR that induces antibody-dependent cell cytotoxicity (ADCC) through Fcγ receptors on immune cells. Although SNPs in genes encoding Fcγ receptors are functionally relevant to cetuximab-mediated ADCC in colorectal cancer, a direct correlation between in vitro ADCC and clinical response to cetuximab is not defined. We therefore enrolled 96 consecutive metastatic colorectal cancer (mCRC) patients at diagnosis in a study that assessed FcγR status and cetuximab-mediated ADCC. Patients carrying the FcγRIIa H alleles 131H/Hand 131H/R had significantly higher ADCC compared with patients with the 131R/R alleles (P= 0.013). Patients carrying FcγRIIIa genotypes with the V alleles 158V/V and 158V/F displayed higher ADCC compared with patients carrying the 158F/F genotype (P= 0.001). Progression-free survival of patients with an FcγRIIIa 158V allele was significantly longer compared with patients carrying 158F/F (P= 0.05), whereas no significant difference was observed for overall survival. Twenty-eight of 50 mCRC patients with wild-type KRAS received cetuximab. The average ADCC-mediated killing was 30% of assay targets for patients who experienced cetuximab complete or partial response, 21% in patients with stable disease and 9% in patients with progressive disease. To characterize basal natural killer (NK) activity, cytotoxicity was evaluated in 39 of 96 mCRC patients. Patients who responded to first-line treatment had higher NK-cell cytotoxicity. Thus, although limited to this cohort of patients, in vitro cetuximab-mediated ADCC correlated with FcγR polymorphisms and predicted cetuximab responsiveness. ©2016 American Association for Cancer Research.

  2. B cell receptor-mediated internalization of salmonella: a novel pathway for autonomous B cell activation and antibody production.

    PubMed

    Souwer, Yuri; Griekspoor, Alexander; Jorritsma, Tineke; de Wit, Jelle; Janssen, Hans; Neefjes, Jacques; van Ham, S Marieke

    2009-06-15

    The present paradigm is that primary B cells are nonphagocytosing cells. In this study, we demonstrate that human primary B cells are able to internalize bacteria when the bacteria are recognized by the BCR. BCR-mediated internalization of Salmonella typhimurium results in B cell differentiation and secretion of anti-Salmonella Ab by the Salmonella-specific B cells. In addition, BCR-mediated internalization leads to efficient Ag delivery to the MHC class II Ag-loading compartments, even though Salmonella remains vital intracellularly in primary B cells. Consequently, BCR-mediated bacterial uptake induces efficient CD4(+) T cell help, which boosts Salmonella-specific Ab production. BCR-mediated internalization of Salmonella by B cells is superior over extracellular Ag extraction to induce rapid and specific humoral immune responses and efficiently combat infection.

  3. Thaw-and-use target cells pre-labeled with calcein AM for antibody-dependent cell-mediated cytotoxicity assays.

    PubMed

    Chung, Shan; Nguyen, Van; Lin, Yuwen Linda; Kamen, Lynn; Song, An

    2017-08-01

    In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) assays are routinely performed to support the research and development of therapeutic antibodies. In ADCC assays, target cells bound by the antibodies are lysed by activated effector cells following interactions between the Fc region of the bound antibody and Fcγ receptors on effector cells. Target cell lysis is typically measured by quantification of released endogenous enzymes, e.g., lactate dehydrogenase, or measurement of released exogenous labels, e.g., (51)Cr, europium or calcein. ADCC assays based on the detection of exogenous labels released from lysed target cells generally show higher sensitivity and require shorter incubation times. However, target cells are usually labeled immediately prior to assay, which inadvertently introduces additional assay variations due to differences in target cell conditions and labeling/handling processes. In this report, we describe the use of thaw-and-use pre-labeled target cells for ADCC assays. Thaw-and-use target cells in our experiments were pre-labeled with the fluorescent dye calcein AM, cryopreserved in single-use aliquots and used directly in assays after thawing. Upon thaw, the pre-labeled cells displayed viability and label retention comparable to freshly labeled cells, responded to ADCC mediated by both peripheral blood mononuclear cells and engineered natural killer cells, performed stably for at least 3 years and provided favorable precision and accuracy to ADCC assays. Implementation of thaw-and-use pre-labeled target cells in ADCC assays can help to alleviate both cell culture and dye labeling derived variability, increase the flexibility of assay scheduling and improve assay consistency and robustness. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Myelin-reactive antibodies initiate T cell-mediated CNS autoimmune disease by opsonization of endogenous antigen.

    PubMed

    Kinzel, Silke; Lehmann-Horn, Klaus; Torke, Sebastian; Häusler, Darius; Winkler, Anne; Stadelmann, Christine; Payne, Natalie; Feldmann, Linda; Saiz, Albert; Reindl, Markus; Lalive, Patrice H; Bernard, Claude C; Brück, Wolfgang; Weber, Martin S

    2016-07-01

    In the pathogenesis of central nervous system (CNS) demyelinating disorders, antigen-specific B cells are implicated to act as potent antigen-presenting cells (APC), eliciting waves of inflammatory CNS infiltration. Here, we provide the first evidence that CNS-reactive antibodies (Ab) are similarly capable of initiating an encephalitogenic immune response by targeting endogenous CNS antigen to otherwise inert myeloid APC. In a transgenic mouse model, constitutive production of Ab against myelin oligodendrocyte glycoprotein (MOG) was sufficient to promote spontaneous experimental autoimmune encephalomyelitis (EAE) in the absence of B cells, when mice endogenously contained MOG-recognizing T cells. Adoptive transfer studies corroborated that anti-MOG Ab triggered activation and expansion of peripheral MOG-specific T cells in an Fc-dependent manner, subsequently causing EAE. To evaluate the underlying mechanism, anti-MOG Ab were added to a co-culture of myeloid APC and MOG-specific T cells. At otherwise undetected concentrations, anti-MOG Ab enabled Fc-mediated APC recognition of intact MOG; internalized, processed and presented MOG activated naïve T cells to differentiate in an encephalitogenic manner. In a series of translational experiments, anti-MOG Ab from two patients with an acute flare of CNS inflammation likewise facilitated detection of human MOG. Jointly, these observations highlight Ab-mediated opsonization of endogenous CNS auto-antigen as a novel disease- and/or relapse-triggering mechanism in CNS demyelinating disorders.

  5. Characterization of a novel bispecific antibody that mediates Fcgamma receptor type I-dependent killing of tumor-associated glycoprotein-72-expressing tumor cells.

    PubMed

    Russoniello, C; Somasundaram, C; Schlom, J; Deo, Y M; Keler, T

    1998-09-01

    A bispecific antibody was made by chemical conjugation of Fab' fragments from humanized antibodies specific for tumor-associated glycoprotein-72 (TAG-72) and high-affinity immunoglobulin receptor, FcgammaA receptor type I (FcgammaRI). The purified anti-TAG-72 x anti-FcgammaRI (HCC49xH22) bispecific antibody had an approximate Mr of 111,000, consistent with a F(ab')2, and bound specifically to KLEB and LS174T tumor cell lines, which express the TAG-72 tumor antigen. Furthermore, HCC49x H22 was shown to simultaneously bind to KLEB cells and a soluble FcgammaRI fusion protein, demonstrating the bifunctional nature of the molecule. Using IFN-gamma-treated monocytes as effector cells, concentrations of the bispecific antibody in the range of 1-10,000 ng/ml mediated specific lysis of TAG-72-positive tumor cells. In contrast, the bispecific antibody did not promote antibody-dependent cellular cytotoxicity of a cell line that was negative for TAG-72 antigen. Importantly, the antibody-dependent cellular cytotoxicity activity of the bispecific antibody was significantly greater than that of the monoclonal antibody HCC49. These in vitro data indicate that the humanized bispecific antibody HCC49xH22 has the appropriate specificity and functional activity for further evaluation as potential immunotherapy for TAG-72-positive malignancies.

  6. The Impact of HLA Class I-Specific Killer Cell Immunoglobulin-Like Receptors on Antibody-Dependent Natural Killer Cell-Mediated Cytotoxicity and Organ Allograft Rejection

    PubMed Central

    Rajalingam, Raja

    2016-01-01

    Natural killer (NK) cells of the innate immune system are cytotoxic lymphocytes that play an important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self-human leukocyte antigen (HLA) class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIRs) is involved in the calibration of NK cell effector capacities during the developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self-HLA class I (due to virus infection or tumor transformation) or HLA class I disparities (in the setting of allogeneic transplantation). NK cells expressing an inhibitory KIR-binding self-HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC), triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR–HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants. PMID:28066408

  7. The Impact of HLA Class I-Specific Killer Cell Immunoglobulin-Like Receptors on Antibody-Dependent Natural Killer Cell-Mediated Cytotoxicity and Organ Allograft Rejection.

    PubMed

    Rajalingam, Raja

    2016-01-01

    Natural killer (NK) cells of the innate immune system are cytotoxic lymphocytes that play an important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self-human leukocyte antigen (HLA) class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIRs) is involved in the calibration of NK cell effector capacities during the developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self-HLA class I (due to virus infection or tumor transformation) or HLA class I disparities (in the setting of allogeneic transplantation). NK cells expressing an inhibitory KIR-binding self-HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC), triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR-HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants.

  8. Antibody and cell-mediated immunity to Streptococcus pneumoniae: implications for vaccine development.

    PubMed

    Malley, Richard

    2010-02-01

    It has long been assumed that children develop natural immunity to pneumococci via the acquisition of anticapsular antibodies, which confers serotype-specific immunity to the organism. This view has been further reinforced by the recent success of capsular polysaccharide conjugate vaccines in children in reducing colonization and disease caused by vaccine-type strains. Less clear, however, is whether this mechanism is responsible for the age-related gradual increased resistance to pneumococcal carriage and disease. Recent epidemiologic and experimental evidence point to the possibility that another mechanism may be involved. Here, an alternative possibility is presented, whereby it is proposed that acquired immunity to this common human pathogen is derived not only from natural acquisition of antibodies (capsular and noncapsular) that provides protection against invasive disease but also from the development of pneumococcus-specific CD4+ T(H)17 cells that reduces the duration of carriage and may also impact mucosal disease. This review focuses on the experimental and clinical evidence in support of this hypothesis. The implications for future vaccine development against Streptococcus pneumoniae are also discussed.

  9. Early membrane rupture events during neutrophil-mediated antibody-dependent tumor cell cytolysis.

    PubMed

    Kindzelskii, A L; Petty, H R

    1999-03-15

    Although cell-mediated cytolysis is a fundamental immune effector response, its mechanism remains poorly understood at the cellular level. In this report, we image for the first time transient ruptures, as inferred by cytoplasmic marker release, in tumor cell membranes during Ab-dependent cellular cytolysis. The cytosol of IgG-opsonized YAC tumor cells was labeled with tetra-methylrhodamine diacetate followed by the formation of tumor cell-neutrophil conjugates. We hypothesized that tumor cell cytolysis proceeds via a series of discrete membrane rupture/resealing events that contribute to marker release. To test this hypothesis, we occluded the fluorescence image of the labeled tumor cells by passing an opaque disk into a field-conjugated plane between the light source and the sample. Multiple small bursts of fluorescent label release from tumor cells could be detected using a photomultiplier tube. Similarly, multiple fluorescent plumes were observed at various sites around the perimeter of a target. These findings support a multihit model of target cytolysis and suggest that cytolytic release is not focused at specific sites. Cytolytic bursts were generally observed at 20-s intervals, which match the previously described reduced nicotinamide-adenine dinucleotide phosphate and superoxide release oscillation periods for neutrophils; we speculate that metabolic oscillations of the effector cell drive the membrane damage of the target.

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

    PubMed

    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.

  11. The Strong In Vivo Anti-Tumor Effect of the UIC2 Monoclonal Antibody Is the Combined Result of Pgp Inhibition and Antibody Dependent Cell-Mediated Cytotoxicity

    PubMed Central

    Szalóki, Gábor; Krasznai, Zoárd T.; Tóth, Ágnes; Vízkeleti, Laura; Szöllősi, Attila G.; Trencsényi, György; Lajtos, Imre; Juhász, István; Krasznai, Zoltán; Márián, Teréz; Balázs, Margit; Szabó, Gábor; Goda, Katalin

    2014-01-01

    P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10–40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC50 of doxorubicin (DOX) in KB-V1 (Pgp+) cells in vitro almost to the level of KB-3-1 (Pgp-) cells. At the same time, UIC2 alone did not affect the EC50 value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp+ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp+ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased 18FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC). PMID:25238617

  12. Subclinical antibody-mediated rejection due to anti-human-leukocyte-antigen-DR53 antibody accompanied by plasma cell-rich acute rejection in a patient with cadaveric kidney transplantation.

    PubMed

    Katsuma, Ai; Yamamoto, Izumi; Komatsuzaki, Yo; Niikura, Takahito; Kawabe, Mayuko; Okabayashi, Yusuke; Yamakawa, Takafumi; Katsumata, Haruki; Nakada, Yasuyuki; Kobayashi, Akimitsu; Tanno, Yudo; Miki, Jun; Yamada, Hiroki; Ohkido, Ichiro; Tsuboi, Nobuo; Yamamoto, Hiroyasu; Yokoo, Takashi

    2016-07-01

    A 56-year-old man who had undergone cadaveric kidney transplantation 21 months earlier was admitted to our hospital for a protocol biopsy; he had a serum creatinine level of 1.2 mg/dL and no proteinuria. Histological features showed two distinct entities: (i) inflammatory cell infiltration, in the glomerular and peritubular capillaries and (ii) focal, aggressive tubulointerstitial inflammatory cell infiltration, predominantly plasma cells, with mild tubulitis (Banff 13 classification: i2, t1, g2, ptc2, v0, ci1, ct1, cg0, cv0). Immunohistological studies showed mildly positive C4d immunoreactivity in the peritubular capillaries. The patient had donor specific antibody to human-leucocyte-antigen-DR53. We diagnosed him with subclinical antibody-mediated rejection accompanied by plasma cell-rich acute rejection. Both antibody-mediated rejection due to anti- human-leucocyte-antigen -DR53 antibodies and plasma cell-rich acute rejection are known to be refractory and have a poor prognosis. Thus, we started plasma exchange with intravenous immunoglobulin and rituximab for the former and 3 days of consecutive steroid pulse therapy for the latter. Three months after treatment, a follow-up allograft biopsy showed excellent responses to treatment for both histological features. This case report considers the importance of an early diagnosis and appropriate intervention for subclinical antibody-mediated rejection due to donor specific antibody to human-leucocyte-antigen-DR53 and plasma cell-rich acute rejection.

  13. Protooncogene bcl-2 gene transfer abrogates Fas/APO-1 antibody-mediated apoptosis of human malignant glioma cells and confers resistance to chemotherapeutic drugs and therapeutic irradiation.

    PubMed Central

    Weller, M; Malipiero, U; Aguzzi, A; Reed, J C; Fontana, A

    1995-01-01

    The majority of human malignant glioma cells express Fas/APO-1 and are susceptible to Fas/APO-1 antibody-mediated apoptosis in vitro. The sensitivity of Fas/APO-1-positive glioma cell lines to Fas/APO-1 antibody-mediated killing correlates inversely with the constitutive expression of the antiapoptotic protooncogene bcl-2. Here we report that BCL-2 protein expression of human glial tumors in vivo correlates with malignant transformation in that BCL-2 immunoreactive glioma cells were more abundant in WHO grade III/IV gliomas than in grade I/II gliomas. Fas/APO-1 antibody-sensitive human glioma cell lines stably transfected with a murine bcl-2 cDNA acquired resistance to Fas/APO-1 antibody-mediated apoptosis. Forced expression of bcl-2 also attenuated TNF alpha-mediated cytotoxicity of glioma cell lines in the presence of actinomycin D and cycloheximide and conferred partial protection from irradiation and the cancer chemotherapy drugs, cisplatin and BCNU. Preexposure of the glioma cell lines to the cytokines, IFN gamma and TNF alpha, which sensitize for Fas/APO-1-dependent killing, partially overcame bcl-2-mediated rescue from apoptosis, suggesting that multimodality immunotherapy involving cytokines and Fas/APO-1 targeting might eventually provide a promising approach to the treatment of human malignant gliomas. Images PMID:7539458

  14. C-type lectin-like molecule-1 (CLL1)-targeted TRAIL augments the tumoricidal activity of granulocytes and potentiates therapeutic antibody-dependent cell-mediated cytotoxicity.

    PubMed

    Wiersma, Valerie R; de Bruyn, Marco; Shi, Ce; Gooden, Marloes J M; Wouters, Maartje C A; Samplonius, Douwe F; Hendriks, Djoke; Nijman, Hans W; Wei, Yunwei; Zhou, Jin; Helfrich, Wijnand; Bremer, Edwin

    2015-01-01

    The therapeutic effect of anti-cancer monoclonal antibodies stems from their capacity to opsonize targeted cancer cells with subsequent phagocytic removal, induction of antibody-dependent cell-mediated cytotoxicity (ADCC) or induction of complement-mediated cytotoxicity (CDC). The major immune effector cells involved in these processes are natural killer (NK) cells and granulocytes. The latter and most prevalent blood cell population contributes to phagocytosis, but is not effective in inducing ADCC. Here, we report that targeted delivery of the tumoricidal protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to granulocyte marker C-type lectin-like molecule-1 (CLL1), using fusion protein CLL1:TRAIL, equips granulocytes with high levels of TRAIL. Upon CLL1-selective binding of this fusion protein, granulocytes acquire additional TRAIL-mediated cytotoxic activity that, importantly, potentiates antibody-mediated cytotoxicity of clinically used therapeutic antibodies (e.g., rituximab, cetuximab). Thus, CLL1:TRAIL could be used as an adjuvant to optimize the clinical potential of anticancer antibody therapy by augmenting tumoricidal activity of granulocytes.

  15. Pathogenesis and mechanisms of antibody-mediated hemolysis

    PubMed Central

    Flegel, Willy A

    2015-01-01

    Background The clinical consequences of antibodies to red blood cells (RBC) have been studied for a century. Most clinically relevant antibodies can be detected by sensitive in vitro assays. Several mechanisms of antibody-mediated hemolysis are well understood. Such hemolysis following transfusion is reliably avoided in a donor/recipient pair, if one individual is negative for the cognate antigen to which the other has the antibody. Study design and results Mechanisms of antibody-mediated hemolysis were reviewed based on a presentation at the Strategies to Address Hemolytic Complications of Immune Globulin Infusions Workshop addressing intravenous immunoglobulin (IVIG) and ABO antibodies. The presented topics included the rates of intravascular and extravascular hemolysis; IgM and IgG isoagglutinins; auto- and alloantibodies; antibody specificity; A, B, A,B and A1 antigens; A1 versus A2 phenotypes; monocytes/macrophages, other immune cells and complement; monocyte monolayer assay (MMA); antibody-dependent cell-mediated cytotoxicity (ADCC); and transfusion reactions due to ABO and other antibodies. Conclusion Several clinically relevant questions remained unresolved, and diagnostic tools were lacking to routinely and reliably predict the clinical consequences of RBC antibodies. Most hemolytic transfusion reactions associated with IVIG were due to ABO antibodies. Reducing the titers of such antibodies in IVIG may lower the frequency of this kind of adverse event. The only way to stop these events is to have no anti-A or anti-B antibodies in the IVIG products. PMID:26174897

  16. Cutting edge: An antibody recognizing ancestral endogenous virus glycoproteins mediates antibody-dependent cellular cytotoxicity on HIV-1-infected cells.

    PubMed

    Michaud, Henri-Alexandre; SenGupta, Devi; de Mulder, Miguel; Deeks, Steven G; Martin, Jeffrey N; Kobie, James J; Sacha, Jonah B; Nixon, Douglas F

    2014-08-15

    The failure of antiviral vaccines is often associated with rapid viral escape from specific immune responses. In the past, conserved epitope or algorithmic epitope selections, such as mosaic vaccines, have been designed to diversify immunity and to circumvent potential viral escape. An alternative approach is to identify conserved stable non-HIV-1 self-epitopes present exclusively in HIV-1-infected cells. We showed previously that human endogenous retroviral (HERV) mRNA transcripts and protein are found in cells of HIV-1-infected patients and that HERV-K (HML-2)-specific T cells can eliminate HIV-1-infected cells in vitro. In this article, we demonstrate that a human anti-HERV-K (HML-2) transmembrane protein Ab binds specifically to HIV-1-infected cells and eliminates them through an Ab-dependent cellular cytotoxicity mechanism in vitro. Thus, Abs directed against epitopes other than HIV-1 proteins may have a role in eliminating HIV-1-infected cells and could be targeted in novel vaccine approaches or immunotherapeutic modalities.

  17. Macrophage and NK-mediated killing of precursor-B acute lymphoblastic leukemia cells targeted with a-fucosylated anti-CD19 humanized antibodies.

    PubMed

    Matlawska-Wasowska, K; Ward, E; Stevens, S; Wang, Y; Herbst, R; Winter, S S; Wilson, B S

    2013-06-01

    This work reports the tumoricidal effects of a novel investigational humanized anti-CD19 monoclonal antibody (Medi-551). An a-fucosylated antibody with increased affinity for human FcγRIIIA, Medi-551 is shown to mediate both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Medi-551/CD19 complexes internalize slowly (>5 h) and thus remain accessible to effector cells for prolonged periods. We evaluated in vitro ADCC and ADCP activities of primary human natural killer (NK) cells and macrophages against precursor-B (pre-B) acute lymphoblastic leukemia (ALL) cell lines and pediatric patient blasts. Fluorescent imaging studies document immunological synapses formed between anti-CD19-bound target leukemia cells and effector cells and capture the kinetics of both NK-mediated killing and macrophage phagocytosis. Genetic polymorphisms in FcγRIIIA-158F/V modulate in vitro activities of effector cells, with FcγRIIIA-158V homozygotes or heterozygotes showing the strongest activity. Medi-551 treatment of severe combined immunodeficiency (SCID) mice engrafted with human pre-B cells led to prolonged animal survival and markedly reduced disease burden in blood, liver and bone marrow. These data show that anti-CD19 antibodies effectively recruit immune cells to pre-B ALL cells and support a move forward to early phase trials in this disease.

  18. Antibody-mediated pure red cell aplasia (PRCA): ensuring future progress by collecting data from a registry.

    PubMed

    Aljama, Pedro; Carracedo, Julia; Madueño, Juan Antonio; Ramirez, Rafael; Martin-Malo, Alejandro; Martin de Francisco, Angel L

    2005-05-01

    The recent emergence of antibody (Ab)-mediated pure red cell aplasia (PRCA) resulting from administration of certain erythropoiesis-stimulating agents (ESAs) has heightened awareness of the potential for this disorder in patients receiving ESAs for anaemia associated with chronic kidney disease (CKD). The Spanish Society of Nephrology sponsored an independent registry for analysis of patients who developed epoetin-induced, Ab-mediated PRCA in Spain. Twelve patients from 11 regional hospitals were included in the Spanish PRCA registry from November 2000 to December 2002 that met the criteria for Ab-mediated PRCA. Patients were reported using a standardized form specifically developed for PRCA, and serum samples were analysed in a central laboratory at Reina Sofia University Hospital in Cordoba, Spain. The characteristics of these patients, their serological and haematological results, and the outcomes of immunosuppressive therapies are presented. The Spanish PRCA registry serves as a model for establishing an independent global PRCA registry sponsored by the various nephrology societies in European Union countries, and elsewhere and coordinated by key investigators from the respective countries.

  19. Assays for detecting and diagnosing antibody-mediated pure red cell aplasia (PRCA): an assessment of available procedures.

    PubMed

    Thorpe, Robin; Swanson, Steven J

    2005-05-01

    Antibody (Ab)-mediated pure red cell aplasia (PRCA) develops when patients mount a neutralizing Ab response to recombinant erythropoiesis-stimulating agents (ESAs) such as epoetin-alpha (EPO). These neutralizing Abs can also cross-neutralize endogenous EPO, leading to a state of absolute EPO resistance and transfusion dependence. The diagnosis of Ab-mediated PRCA in part relies on the sensitive and specific detection of serum anti-EPO Abs and a confirmatory examination of patients' bone marrow for lack of erythroid precursors. To date, a variety of assays have been used to detect anti-EPO Abs, including radioimmunoprecipitation (RIP) assay, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) and bioassays that measure neutralizing Abs. Each of these assays can yield informative results and possess characteristic benefits and limitations, so it is unclear whether or not a "superior" assay is required or possible. To date, a universal standardized assay has not yet been established that would facilitate the comparison of Ab data derived from different laboratories and retrospective analysis of stored sera. This review evaluates the results of studies measuring anti-EPO Abs with different assays and compares their relative advantages and disadvantages in terms of specificity, sensitivity, ease of use and ability to measure Ab-binding affinities and subclasses. These comparisons provide a basis for determining the optimal assay(s) for screening and/or analysis of patients' serum for anti-EPO Abs during treatment or after onset of Ab-mediated PRCA.

  20. Fenretinide sensitizes multidrug-resistant human neuroblastoma cells to antibody-independent and ch14.18-mediated NK cell cytotoxicity.

    PubMed

    Shibina, Anastasia; Seidel, Diana; Somanchi, Srinivas S; Lee, Dean A; Stermann, Alexander; Maurer, Barry J; Lode, Holger N; Reynolds, C Patrick; Huebener, Nicole

    2013-04-01

    Neuroblastoma (NB) is the most common extracranial solid tumor in children. Combining passive immunotherapy with an antibody to the disialoganglioside GD2 (ch14.18/SP2/0) and cytokines with 13-cis-retinoic acid for post-myeloablative maintenance therapy increased survival in high-risk NB, but the overall prognosis for these children is still in need of improvement. Fenretinide (4-HPR) is a synthetic retinoid that has shown clinical activity in recurrent NB and is cytotoxic to a variety of cancer cells, in part via the accumulation of dihydroceramides, which are precursors of GD2. We investigated the effect of 4-HPR on CHO-derived, ch14.18-mediated anti-NB effector functions, complement-dependent cytotoxicity (CDC), and antibody-dependent and antibody-independent cellular cytotoxicity (ADCC and AICC, respectively). Here, we demonstrate for the first time that pretreatment of fenretinide-resistant NB cells with 4-HPR significantly enhanced ch14.18/CHO-mediated CDC and ADCC and AICC by both human natural killer cells and peripheral blood mononuclear cells. Treatment with 4-HPR increased GD2 and death receptor (DR) expression in resistant NB cells and induced an enhanced granzyme B and perforin production by effector cells. Blocking of ganglioside synthesis with a glucosylceramide synthase inhibitor abrogated the increased ADCC response but had no effect on the AICC, indicating that GD2 induced by 4-HPR mediates the sensitization of NB cells for ADCC. We also showed that 4-HPR induced increased GD2 and DR expression in a resistant NB xenograft model that was associated with an increased ADCC and AICC response using explanted tumor target cells from 4-HPR-treated mice. In summary, these findings provide an important baseline for the combination of 4-HPR and passive immunotherapy with ch14.18/CHO in future clinical trials for high-risk NB patients.

  1. FcRn-mediated antibody transport across epithelial cells revealed by electron tomography

    PubMed Central

    He, Wanzhong; Ladinsky, Mark S.; Huey-Tubman, Kathryn E.; Jensen, Grant J.; McIntosh, J. Richard; Björkman, Pamela J.

    2009-01-01

    The neonatal Fc receptor (FcRn) transports maternal IgG across epithelial barriers1,2, thereby providing the fetus or newborn with humoral immunity before its immune system is fully functional. In newborn rodents, FcRn transfers IgG from milk to blood by apical-to-basolateral transcytosis across intestinal epithelial cells. The pH difference between the apical (pH 6.0-6.5) and basolateral (pH 7.4) sides of intestinal epithelial cells facilitates efficient unidirectional transport of IgG, since FcRn binds IgG at pH 6.0-6.5 but not pH ≥7 1,2. As milk passes through the neonatal intestine, maternal IgG is removed by FcRn-expressing cells in the proximal small intestine (duodenum, jejunum); remaining proteins are absorbed and degraded by FcRn-negative cells in the distal small intestine (ileum)3-6. We used electron tomography to directly visualize jejunal transcytosis in space and time, developing new labeling and detection methods to map individual nanogold-labeled Fc within transport vesicles7 and to simultaneously characterize these vesicles by immunolabeling. Combining electron tomography with a non-perturbing endocytic label allowed us to conclusively identify receptor-bound ligands, resolve interconnecting vesicles, determine if a vesicle was microtubule-associated, and accurately trace FcRn-mediated transport of IgG. Our results present a complex picture in which Fc moved through networks of entangled tubular and irregular vesicles, only some of which were microtubule-associated, as it migrated to the basolateral surface. New features of transcytosis were elucidated, including transport involving multivesicular body inner vesicles/tubules and exocytosis via clathrin-coated pits. Markers for early, late, and recycling endosomes each labeled vesicles in different and overlapping morphological classes, revealing unexpected spatial complexity in endo-lysosomal trafficking. PMID:18818657

  2. Automated in situ measurement of cell-specific antibody secretion and laser-mediated purification for rapid cloning of highly-secreting producers.

    PubMed

    Hanania, Elie G; Fieck, Annabeth; Stevens, Janine; Bodzin, Leon J; Palsson, Bernhard Ø; Koller, Manfred R

    2005-09-30

    Cloning of highly-secreting recombinant cells is critical for biopharmaceutical manufacturing, but faces numerous challenges including the fact that secreted protein does not remain associated with the producing cell. A fundamentally new approach was developed combining in situ capture and measurement of individual cell protein secretion followed by laser-mediated elimination of all non- and poorly-secreting cells, leaving only the highest-secreting cell in a well. Recombinant cells producing humanized antibody were cultured serum-free on a capture matrix, followed by staining with fluorescently-labeled anti-human antibody fragment. A novel, automated, high-throughput instrument (called LEAP) was used to image and locate every cell, quantify the cell-associated and secreted antibody (surrounding each cell), eliminate all undesired cells from a well via targeted laser irradiation, and then track clone outgrowth and stability. Temporarily sparing an island of helper cells around the clone of interest improved cloning efficiency (particularly when using serum-free medium), and helper cells were easily eliminated with the laser after several days. The in situ nature of this process allowed several serial sub-cloning steps to be performed within days of one another, resulting in rapid generation of clonal populations with significantly increased and more stable, homogeneous antibody secretion. Cell lines with specific antibody secretion rates of > 50 pg/cell per day (in static batch culture) were routinely obtained as a result of this cloning approach, often times representing up to 20% of the clones screened.

  3. The CD25-binding antibody Daclizumab High-Yield Process has a distinct glycosylation pattern and reduced antibody-dependent cell-mediated cytotoxicity in comparison to Zenapax®

    PubMed Central

    Ganguly, Bishu; Balasa, Balaji; Efros, Lyubov; Hinton, Paul R.; Hartman, Stephen; Thakur, Archana; Xiong, Joanna M.; Schmidt, Brian; Robinson, Randy R.; Sornasse, Thierry; Vexler, Vladimir; Sheridan, James P.

    2016-01-01

    ABSTRACT The CD25-binding antibody daclizumab high-yield process (DAC HYP) is an interleukin (IL)-2 signal modulating antibody that shares primary amino acid sequence and CD25 binding affinity with Zenapax®, a distinct form of daclizumab, which was approved for the prevention of acute organ rejection in patients receiving renal transplants as part of an immunosuppressive regimen that includes cyclosporine and corticosteroids. Comparison of the physicochemical properties of the two antibody forms revealed the glycosylation profile of DAC HYP differs from Zenapax in both glycan distribution and the types of oligosaccharides, most notably high-mannose, galactosylated and galactose-α-1,3-galactose (α-Gal) oligosaccharides, resulting in a DAC HYP antibody material that is structurally distinct from Zenapax. Although neither antibody elicited complement-dependent cytotoxicity in vitro, DAC HYP antibody had significantly reduced levels of antibody-dependent cell-mediated cytotoxicity (ADCC). The ADCC activity required natural killer (NK) cells, but not monocytes, suggesting the effects were mediated through binding to Fc-gamma RIII (CD16). Incubation of each antibody with peripheral blood mononuclear cells also caused the down-modulation of CD16 expression on NK cells and the CD16 down-modulation was greater for Zenapax in comparison to that observed for DAC HYP. The substantive glycosylation differences between the two antibody forms and corresponding greater Fc-mediated effector activities by Zenapax, including cell killing activity, manifest as a difference in the biological function and pharmacology between DAC HYP and Zenapax. PMID:27367933

  4. Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding.

    PubMed

    Borrok, M Jack; Luheshi, Nadia M; Beyaz, Nurten; Davies, Gareth C; Legg, James W; Wu, Herren; Dall'Acqua, William F; Tsui, Ping

    2015-01-01

    Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.

  5. IL1RAP antibodies block IL-1-induced expansion of candidate CML stem cells and mediate cell killing in xenograft models.

    PubMed

    Ågerstam, Helena; Hansen, Nils; von Palffy, Sofia; Sandén, Carl; Reckzeh, Kristian; Karlsson, Christine; Lilljebjörn, Henrik; Landberg, Niklas; Askmyr, Maria; Högberg, Carl; Rissler, Marianne; Porkka, Kimmo; Wadenvik, Hans; Mustjoki, Satu; Richter, Johan; Järås, Marcus; Fioretos, Thoas

    2016-12-08

    Chronic myeloid leukemia (CML) is currently treated with tyrosine kinase inhibitors, but these do not effectively eliminate the CML stem cells. As a consequence, CML stem cells persist and cause relapse in most patients upon drug discontinuation. Furthermore, no effective therapy exists for the advanced stages of the disease. Interleukin-1 receptor accessory protein (IL1RAP; IL1R3) is a coreceptor of interleukin-1 receptor type 1 and has been found upregulated on CML stem cells. Here, we show that primitive (CD34(+)CD38(-)) CML cells, in contrast to corresponding normal cells, express a functional interleukin-1 (IL-1) receptor complex and respond with NF-κB activation and marked proliferation in response to IL-1. IL1RAP antibodies that inhibit IL-1 signaling could block these effects. In vivo administration of IL1RAP antibodies in mice transplanted with chronic and blast phase CML cells resulted in therapeutic effects mediated by murine effector cells. These results provide novel insights into the role of IL1RAP in CML and a strong rationale for the development of an IL1RAP antibody therapy to target residual CML stem cells. © 2016 by The American Society of Hematology.

  6. Ligation of human Fc receptor like-2 by monoclonal antibodies down-regulates B-cell receptor-mediated signalling

    PubMed Central

    Shabani, Mahdi; Bayat, Ali Ahmad; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Hojjat-Farsangi, Mohammad; Ulivieri, Cristina; Amirghofran, Zahra; Baldari, Cosima Tatiana; Shokri, Fazel

    2014-01-01

    B-cell antigen receptor (BCR) signalling and its regulation through negative and positive regulators are critical for balancing B-cell response and function. Human Fc receptor like-2 (FCRL2), a member of the newly identified FCRL family, could influence B-cell signalling due to possession of both immunoreceptor tyrosine-based activation and inhibitory motifs (ITAM and ITIM). Since the natural ligand of FCRL2 has not been identified, we generated FCRL2-specific monoclonal antibodies (mAbs) and employed them to investigate the influence of FCRL2 stimulation on BCR signalling in an FCRL2-expressing B-cell line. Two anti-FCRL2 mAb-producing hybridoma clones (5A7-E7 and 3D8-G8) were selected. None of the mAbs displayed any cross-reactivity with the other members of the FCRL family including recombinant FCRL1, -3, -4 and -5, as tested by FACS and ELISA techniques. Engagement of the FCRL2 by these mAbs resulted in significant inhibition of BCR signalling mediators such as calcium mobilization and phosphorylation of the mitogen-activated protein kinases Erk, p38 and Jnk. These findings indicate that the FCRL2 ITIM motifs are functional and the anti-FCRL2 mAbs may mimic the natural ligand of FCRL2 by induction of inhibitory signals in B cells. PMID:24797767

  7. Comparison of the effectiveness of antibody and cell-mediated immunity against inhaled and instilled influenza virus challenge.

    PubMed

    Rivers, Katie; Bowen, Larry E; Gao, Jin; Yang, Kevin; Trombley, John E; Bohannon, J Kyle; Eichelberger, Maryna C

    2013-06-19

    To evaluate immunity against influenza, mouse challenge studies are typically performed by intranasal instillation of a virus suspension to anesthetized animals. This results in an unnatural environment in the lower respiratory tract during infection, and therefore there is some concern that immune mechanisms identified in this model may not reflect those that protect against infectious virus particles delivered directly to the lower respiratory tract as an aerosol. To evaluate differences in protection against instilled and inhaled virus, mice were immunized with influenza antigens known to induce antibody or cell-mediated responses and then challenged with 100 LD50 A/PR/8/34 (PR8) in the form of aerosol (inhaled) or liquid suspension (instilled). Mice immunized with recombinant adenovirus (Ad) expressing hemagglutinin were protected against weight loss and death in both challenge models, however immunization with Ad expressing nucleoprotein of influenza A (NPA) or M2 resulted in greater protection against inhaled aerosolized virus than virus instilled in liquid suspension. Ad-M2, but not Ad-NPA-immunized mice were protected against a lower instillation challenge dose. These results demonstrate differences in protection that are dependent on challenge method, and suggest that cell-mediated immunity may be more accurately demonstrated in mouse inhalation studies. Furthermore, the data suggest immune mechanisms generally characterized as incomplete or weak in mouse models using liquid intranasal challenge may offer greater immunity against influenza infection than previously thought.

  8. Comparison of the effectiveness of antibody and cell-mediated immunity against inhaled and instilled influenza virus challenge

    PubMed Central

    2013-01-01

    Background To evaluate immunity against influenza, mouse challenge studies are typically performed by intranasal instillation of a virus suspension to anesthetized animals. This results in an unnatural environment in the lower respiratory tract during infection, and therefore there is some concern that immune mechanisms identified in this model may not reflect those that protect against infectious virus particles delivered directly to the lower respiratory tract as an aerosol. Method To evaluate differences in protection against instilled and inhaled virus, mice were immunized with influenza antigens known to induce antibody or cell-mediated responses and then challenged with 100 LD50 A/PR/8/34 (PR8) in the form of aerosol (inhaled) or liquid suspension (instilled). Results Mice immunized with recombinant adenovirus (Ad) expressing hemagglutinin were protected against weight loss and death in both challenge models, however immunization with Ad expressing nucleoprotein of influenza A (NPA) or M2 resulted in greater protection against inhaled aerosolized virus than virus instilled in liquid suspension. Ad-M2, but not Ad-NPA-immunized mice were protected against a lower instillation challenge dose. Conclusions These results demonstrate differences in protection that are dependent on challenge method, and suggest that cell-mediated immunity may be more accurately demonstrated in mouse inhalation studies. Furthermore, the data suggest immune mechanisms generally characterized as incomplete or weak in mouse models using liquid intranasal challenge may offer greater immunity against influenza infection than previously thought. PMID:23777453

  9. In vivo gene targeting of IL-3 into immature hematopoietic cells through CD117 receptor mediated antibody gene delivery

    PubMed Central

    Chapel, Alain; Deas, Olivier; Bensidhoum, Morad; François, Sabine; Mouiseddine, Moubarak; Poncet, Pascal; Dürrbach, Antoine; Aigueperse, Jocelyne; Gourmelon, Patrick; Gorin, Norbert C; Hirsch, François; Thierry, Dominique

    2004-01-01

    Background Targeted gene transfection remains a crucial issue to permit the real development of genetic therapy. As such, in vivo targeted transfection of specific subsets of hematopoietic stem cells might help to sustain hematopoietic recovery from bone marrow aplasia by providing local production of growth factors. Methods Balb/C mice were injected intravenously, with an anti-mouse c-kit (CD117) monoclonal antibody chemically coupled to a human IL-3 gene-containing plasmid DNA. Mice were sacrificed for tissue analyses at various days after injection of the conjugates. Results By ELISA, the production of human IL-3 was evidenced in the sera of animals 5 days after treatment. Cytofluorometric analysis after in vivo transfection of a reporter gene eGFP demonstrated transfection of CD117+/Sca1+ hematopoietic immature cells. By PCR analysis of genomic DNA and RNA using primer specific pIL3 sequences, presence and expression of the human IL-3-transgene were detected in the bone marrow up to 10 days in transfected mice but not in control animals. Conclusions These data clearly indicate that antibody-mediated endocytosis gene transfer allows the expression of the IL-3 transgene into hematopoietic immature cells, in vivo. While availability of marketed recombinant growth factors is restricted, this targeting strategy should permit delivery of therapeutic genes to tissues of interest through systemic delivery. In particular, the ability to specifically target growth factor expression into repopulating hematopoietic stem cells may create new opportunities for the treatment of primary or radiation-induced marrow failures. PMID:15509303

  10. Matrix Metalloprotease Inhibitors Restore Impaired NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Human Immunodeficiency Virus Type 1 Infection ▿

    PubMed Central

    Liu, Qingquan; Sun, Yongtao; Rihn, Suzannah; Nolting, Anne; Tsoukas, Peter Nicholas; Jost, Stephanie; Cohen, Kristen; Walker, Bruce; Alter, Galit

    2009-01-01

    Increasing evidence suggests that NK cells not only are critical in the initial host defense against pathogens but also may contribute to continued protection from human immunodeficiency virus type 1 (HIV-1) disease progression. NK cell cytolysis can be induced directly through diverse receptor families or can be induced indirectly through Fc receptors by antibodies mediating antibody-dependent cellular cytotoxicity (ADCC). ADCC has been implicated in both protection from simian immunodeficiency virus infection and slower progression of HIV-1 disease. ADCC activity declines with advancing infection, and yet the underlying mechanism for this dysfunction has not been defined, nor has it been determined whether the activity can be reconstituted. Here we demonstrate that NK cell-mediated ADCC is severely compromised in chronic HIV infection. The potency of ADCC function was directly correlated with baseline FcγRIIIa receptor (CD16) expression on NK cells. CD16 expression was negatively influenced by elevated expression of a group of enzymes, the matrix metalloproteinases (MMPs), normally involved in tissue/receptor remodeling. Inhibition of MMPs resulted in increased CD16 expression and augmented ADCC activity in response to antibody-coated target cells. These data suggest that MMP inhibitors may improve NK cell-mediated ADCC, which may provide subjects with an opportunity to harness the cytolytic power of NK cells through naturally occurring nonneutralizing HIV-specific antibodies. PMID:19553339

  11. Matrix metalloprotease inhibitors restore impaired NK cell-mediated antibody-dependent cellular cytotoxicity in human immunodeficiency virus type 1 infection.

    PubMed

    Liu, Qingquan; Sun, Yongtao; Rihn, Suzannah; Nolting, Anne; Tsoukas, Peter Nicholas; Jost, Stephanie; Cohen, Kristen; Walker, Bruce; Alter, Galit

    2009-09-01

    Increasing evidence suggests that NK cells not only are critical in the initial host defense against pathogens but also may contribute to continued protection from human immunodeficiency virus type 1 (HIV-1) disease progression. NK cell cytolysis can be induced directly through diverse receptor families or can be induced indirectly through Fc receptors by antibodies mediating antibody-dependent cellular cytotoxicity (ADCC). ADCC has been implicated in both protection from simian immunodeficiency virus infection and slower progression of HIV-1 disease. ADCC activity declines with advancing infection, and yet the underlying mechanism for this dysfunction has not been defined, nor has it been determined whether the activity can be reconstituted. Here we demonstrate that NK cell-mediated ADCC is severely compromised in chronic HIV infection. The potency of ADCC function was directly correlated with baseline Fc gammaRIIIa receptor (CD16) expression on NK cells. CD16 expression was negatively influenced by elevated expression of a group of enzymes, the matrix metalloproteinases (MMPs), normally involved in tissue/receptor remodeling. Inhibition of MMPs resulted in increased CD16 expression and augmented ADCC activity in response to antibody-coated target cells. These data suggest that MMP inhibitors may improve NK cell-mediated ADCC, which may provide subjects with an opportunity to harness the cytolytic power of NK cells through naturally occurring nonneutralizing HIV-specific antibodies.

  12. Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia.

    PubMed

    Paszkiewicz, Paulina J; Fräßle, Simon P; Srivastava, Shivani; Sommermeyer, Daniel; Hudecek, Michael; Drexler, Ingo; Sadelain, Michel; Liu, Lingfeng; Jensen, Michael C; Riddell, Stanley R; Busch, Dirk H

    2016-11-01

    The adoptive transfer of T cells that have been genetically modified to express a CD19-specific chimeric antigen receptor (CAR) is effective for treating human B cell malignancies. However, the persistence of functional CD19 CAR T cells causes sustained depletion of endogenous CD19+ B cells and hypogammaglobulinemia. Thus, there is a need for a mechanism to ablate transferred T cells after tumor eradication is complete to allow recovery of normal B cells. Previously, we developed a truncated version of the epidermal growth factor receptor (EGFRt) that is coexpressed with the CAR on the T cell surface. Here, we show that targeting EGFRt with the IgG1 monoclonal antibody cetuximab eliminates CD19 CAR T cells both early and late after adoptive transfer in mice, resulting in complete and permanent recovery of normal functional B cells, without tumor relapse. EGFRt can be incorporated into many clinical applications to regulate the survival of gene-engineered cells. These results support the concept that EGFRt represents a promising approach to improve safety of cell-based therapies.

  13. Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia

    PubMed Central

    Paszkiewicz, Paulina J.; Fräßle, Simon P.; Srivastava, Shivani; Sommermeyer, Daniel; Hudecek, Michael; Sadelain, Michel; Liu, Lingfeng; Jensen, Michael C.; Riddell, Stanley R.; Busch, Dirk H.

    2016-01-01

    The adoptive transfer of T cells that have been genetically modified to express a CD19-specific chimeric antigen receptor (CAR) is effective for treating human B cell malignancies. However, the persistence of functional CD19 CAR T cells causes sustained depletion of endogenous CD19+ B cells and hypogammaglobulinemia. Thus, there is a need for a mechanism to ablate transferred T cells after tumor eradication is complete to allow recovery of normal B cells. Previously, we developed a truncated version of the epidermal growth factor receptor (EGFRt) that is coexpressed with the CAR on the T cell surface. Here, we show that targeting EGFRt with the IgG1 monoclonal antibody cetuximab eliminates CD19 CAR T cells both early and late after adoptive transfer in mice, resulting in complete and permanent recovery of normal functional B cells, without tumor relapse. EGFRt can be incorporated into many clinical applications to regulate the survival of gene-engineered cells. These results support the concept that EGFRt represents a promising approach to improve safety of cell-based therapies. PMID:27760047

  14. Mechanisms involved in antibody- and complement-mediated allograft rejection

    PubMed Central

    2010-01-01

    Antibody-mediated rejection has become critical clinically because this form of rejection is usually unresponsive to conventional anti-rejection therapy, and therefore, it has been recognized as a major cause of allograft loss. Our group developed experimental animal models of vascularized organ transplantation to study pathogenesis of antibody- and complement-mediated endothelial cell injury leading to graft rejection. In this review, we discuss mechanisms of antibody-mediated graft rejection resulting from activation of complement by C1q- and MBL (mannose-binding lectin)-dependent pathways and interactions with a variety of effector cells, including macrophages and monocytes through Fcγ receptors and complement receptors. PMID:20135240

  15. Correlation Between Rubella Antibody Levels and Cytokine Measures of Cell-Mediated Immunity

    PubMed Central

    Tosh, Pritish K.; Kennedy, Richard B.; Vierkant, Robert A.; Jacobson, Robert M.

    2009-01-01

    Abstract Despite a safe and effective vaccine, endemic rubella remains a problem in developing countries. Isolated cases and outbreaks can occur in areas with high vaccine coverage. Individuals, especially pregnant women who remain unimmunized or do not seroconvert, are susceptible to infection and their infants are at risk for congenital rubella syndrome (CRS). Both humoral and cellular immune responses contribute to immune protection. Classically, immunity to rubella has been assessed through the detection of rubella-specific antibody titers. In this study we examined correlates of both humoral and cellular immunity in a large population of immunized young adults in Olmsted County, MN. We were unable to find any significant correlation between cytokine production after in-vitro rubella stimulation and serum antibody titers. PMID:19951182

  16. Plasma cell-rich rejection accompanied by acute antibody-mediated rejection in a patient with ABO-incompatible kidney transplantation.

    PubMed

    Furuya, Maiko; Yamamoto, Izumi; Kobayashi, Akimitsu; Nakada, Yasuyuki; Sugano, Naoki; Tanno, Yudo; Ohkido, Ichiro; Tsuboi, Nobuo; Yamamoto, Hiroyasu; Yokoyama, Keitaro; Yokoo, Takashi

    2014-06-01

    We report a case of plasma cell-rich rejection accompanied by acute antibody-mediated rejection in a patient with ABO-incompatible kidney transplantation. A 33-year-old man was admitted for an episode biopsy; he had a serum creatinine (S-Cr) level of 5.7 mg/dL 1 year following primary kidney transplantation. Histological features included two distinct entities: (1) a focal, aggressive tubulointerstitial inflammatory cell (predominantly plasma cells) infiltration with moderate tubulitis; and (2) inflammatory cell infiltration (including neutrophils) in peritubular capillaries. Substantial laboratory examination showed that the patient had donor-specific antibodies for DQ4 and DQ6. Considering both the histological and laboratory findings, we diagnosed him with plasma cell-rich rejection accompanied by acute antibody-mediated rejection. We started 3 days of consecutive steroid pulse therapy three times every 2 weeks for the former and plasma exchange with intravenous immunoglobulin (IVIG) for the latter histological feature. One month after treatment, a second allograft biopsy showed excellent responses to treatment for plasma cell-rich rejection, but moderate, acute antibody-mediated rejection remained. Therefore, we added plasma exchange with IVIG again. After treatment, allograft function was stable, with an S-Cr level of 2.8 mg/dL. This case report demonstrates the difficulty of the diagnosis of, and treatment for, plasma cell-rich rejection accompanied by acute antibody-mediated rejection in a patient with ABO-incompatible kidney transplantation. We also include a review of the related literature.

  17. Antibody-mediated rejection, T cell-mediated rejection, and the injury-repair response: new insights from the Genome Canada studies of kidney transplant biopsies.

    PubMed

    Halloran, Philip F; Reeve, Jeff P; Pereira, Andre B; Hidalgo, Luis G; Famulski, Konrad S

    2014-02-01

    Prospective studies of unselected indication biopsies from kidney transplants, combining conventional assessment with molecular analysis, have created a new understanding of transplant disease states and their outcomes. A large-scale Genome Canada grant permitted us to use conventional and molecular phenotypes to create a new disease classification. T cell-mediated rejection (TCMR), characterized histologically or molecularly, has little effect on outcomes. Antibody-mediated rejection (ABMR) manifests as microcirculation lesions and transcript changes reflecting endothelial injury, interferon-γ effects, and natural killer cells. ABMR is frequently C4d negative and has been greatly underestimated by conventional criteria. Indeed, ABMR, triggered in some cases by non-adherence, is the major disease causing failure. Progressive dysfunction is usually attributable to specific diseases, and pure calcineurin inhibitor toxicity rarely explains failure. The importance of ABMR argues against immunosuppressive drug minimization and stands as a barrier to tolerance induction. Microarrays also defined the transcripts induced by acute kidney injury (AKI), which correlate with reduced function, whereas histologic changes of acute tubular injury do not. AKI transcripts are induced in kidneys with late dysfunction, and are better predictors of failure than fibrosis and inflammation. Thus progression reflects ongoing parenchymal injury, usually from identifiable diseases such as ABMR, not destructive fibrosis.

  18. Antibody induced CD4 down-modulation of T cells is site-specifically mediated by CD64+ cells

    PubMed Central

    Vogel, Stephanie; Grabski, Elena; Buschjäger, Daniela; Klawonn, Frank; Döring, Marius; Wang, Junxi; Fletcher, Erika; Bechmann, Ingo; Witte, Torsten; Durisin, Martin; Schraven, Burkhart; Mangsbo, Sara M.; Schönfeld, Kurt; Czeloth, Niklas; Kalinke, Ulrich

    2015-01-01

    Treatment of PBMC with the CD4-specific mAb BT-061 induces CD4 down-modulation of T cells. Here we report that addition of BT-061 to purified T cells did not confer this effect, whereas incubation of T cells in BT-061 coated wells restored CD4 down-modulation. These results implied that Fcγ receptor mediated cell-cell interactions played a role. In consistence with this hypothesis PBMC depleted of CD64+ monocytes did not confer CD4 down-modulation of BT-061 decorated T cells. Strikingly, CD4 down-modulation was observed in BT-061 treated synovial fluid punctuated from patients’ inflamed joints that comprised enhanced numbers of CD64+ cells. In contrast, in a circulating whole blood system injection of BT-061 did not induce CD4 down-modulation, due to CD64 saturation by serum IgG. Similarly, tonsil derived mononuclear cells devoid of CD64+ cells did not show CD4 down-modulation, whereas addition of blood derived monocytes restored the effect. Thus, the interaction of BT-061 decorated T cells with CD64+ cells is needed for CD4 down-modulation, implying that in patients BT-061 would primarily induce CD4 down-modulation at inflammatory sites. These results highlight the need not only to examine the interaction of a given mAb with single FcγR, but also the immunological environment that is appropriate to support such interactions. PMID:26670584

  19. Antibody induced CD4 down-modulation of T cells is site-specifically mediated by CD64(+) cells.

    PubMed

    Vogel, Stephanie; Grabski, Elena; Buschjäger, Daniela; Klawonn, Frank; Döring, Marius; Wang, Junxi; Fletcher, Erika; Bechmann, Ingo; Witte, Torsten; Durisin, Martin; Schraven, Burkhart; Mangsbo, Sara M; Schönfeld, Kurt; Czeloth, Niklas; Kalinke, Ulrich

    2015-12-16

    Treatment of PBMC with the CD4-specific mAb BT-061 induces CD4 down-modulation of T cells. Here we report that addition of BT-061 to purified T cells did not confer this effect, whereas incubation of T cells in BT-061 coated wells restored CD4 down-modulation. These results implied that Fcγ receptor mediated cell-cell interactions played a role. In consistence with this hypothesis PBMC depleted of CD64(+) monocytes did not confer CD4 down-modulation of BT-061 decorated T cells. Strikingly, CD4 down-modulation was observed in BT-061 treated synovial fluid punctuated from patients' inflamed joints that comprised enhanced numbers of CD64(+) cells. In contrast, in a circulating whole blood system injection of BT-061 did not induce CD4 down-modulation, due to CD64 saturation by serum IgG. Similarly, tonsil derived mononuclear cells devoid of CD64(+) cells did not show CD4 down-modulation, whereas addition of blood derived monocytes restored the effect. Thus, the interaction of BT-061 decorated T cells with CD64(+) cells is needed for CD4 down-modulation, implying that in patients BT-061 would primarily induce CD4 down-modulation at inflammatory sites. These results highlight the need not only to examine the interaction of a given mAb with single FcγR, but also the immunological environment that is appropriate to support such interactions.

  20. The combination of trastuzumab and pertuzumab administered at approved doses may delay development of trastuzumab resistance by additively enhancing antibody-dependent cell-mediated cytotoxicity.

    PubMed

    Tóth, Gábor; Szöőr, Árpád; Simon, László; Yarden, Yosef; Szöllősi, János; Vereb, György

    2016-10-01

    Although the recently concluded CLEOPATRA trial showed clinical benefits of combining trastuzumab and pertuzumab for treating HER2-positive metastatic breast cancer, trastuzumab monotherapy is still the mainstay in adjuvant settings. Since trastuzumab resistance occurs in over half of these cancers, we examined the mechanisms by which treatment of intrinsically trastuzumab-resistant and -sensitive tumors can benefit from the combination of these antibodies. F(ab')2 of both trastuzumab and pertuzumab were generated and validated in order to separately analyze antibody-dependent cell-mediated cytotoxicity (ADCC)-based and direct biological effects of the antibodies. Compared to monotherapy, combination of the two antibodies at clinically permitted doses enhanced the recruitment of natural killer cells responsible for ADCC, and significantly delayed the outgrowth of xenografts from intrinsically trastuzumab-resistant JIMT-1 cells. Antibody dose-response curves of in vitro ADCC showed that antibody-mediated killing can be saturated, and the two antibodies exert an additive effect at sub-saturation doses. Thus, the additive effect in vivo indicates that therapeutic tissue levels likely do not saturate ADCC. Additionally, isobole studies with the in vitro trastuzumab-sensitive BT-474 cells showed that the direct biological effect of combined treatment is additive, and surpasses the maximum effect of either monotherapy. Our results suggest the combined therapy is expected to give results that are superior to monotherapy, whatever the type of HER2-positive tumor may be. The combination of both antibodies at maximum clinically approved doses should thus be administered to patients to recruit maximum ADCC and cause maximum direct biological growth inhibition.

  1. The combination of trastuzumab and pertuzumab administered at approved doses may delay development of trastuzumab resistance by additively enhancing antibody-dependent cell-mediated cytotoxicity

    PubMed Central

    Tóth, Gábor; Szöőr, Árpád; Simon, László; Yarden, Yosef; Szöllősi, János; Vereb, György

    2016-01-01

    ABSTRACT Although the recently concluded CLEOPATRA trial showed clinical benefits of combining trastuzumab and pertuzumab for treating HER2-positive metastatic breast cancer, trastuzumab monotherapy is still the mainstay in adjuvant settings. Since trastuzumab resistance occurs in over half of these cancers, we examined the mechanisms by which treatment of intrinsically trastuzumab-resistant and -sensitive tumors can benefit from the combination of these antibodies. F(ab′)2 of both trastuzumab and pertuzumab were generated and validated in order to separately analyze antibody-dependent cell-mediated cytotoxicity (ADCC)-based and direct biological effects of the antibodies. Compared to monotherapy, combination of the two antibodies at clinically permitted doses enhanced the recruitment of natural killer cells responsible for ADCC, and significantly delayed the outgrowth of xenografts from intrinsically trastuzumab-resistant JIMT-1 cells. Antibody dose-response curves of in vitro ADCC showed that antibody-mediated killing can be saturated, and the two antibodies exert an additive effect at sub-saturation doses. Thus, the additive effect in vivo indicates that therapeutic tissue levels likely do not saturate ADCC. Additionally, isobole studies with the in vitro trastuzumab-sensitive BT-474 cells showed that the direct biological effect of combined treatment is additive, and surpasses the maximum effect of either monotherapy. Our results suggest the combined therapy is expected to give results that are superior to monotherapy, whatever the type of HER2-positive tumor may be. The combination of both antibodies at maximum clinically approved doses should thus be administered to patients to recruit maximum ADCC and cause maximum direct biological growth inhibition. PMID:27380003

  2. Anti-Fas/APO-1 antibody-mediated apoptosis of cultured human glioma cells. Induction and modulation of sensitivity by cytokines.

    PubMed Central

    Weller, M; Frei, K; Groscurth, P; Krammer, P H; Yonekawa, Y; Fontana, A

    1994-01-01

    Fas/APO-1 is a transmembrane protein of the nerve growth factor/TNF alpha receptor family which signals apoptotic cell death in susceptible target cells. We have investigated the susceptibility of seven human malignant glioma cell lines to Fas/APO-1-dependent apoptosis. Sensitivity to Fas/APO-1 antibody-mediated cell killing correlated with cell surface expression of Fas/APO-1. Expression of Fas/APO-1 as well as Fas/APO-1-dependent cytotoxicity were augmented by preexposure of human malignant glioma cells to IFN gamma and TNF alpha. Further, pretreatment with TGF beta 2, IL1 and IL8 enhanced Fas/APO-1 antibody-induced glioma cell apoptosis whereas other cytokines including TNF beta, IL6, macrophage colony-stimulating factor, IL10 and IL13 had no such effect. None of the human malignant glioma cell lines was susceptible to TNF alpha-induced cytotoxicity. Fas/APO-1 antibody-sensitive glioma cell lines (n = 5), but not Fas/APO-1 antibody-resistant glioma cell lines (n = 2), became sensitive to TNF alpha when co-treated with inhibitors of RNA and protein synthesis. Resistance of human glioma cells to Fas/APO-1 antibody-mediated apoptosis was mainly related to low level expression of Fas/APO-1 and appeared not to be linked to overexpression of the anti-apoptotic protooncogene, bcl-2. Given the resistance of human malignant glioma to surgery, irradiation, chemotherapy and immunotherapy, we propose that Fas/APO-1 may be a promising target for a novel locoregionary approach to human malignant glioma. This strategy gains support from the demonstration of Fas/APO-1 expression in ex vivo human malignant glioma specimens and from the absence of Fas/APO-1 in normal human brain parenchyma. Images PMID:7521890

  3. Phenotypical and functional profiles of natural killer cells exhibiting matrix metalloproteinase-mediated CD16 cleavage after anti-HIV antibody-dependent activation.

    PubMed

    Tang, C-C; Isitman, G; Bruneau, J; Tremblay, C; Bernard, N F; Kent, S J; Parsons, M S

    2015-08-01

    Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) has been linked to protection from HIV infection and slower progression towards AIDS. However, antibody-dependent activation of NK cells results in phenotypical alterations similar to those observed on NK cells from individuals with progressive HIV infection. Activation of NK cells induces matrix metalloproteinase (MMP)-mediated cleavage of cell surface CD16. In the present study we assessed the phenotype and functional profile of NK cells exhibiting post-activation MMP-mediated CD16 cleavage. We found that NK cells achieving the highest levels of activation during stimulation exhibit the most profound decreases in CD16 expression. Further, we observed that educated KIR3DL1(+) NK cells from human leucocyte antigen (HLA)-Bw4-carrying donors exhibit larger decreases in CD16 expression post-activation than the KIR3DL1(-) NK cell subset containing cells educated via other inhibitory receptor/ligand combinations and non-educated NK cells. Lastly, we assessed the ex-vivo expression of CD16 on educated KIR3DL1(+) NK cells and the KIR3DL1(-) NK cell subset from HLA-Bw4-carrying HIV-uninfected and HIV-infected donors. Suggestive of in-vivo activation of KIR3DL1(+) NK cells during HIV infection, CD16 expression was higher on KIR3DL1(+) than KIR3DL1(-) NK cells in uninfected donors but similar on both subsets in HIV-infected donors. These results are discussed in the context of how they may assist with understanding HIV disease progression and the design of immunotherapies that utilize antibody-dependent NK cell responses.

  4. Control of Immune Response to Allogeneic Embryonic Stem Cells by CD3 Antibody-Mediated Operational Tolerance Induction.

    PubMed

    Calderon, D; Prot, M; You, S; Marquet, C; Bellamy, V; Bruneval, P; Valette, F; de Almeida, P; Wu, J C; Pucéat, M; Menasché, P; Chatenoud, L

    2016-02-01

    Implantation of embryonic stem cells (ESCs) and their differentiated derivatives into allogeneic hosts triggers an immune response that represents a hurdle to clinical application. We established in autoimmunity and in transplantation that CD3 antibody therapy induces a state of immune tolerance. Promising results have been obtained with CD3 antibodies in the clinic. In this study, we tested whether this strategy can prolong the survival of undifferentiated ESCs and their differentiated derivatives in histoincompatible hosts. Recipients of either mouse ESC-derived embryoid bodies (EBs) or cardiac progenitors received a single short tolerogenic regimen of CD3 antibody. In immunocompetent mice, allogeneic EBs and cardiac progenitors were rejected within 20-25 days. Recipients treated with CD3 antibody showed long-term survival of implanted cardiac progenitors or EBs. In due course, EBs became teratomas, the growth of which was self-limited. Regulatory CD4(+)FoxP3(+) T cells and signaling through the PD1/PDL1 pathway played key roles in the CD3 antibody therapeutic effect. Gene profiling emphasized the importance of TGF-β and the inhibitory T cell coreceptor Tim3 to the observed effect. These results demonstrate that CD3 antibody administered alone promotes prolonged survival of allogeneic ESC derivatives and thus could prove useful for enhancing cell engraftment in the absence of chronic immunosuppression. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

  5. Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

    PubMed Central

    Shen, Yang; Zeng, Lin; Zhu, Aiping; Blanc, Tim; Patel, Dipa; Pennello, Anthony; Bari, Amtul; Ng, Stanley; Persaud, Kris; Kang, Yun (Kenneth); Balderes, Paul; Surguladze, David; Hindi, Sagit; Zhou, Qinwei; Ludwig, Dale L.; Snavely, Marshall

    2013-01-01

    Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies. PMID:23567210

  6. Tissue resident NK cells mediate ischemic kidney injury and are not depleted by anti-Asialo GM1 antibody

    PubMed Central

    Victorino, Francisco; Sojka, Dorothy K.; Brodsky, Kelley S.; McNamee, Eoin N.; Masterson, Joanne C.; Homann, Dirk; Yokoyama, Wayne M.; Eltzschig, Holger K.; Clambey, Eric T.

    2015-01-01

    NK cells are innate lymphoid cells important for immune surveillance, identifying and responding to stress, infection, and/or transformation. While conventional NK (cNK) cells circulate systemically, many NK cells reside in tissues where they appear to be poised to locally regulate tissue function. Here we tested the contribution of tissue-resident NK (trNK) cells to tissue homeostasis by studying ischemic injury in the mouse kidney. Parabiosis experiments demonstrate that the kidney contains a significant fraction of trNK cells under homeostatic conditions. Kidney trNK cells developed independent of NFIL3 and Tbet, and expressed a distinct cell surface phenotype as compared to cNK cells. Among these, trNK cells had reduced asialo-GM1 (AsGM1) expression relative to cNK cells, a phenotype observed in trNK cells across multiple organs and mouse strains. Strikingly, anti-AsGM1 antibody treatment, commonly used as NK cell-depleting regimen, resulted in a robust and selective depletion of cNKs, leaving trNKs largely intact. Using this differential depletion, we tested the relative contribution of cNK and trNK cells in ischemic kidney injury. Whereas anti-NK1.1 antibody effectively depleted both trNK and cNK cells and protected against ischemic-reperfusion injury, anti-AsGM1 antibody preferentially depleted cNK cells and failed to protect against injury. These data demonstrate unanticipated specificity of anti-AsGM1 antibody depletion on NK cell subsets and reveal a new approach to study the contributions of cNK and trNK cells in vivo. In total, these data demonstrate that trNK cells play a key role in modulating local responses to ischemic tissue injury in the kidney and potentially other organs. PMID:26453755

  7. Immobilized Surfactant-Nanotube Complexes Support Selectin-Mediated Capture of Viable Circulating Tumor Cells in the Absence of Capture Antibodies

    PubMed Central

    Mitchell, Michael J.; Castellanos, Carlos A.; King, Michael R.

    2015-01-01

    The metastatic spread of tumor cells from the primary site to anatomically distant organs leads to a poor patient prognosis. Increasing evidence has linked adhesive interactions between circulating tumor cells (CTCs) and endothelial cells to metastatic dissemination. Microscale biomimetic flow devices hold promise as a diagnostic tool to isolate CTCs and develop metastatic therapies, utilizing E-selectin (ES) to trigger the initial rolling adhesion of tumor cells under flow. To trigger firm adhesion and capture under flow, such devices also typically require antibodies against biomarkers thought to be expressed on CTCs. This approach is challenged by the fact that CTCs are now known to exhibit heterogeneous expression of conventional biomarkers. Here, we describe surfactant-nanotube complexes to enhance ES-mediated capture and isolation of tumor cells without the use of capture antibodies. While the majority of tumor cells exhibited weaker rolling adhesion on halloysite nanotubes (HNT) coated with ES, HNT functionalization with the sodium dodecanoate (NaL) surfactant induced a switch to firm cellular adhesion under flow. Conversely, surfactant-nanotube complexes significantly reduced the number of primary human leukocytes captured via ES-mediated adhesion under flow. The switch in tumor cell adhesion was exploited to capture and isolate tumor cells in the absence of EpCAM antibodies, commonly utilized as the gold standard for CTC isolation. Additionally, HNT-NaL complexes were shown to capture tumor cells with low to negligible EpCAM expression, that are not efficiently captured using conventional approaches. PMID:25761664

  8. Enhancing Virion Tethering by BST2 Sensitizes Productively and Latently HIV-infected T cells to ADCC Mediated by Broadly Neutralizing Antibodies

    PubMed Central

    Pham, Tram N. Q.; Lukhele, Sabelo; Dallaire, Frédéric; Perron, Gabrielle; Cohen, Éric A.

    2016-01-01

    Binding of anti-HIV antibodies (Abs) to envelope (Env) glycoproteins on infected cells can mark them for elimination via antibody-dependent cell-mediated cytotoxicity (ADCC). BST2, a type I interferon (IFN)-stimulated restriction factor that anchors nascent Env-containing virions at the surface of infected cells has been shown to enhance ADCC functions. In a comprehensive analysis of ADCC potency by neutralizing anti-HIV Abs (NAbs), we show in this study that NAbs are capable of mediating ADCC against HIV-infected T cells with 3BNC117, PGT126 and PG9 being most efficient. We demonstrate that HIV-induced BST2 antagonism effectively attenuates Ab binding and ADCC responses mediated by all classes of NAbs that were tested. Interestingly, IFNα treatment can reverse this effect in a BST2-dependent manner. Importantly, while reactivated latent T cell lines display some susceptibility to ADCC mediated by broadly NAbs, inactivating BST2 viral countermeasures and/or exogenous IFNα augment their elimination. Overall, our findings support the notion that NAbs can induce ADCC. They highlight that while BST2 antagonism by HIV promotes ADCC evasion, strategies aimed at restoring BST2 restriction could improve anti-HIV responses and potentially provide a means to eliminate reactivated cells in latent reservoirs. PMID:27853288

  9. Antibody-Mediated Lung Transplant Rejection

    PubMed Central

    Hachem, Ramsey

    2012-01-01

    Antibody-mediated rejection after lung transplantation remains enigmatic. However, emerging evidence over the past several years suggests that humoral immunity plays an important role in allograft rejection. Indeed, the development of donor-specific antibodies after transplantation has been identified as an independent risk factor for acute cellular rejection and bronchiolitis obliterans syndrome. Furthermore, cases of acute antibody-mediated rejection resulting in severe allograft dysfunction have been reported, and these demonstrate that antibodies can directly injure the allograft. However, the incidence and toll of antibody-mediated rejection are unknown because there is no widely accepted definition and some cases may be unrecognized. Clearly, humoral immunity has become an important area for research and clinical investigation. PMID:23002428

  10. Preclinical characterization of 1-7F9, a novel human anti–KIR receptor therapeutic antibody that augments natural killer–mediated killing of tumor cells

    PubMed Central

    André, Pascale; Spee, Pieter; Zahn, Stefan; Anfossi, Nicolas; Gauthier, Laurent; Capanni, Marusca; Ruggeri, Loredana; Benson, Don M.; Blaser, Bradley W.; Della Chiesa, Mariella; Moretta, Alessandro; Vivier, Eric; Caligiuri, Michael A.; Velardi, Andrea

    2009-01-01

    Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK) cell–mediated killing of HLA class I–expressing tumors. Lack of KIR-HLA class I interactions has been associated with potent NK-mediated antitumor efficacy and increased survival in acute myeloid leukemia (AML) patients upon haploidentical stem cell transplantation from KIR-mismatched donors. To exploit this pathway pharmacologically, we generated a fully human monoclonal antibody, 1-7F9, which cross-reacts with KIR2DL1, -2, and -3 receptors, and prevents their inhibitory signaling. The 1-7F9 monoclonal antibody augmented NK cell–mediated lysis of HLA-C–expressing tumor cells, including autologous AML blasts, but did not induce killing of normal peripheral blood mononuclear cells, suggesting a therapeutic window for preferential enhancement of NK-cell cytotoxicity against malignant target cells. Administration of 1-7F9 to KIR2DL3-transgenic mice resulted in dose-dependent rejection of HLA-Cw3–positive target cells. In an immunodeficient mouse model in which inoculation of human NK cells alone was unable to protect against lethal, autologous AML, preadministration of 1-7F9 resulted in long-term survival. These data show that 1-7F9 confers specific, stable blockade of KIR, boosting NK-mediated killing of HLA-matched AML blasts in vitro and in vivo, providing a preclinical basis for initiating phase 1 clinical trials with this candidate therapeutic antibody. PMID:19553639

  11. An antibody against the surfactant protein A (SP-A)-binding domain of the SP-A receptor inhibits T cell-mediated immune responses to Mycobacterium tuberculosis.

    PubMed

    Samten, Buka; Townsend, James C; Sever-Chroneos, Zvjezdana; Pasquinelli, Virginia; Barnes, Peter F; Chroneos, Zissis C

    2008-07-01

    Surfactant protein A (SP-A) suppresses lymphocyte proliferation and IL-2 secretion, in part, by binding to its receptor, SP-R210. However, the mechanisms underlying this effect are not well understood. Here, we studied the effect of antibodies against the SP-A-binding (neck) domain (alpha-SP-R210n) or nonbinding C-terminal domain (alpha-SP-R210ct) of SP-R210 on human peripheral blood T cell immune responses against Mycobacterium tuberculosis. We demonstrated that both antibodies bind to more than 90% of monocytes and 5-10% of CD3+ T cells in freshly isolated PBMC. Stimulation of PBMC from healthy tuberculin reactors [purified protein derivative-positive (PPD+)] with heat-killed M. tuberculosis induced increased antibody binding to CD3+ cells. Increased antibody binding suggested enhanced expression of SP-R210, and this was confirmed by Western blotting. The antibodies (alpha-SP-R210n) cross-linking the SP-R210 through the SP-A-binding domain markedly inhibited cell proliferation and IFN-gamma secretion by PBMC from PPD+ donors in response to heat-killed M. tuberculosis, whereas preimmune IgG and antibodies (alpha-SP-R210ct) cross-linking SP-R210 through the non-SP-A-binding, C-terminal domain had no effect. Anti-SP-R210n also decreased M. tuberculosis-induced production of TNF-alpha but increased production of IL-10. Inhibition of IFN-gamma production by alpha-SP-R210n was abrogated by the combination of neutralizing antibodies to IL-10 and TGF-beta1. Together, these findings support the hypothesis that SP-A, via SP-R210, suppresses cell-mediated immunity against M. tuberculosis via a mechanism that up-regulates secretion of IL-10 and TGF-beta1.

  12. Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003

    PubMed Central

    Valenzuela, Nicole M.; Thomas, Kimberly A.; Mulder, Arend; Parry, Graham C.; Panicker, Sandip; Reed, Elaine F.

    2017-01-01

    Background Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. Methods Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). Results Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. Conclusions Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR. PMID:28640789

  13. Red Blood Cell Antibody Identification

    MedlinePlus

    ... name: Red Blood Cell Antibody Identification Related tests: Direct Antiglobulin Test ; RBC Antibody Screen ; Blood Typing ; Type ... a positive RBC antibody screen or a positive direct antiglobulin test (DAT) . It is used to identify ...

  14. A Humanized Anti-CD22-Onconase Antibody-Drug Conjugate Mediates Highly Potent Destruction of Targeted Tumor Cells

    PubMed Central

    Weber, Tobias; Mavratzas, Athanasios; Kiesgen, Stefan; Haase, Stephanie; Bötticher, Benedikt; Exner, Evelyn; Mier, Walter; Grosse-Hovest, Ludger; Jäger, Dirk; Arndt, Michaela A. E.; Krauss, Jürgen

    2015-01-01

    Antibody-drug conjugates (ADCs) have evolved as a new class of potent cancer therapeutics. We here report on the development of ADCs with specificity for the B-cell lineage specific (surface) antigen CD22 being expressed in the majority of hematological malignancies. As targeting moiety a previously generated humanized anti-CD22 single-chain variable fragment (scFv) derivative from the monoclonal antibody RFB4 was reengineered into a humanized IgG1 antibody format (huRFB4). Onconase (ranpirnase), a clinically active pancreatic-type ribonuclease, was employed as cytotoxic payload moiety. Chemical conjugation via thiol-cleavable disulfide linkage retained full enzymatic activity and full binding affinity of the ADC. Development of sophisticated purification procedures using size exclusion and ion exchange chromatography allowed the separation of immunoconjugate species with stoichiometrically defined number of Onconase cargos. A minimum of two Onconase molecules per IgG was required for achieving significant in vitro cytotoxicity towards lymphoma and leukemia cell lines. Antibody-drug conjugates with an Onconase to antibody ratio of 3 : 1 exhibited an IC50 of 0.08 nM, corresponding to more than 18,400-fold increased cytotoxicity of the ADC when compared with unconjugated Onconase. These results justify further development of this ADC as a promising first-in-class compound for the treatment of CD22-positive malignancies. PMID:26605343

  15. Measles Virus Neutralizing Antibody Response, Cell-Mediated Immunity, and Immunoglobulin G Antibody Avidity Before and After Receipt of a Third Dose of Measles, Mumps, and Rubella Vaccine in Young Adults.

    PubMed

    Fiebelkorn, Amy Parker; Coleman, Laura A; Belongia, Edward A; Freeman, Sandra K; York, Daphne; Bi, Daoling; Kulkarni, Ashwin; Audet, Susette; Mercader, Sara; McGrew, Marcia; Hickman, Carole J; Bellini, William J; Shivakoti, Rupak; Griffin, Diane E; Beeler, Judith

    2016-04-01

    Two doses of measles, mumps, and rubella (MMR) vaccine are 97% effective against measles, but waning antibody immunity to measles and failure of the 2-dose vaccine occur. We administered a third MMR dose (MMR3) to young adults and assessed immunogenicity over 1 year. Measles virus (MeV) neutralizing antibody concentrations, cell-mediated immunity (CMI), and immunoglobulin G (IgG) antibody avidity were assessed at baseline and 1 month and 1 year after MMR3 receipt. Of 662 subjects at baseline, 1 (0.2%) was seronegative for MeV-neutralizing antibodies (level, <8 mIU/mL), and 23 (3.5%) had low antibody levels (8-120 mIU/mL). One month after MMR3 receipt, 1 subject (0.2%) was seronegative, and 6 (0.9%) had low neutralizing antibodies, with only 21 of 662 (3.2%) showing a ≥ 4-fold rise in neutralizing antibodies. One year after MMR3 receipt, no subject was seronegative, and 10 of 617 (1.6%) had low neutralizing antibody levels. CMI analyses showed low levels of spot-forming cells after stimulation, suggesting the presence of T-cell memory, but the response was minimal after MMR3 receipt. MeV IgG avidity did not correlate with findings of neutralization analyses. Most subjects were seropositive before MMR3 receipt, and very few had a secondary immune response after MMR3 receipt. Similarly, CMI and avidity analyses showed minimal qualitative improvements in immune response after MMR3 receipt. We did not find compelling data to support a routine third dose of MMR vaccine. Published by Oxford University Press for the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  16. Intravital imaging reveals improved Kupffer cell-mediated phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies

    PubMed Central

    Grandjean, Capucine L.; Montalvao, Fabricio; Celli, Susanna; Michonneau, David; Breart, Beatrice; Garcia, Zacarias; Perro, Mario; Freytag, Olivier; Gerdes, Christian A.; Bousso, Philippe

    2016-01-01

    Anti-CD20 monoclonal antibodies (mAbs) represent an effective treatment for a number of B cell malignancies and autoimmune disorders. Glycoengineering of anti-CD20mAb may contribute to increased anti-tumor efficacy through enhanced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) as reported by in vitro studies. However, where and how glycoengineered Ab may potentiate therapeutic responses in vivo is yet to be elucidated. Here, we have performed mouse liver transplants to demonstrate that the liver is sufficient to mediate systemic B cells depletion after anti-CD20 treatment. Relying on intravital two-photon imaging of human CD20-expressing mice, we provide evidence that ADP by Kupffer cells (KC) is a major mechanism for rituximab-mediated B cell depletion. Notably, a glycoengineered anti-mouse CD20 Ab but not its wild-type counterpart triggered potent KC-mediated B cell depletion at low doses. Finally, distinct thresholds for KC phagocytosis were also observed for GA101 (obinutuzumab), a humanized glycoengineered type II anti-CD20 Ab and rituximab. Thus, we propose that enhanced phagocytosis of circulating B cells by KC represents an important in vivo mechanism underlying the improved activity of glycoengineered anti-CD20 mAbs. PMID:27698437

  17. Intravital imaging reveals improved Kupffer cell-mediated phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies.

    PubMed

    Grandjean, Capucine L; Montalvao, Fabricio; Celli, Susanna; Michonneau, David; Breart, Beatrice; Garcia, Zacarias; Perro, Mario; Freytag, Olivier; Gerdes, Christian A; Bousso, Philippe

    2016-10-04

    Anti-CD20 monoclonal antibodies (mAbs) represent an effective treatment for a number of B cell malignancies and autoimmune disorders. Glycoengineering of anti-CD20mAb may contribute to increased anti-tumor efficacy through enhanced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) as reported by in vitro studies. However, where and how glycoengineered Ab may potentiate therapeutic responses in vivo is yet to be elucidated. Here, we have performed mouse liver transplants to demonstrate that the liver is sufficient to mediate systemic B cells depletion after anti-CD20 treatment. Relying on intravital two-photon imaging of human CD20-expressing mice, we provide evidence that ADP by Kupffer cells (KC) is a major mechanism for rituximab-mediated B cell depletion. Notably, a glycoengineered anti-mouse CD20 Ab but not its wild-type counterpart triggered potent KC-mediated B cell depletion at low doses. Finally, distinct thresholds for KC phagocytosis were also observed for GA101 (obinutuzumab), a humanized glycoengineered type II anti-CD20 Ab and rituximab. Thus, we propose that enhanced phagocytosis of circulating B cells by KC represents an important in vivo mechanism underlying the improved activity of glycoengineered anti-CD20 mAbs.

  18. Comparability of Antibody-Mediated Cell Killing Activity Between a Proposed Biosimilar RTXM83 and the Originator Rituximab.

    PubMed

    Cuello, Hector A; Segatori, Valeria I; Alberto, Marina; Pesce, Analía; Alonso, Daniel F; Gabri, Mariano R

    2016-06-01

    Biosimilars are described as biological products that resemble the structure of original biologic therapeutic products, with no clinically meaningful differences in terms of safety and effectiveness from the original. A wide range of biosimilars are under development or are already licensed in many countries. Biosimilars are earning acceptance and becoming a reality for immunotherapy treatments mainly based on the alternatives for the commercial anti-CD20 monoclonal antibody rituximab. The most important mechanism of action reported for this antibody is the induction of antibody-dependent cell cytotoxicity (ADCC), which is associated with the polymorphisms present at the 158 position in the IgG receptor FcγRIIIa. The aim of the study was to validate the functional comparability between the proposed rituximab biosimilar RTXM83 and the original product. To achieve this we assessed the binding capacity and ADCC induction of this biosimilar, taking into account the different FcγRIIIa-158 polymorphisms. Binding capacity was evaluated by flow cytometry using CD20 positive cells and a wide range of antibody concentrations. The FcγRIIIa-158 polymorphisms were analyzed by polymerase chain reaction (PCR) followed by allele-specific restriction enzyme digestion. ADCC was measured by a colorimetric lactate dehydrogenase-release assay, using effector cells from donors with different FcγRIIIa-158 polymorphisms. Binding capacity assay showed no differences between both products. Regarding ADCC, a similar relative potency was obtained between both antibodies, showing a higher response for the FcγRIIIa-158 valine/valine (V/V) polymorphism compared to the phenylalanine/phenylalanine (F/F), for both rituximab and RTXM83. Our data strongly suggest the biocomparability between the proposed biosimilar and the originator rituximab, in antibody recognition and ADCC activity. Additionally, our results suggest that donors with the FcγRIIIa-158V/V polymorphism induce a higher ADCC

  19. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses

    PubMed Central

    McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T.; Dennison, S. Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S. Munir; Haynes, Barton F.; Tomaras, Georgia D.

    2016-01-01

    Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine

  20. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses.

    PubMed

    Tay, Matthew Zirui; Liu, Pinghuang; Williams, LaTonya D; McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T; Dennison, S Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S Munir; Moody, M Anthony; Hope, Thomas J; Haynes, Barton F; Tomaras, Georgia D

    2016-08-01

    Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine

  1. Antibody-mediated immunity against tuberculosis: implications for vaccine development.

    PubMed

    Achkar, Jacqueline M; Casadevall, Arturo

    2013-03-13

    There is an urgent need for new and better vaccines against tuberculosis (TB). Current vaccine design strategies are generally focused on the enhancement of cell-mediated immunity. Antibody-based approaches are not being considered, mostly due to the paradigm that humoral immunity plays little role in the protection against intracellular pathogens. Here, we reappraise and update the increasing evidence for antibody-mediated immunity against Mycobacterium tuberculosis, discuss the complexity of antibody responses to mycobacteria, and address mechanism of protection. Based on these findings and discussions, we challenge the common belief that immunity against M. tuberculosis relies solely on cellular defense mechanisms, and posit that induction of antibody-mediated immunity should be included in TB vaccine development strategies.

  2. Increased infectivity in human cells and resistance to antibody-mediated neutralization by truncation of the SIV gp41 cytoplasmic tail.

    PubMed

    Kuwata, Takeo; Kaori, Takaki; Enomoto, Ikumi; Yoshimura, Kazuhisa; Matsushita, Shuzo

    2013-01-01

    The role of antibodies in protecting the host from human immunodeficiency virus type 1 (HIV-1) infection is of considerable interest, particularly because the RV144 trial results suggest that antibodies contribute to protection. Although infection of non-human primates with simian immunodeficiency virus (SIV) is commonly used as an animal model of HIV-1 infection, the viral epitopes that elicit potent and broad neutralizing antibodies to SIV have not been identified. We isolated a monoclonal antibody (MAb) B404 that potently and broadly neutralizes various SIV strains. B404 targets a conformational epitope comprising the V3 and V4 loops of Env that intensely exposed when Env binds CD4. B404-resistant variants were obtained by passaging viruses in the presence of increasing concentration of B404 in PM1/CCR5 cells. Genetic analysis revealed that the Q733stop mutation, which truncates the cytoplasmic tail of gp41, was the first major substitution in Env during passage. The maximal inhibition by B404 and other MAbs were significantly decreased against a recombinant virus with a gp41 truncation compared with the parental SIVmac316. This indicates that the gp41 truncation was associated with resistance to antibody-mediated neutralization. The infectivities of the recombinant virus with the gp41 truncation were 7,900-, 1,000-, and 140-fold higher than those of SIVmac316 in PM1, PM1/CCR5, and TZM-bl cells, respectively. Immunoblotting analysis revealed that the gp41 truncation enhanced the incorporation of Env into virions. The effect of the gp41 truncation on infectivity was not obvious in the HSC-F macaque cell line, although the resistance of viruses harboring the gp41 truncation to neutralization was maintained. These results suggest that viruses with a truncated gp41 cytoplasmic tail were selected by increased infectivity in human cells and by acquiring resistance to neutralizing antibody.

  3. Identification of Dominant Antibody-Dependent Cell-Mediated Cytotoxicity Epitopes on the Hemagglutinin Antigen of Pandemic H1N1 Influenza Virus

    PubMed Central

    Srivastava, Vikram; Yang, Zheng; Hung, Ivan Fan Ngai; Xu, Jianqing; Zheng, Bojian

    2013-01-01

    Antibody-dependent cell-mediated cytotoxicity (ADCC) bridges innate and adaptive immunity, and it involves both humoral and cellular immune responses. ADCC has been found to be a main route of immune protection against viral infections in vivo. Hemagglutinin (HA) of influenza virus is highly immunogenic and considered the most important target for immune protection. Several potent cross-reactive HA-specific neutralizing monoclonal antibodies (MAbs) have been reported, and their conserved neutralizing epitopes have been revealed, but there has been no report so far about ADCC epitopes on HA. Here we identified two dominant ADCC epitopes, designated E1 (amino acids [aa] 92 to 117) and E2 (aa 124 to 159), on HA of pandemic H1N1 influenza virus by epitope mapping of convalescent-phase plasma IgG antibodies from six H1N1-infected human subjects in China that exhibited different levels of ADCC activity. The E1 and E2 ADCC epitopes overlapped with immunodominant epitopes of HA. Depletion of purified patient plasma IgG antibodies with EBY100 yeast cells expressing E1 or E2 decreased the ADCC activity of the IgG antibodies. E1 and E2 sequences were found to be highly conserved in H1N1 strains but less so in other subtypes of influenza A viruses. Our study may aid in designing immunogens that can elicit antibodies with high ADCC activity. Vaccine immunogens designed to include the structural determinants of potent broadly neutralizing antibodies and ADCC epitopes may confer comprehensive immune protection against influenza virus infection. PMID:23487456

  4. Vector-Mediated In Vivo Antibody Expression.

    PubMed

    Schnepp, Bruce C; Johnson, Philip R

    2014-08-01

    This article focuses on a novel vaccine strategy known as vector-mediated antibody gene transfer, with a particular focus on human immunodeficiency virus (HIV). This strategy provides a solution to the problem of current vaccines that fail to generate neutralizing antibodies to prevent HIV-1 infection and AIDS. Antibody gene transfer allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein. This approach uses recombinant adeno-associated viral (rAAV) vectors, which have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes, to deliver the gene encoding a broadly neutralizing antibody into the muscle. Following rAAV vector gene delivery, the broadly neutralizing antibodies are endogenously synthesized in myofibers and passively distributed to the circulatory system. This is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. Vector-mediated gene transfer studies in mice and monkeys with anti-HIV and simian immunodeficiency virus (SIV)-neutralizing antibodies demonstrated long-lasting neutralizing activity in serum with complete protection against intravenous challenge with virulent HIV and SIV. These results indicate that existing potent anti-HIV antibodies can be rapidly moved into the clinic. However, this methodology need not be confined to HIV. The general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets such as hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis.

  5. Anti-cancer Activity of Novel TM4SF5-Targeting Antibodies through TM4SF5 Neutralization and Immune Cell-Mediated Cytotoxicity

    PubMed Central

    Ahn, Hye-Mi; Ryu, Jihye; Song, Jin Myeong; Lee, Yunhee; Kim, Hye-Jin; Ko, Dongjoon; Choi, Inpyo; Kim, Sang Jick; Lee, Jung Weon; Kim, Semi

    2017-01-01

    The transmembrane four L6 family member 5 (TM4SF5) protein is a novel molecular target for the prevention and treatment of hepatocellular carcinoma. TM4SF5 is highly expressed in liver, colon, esophageal, and pancreatic cancers and is implicated in tumor progression. Here, we screened monoclonal antibodies that specifically bound to the extracellular loop 2 (EC2) of TM4SF5 from a phage-displayed murine antibody (single-chain variable fragment; scFv) library. We constructed and characterized chimeric antibodies, Ab27 and Ab79, of scFv fused with Fc domain of human IgG1. The affinity (KD) of Ab27 and Ab79 for soluble EC2 was approximately 9.2 nM and 16.9 nM, respectively, as determined by surface plasmon resonance analysis. Ab27 and Ab79 efficiently bound to native TM4SF5 on the cell surface were internalized into the cancer cells, leading to a decrease in cell surface TM4SF5. Ab27 and Ab79 inhibited the proliferation and invasion of TM4SF5-positive liver and colon cancer cells and reduced FAK and c-Src phosphorylation. Ab27 and Ab79 also enhanced anoikis sensitivity and reduced survivin. Ab27 mediated antibody-dependent cell-mediated cytotoxicity in vitro. Ab27 and Ab79 efficiently inhibited tumor growth in a liver cancer xenograft model. These results strongly support the further development of Ab27 as a novel anti-cancer agent in the clinic. PMID:28255353

  6. Rational clinical trial design for antibody mediated renal allograft injury

    PubMed Central

    Sandal, Shaifali; Zand, Martin S.

    2015-01-01

    Antibody mediated renal allograft rejection is a significant cause of acute and chronic graft loss. Recent work has revealed that AMR is a complex processes, involving B and plasma cells, donor-specific antibodies, complement, vascular endothelial cells, NK cells, Fc receptors, cytokines and chemokines. These insights have led to the development of numerous new therapies, and adaptation of others originally developed for treatment of hemetologic malignancies, autoimmune and complement mediated conditions. Here we review emerging insights into the pathophysiology of AMR as well as current and emerging therapies for both acute and chronic AMR. Finally, we discuss rational clinical trial design in light of antibody and B cell immunobiology, as well as appropriate efficacy metrics to identify robust protocols and therapeutic agents. PMID:25553476

  7. Human neutrophils mediate trogocytosis rather than phagocytosis of CLL B-cells opsonized with anti-CD20 antibodies.

    PubMed

    Valgardsdottir, Rut; Cattaneo, Irene; Klein, Christian; Introna, Martino; Figliuzzi, Marina; Golay, Josée

    2017-03-13

    Polymorphonuclear neutrophils (PMN) have previously been reported to mediate phagocytosis of anti-CD20 opsonized B-cells from CLL patients. However recent data have suggested that PMN, like macrophages, can also mediate trogocytosis. We have performed experiments to more precisely investigate this point and discriminate between trogocytosis and phagocytosis. In live cell time-lapse microscopy experiments, we could not detect any significant phagocytosis by purified PMN of anti-CD20-opsonized CLL B-cells, but only the repeated close contact between effectors and targets, suggesting trogocytosis. Similarly, in flow cytometry assays using CLL B-cell targets labeled with the membrane dye PKH67 and opsonized with rituximab or obinutuzumab, we could show that a mean of 50% and 75% of PMN had taken a fraction of the dye from CLL B-cells at 3 and 20 hours, respectively, with no significant decrease in absolute live or total CLL B-cell numbers, confirming that trogocytosis takes place, rather than phagocytosis. Trogocytosis was accompanied by loss of membrane CD20 from CLL B-cells, which was evident with rituximab but not obinutuzumab. We conclude that PMN mediate mostly trogocytosis rather than phagocytosis of anti-CD20-opsonized CLL B-cells and discuss the implications of this finding in CLL patients treated with rituximab or obinutuzumab in vivo.

  8. Changes in plasma membrane phospholipids inhibit antibody-mediated lysis.

    PubMed

    Harris, David T

    2012-01-06

    A variety of mechanisms have been proposed to explain how tumors evade immune destruction. This work has identified one such mechanism that determines susceptibility to immune lysis; membrane phospholipid composition altered susceptibility to antibody plus complement (Ab+C)-mediated lysis. Effects on antibody plus complement-mediated lysis were correlated with levels of major histocompatibility complex (MHC) molecules but not inherent resistance to complement damage. This cellular mechanism could be a means by which tumor cells escape immune detection and destruction. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. A novel monoclonal antibody to human laminin α5 chain strongly inhibits integrin-mediated cell adhesion and migration on laminins 511 and 521.

    PubMed

    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.

  10. A Novel Monoclonal Antibody to Human Laminin α5 Chain Strongly Inhibits Integrin-Mediated Cell Adhesion and Migration on Laminins 511 and 521

    PubMed Central

    Wondimu, Zenebech; Omrani, Shahin; Ishikawa, Taichi; Javed, Fawad; Oikawa, Yuko; 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. PMID:23308268

  11. IgE and IgGa antibody-mediated release of histamine from rat peritoneal cells. II. Interaction of IgGa and IgE at the targe cell.

    PubMed

    Bach, M K; Block, K J; Austen, K F

    1971-04-01

    IgGa, in contrast to IgE, antibodies mediated the antigen-induced release of histamine from rat peritoneal mast cells without a requirement for a latent period and without the capacity to bind firmly to the target cell. Nonetheless, IgGa anti-DNP antibody interfered with the capacity of rat anti-N. brasiliensis antiserum rich in IgE antibodies to prepare the target cells for histamine release by worm antigen. Further, interaction of IgE antibody-prepared cells with IgGa anti-DNP antibody and DNP-BSA at 0 degrees C so as to achieve sterile activation, or at 30 degrees C to permit histamine release, inactivated such cells as determined by the subsequent failure to release histamine upon challenge with worm antigen. Thus, although IgE and IgGa antibodies are immunochemically distinct homologous immunoglobulins and exhibit different functional characteristics, their interaction at the target cell involves a common receptor and at least one common point in the pathway to the release of pharmacologic agents from the cell.

  12. Decreased Fc receptor expression on innate immune cells is associated with impaired antibody-mediated cellular phagocytic activity in chronically HIV-1 infected individuals.

    PubMed

    Dugast, Anne-Sophie; Tonelli, Andrew; Berger, Christoph T; Ackerman, Margaret E; Sciaranghella, Gaia; Liu, Qingquan; Sips, Magdalena; Toth, Ildiko; Piechocka-Trocha, Alicja; Ghebremichael, Musie; Alter, Galit

    2011-07-05

    In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection.

  13. Provirus activation plus CD59 blockage triggers antibody-dependent complement-mediated lysis of latently HIV-1-infected cells.

    PubMed

    Lan, Jie; Yang, Kai; Byrd, Daniel; Hu, Ningjie; Amet, Tohti; Shepherd, Nicole; Desai, Mona; Gao, Jimin; Gupta, Samir; Sun, Yongtao; Yu, Qigui

    2014-10-01

    Latently HIV-1-infected cells are recognized as the last barrier toward viral eradication and cure. To purge these cells, we combined a provirus stimulant with a blocker of human CD59, a key member of the regulators of complement activation, to trigger Ab-dependent complement-mediated lysis. Provirus stimulants including prostratin and histone deacetylase inhibitors such as romidepsin and suberoylanilide hydroxamic acid activated proviruses in the latently HIV-1-infected T cell line ACH-2 as virion production and viral protein expression on the cell surface were induced. Romidepsin was the most attractive provirus stimulant as it effectively activated proviruses at nanomolar concentrations that can be achieved clinically. Antiretroviral drugs including two protease inhibitors (atazanavir and darunavir) and an RT inhibitor (emtricitabine) did not affect the activity of provirus stimulants in the activation of proviruses. However, saquinavir (a protease inhibitor) markedly suppressed virus production, although it did not affect the percentage of cells expressing viral Env on the cell surface. Provirus-activated ACH-2 cells expressed HIV-1 Env that colocalized with CD59 in lipid rafts on the cell surface, facilitating direct interaction between them. Blockage of CD59 rendered provirus-activated ACH-2 cells and primary human CD4(+) T cells that were latently infected with HIV-1 sensitive to Ab-dependent complement-mediated lysis by anti-HIV-1 polyclonal Abs or plasma from HIV-1-infected patients. Therefore, a combination of provirus stimulants with regulators of complement activation blockers represents a novel approach to eliminate HIV-1. Copyright © 2014 by The American Association of Immunologists, Inc.

  14. Nanoparticle mediated drug delivery of rolipram to tyrosine kinase B positive cells in the inner ear with targeting peptides and agonistic antibodies

    PubMed Central

    Glueckert, Rudolf; Pritz, Christian O.; Roy, Soumen; Dudas, Jozsef; Schrott-Fischer, Anneliese

    2015-01-01

    Aim: Systemic pharmacotherapies have limitation due to blood-labyrinth barrier, so local delivery via the round window membrane opens a path for effective treatment. Multifunctional nanoparticle (NP)-mediated cell specific drug delivery may enhance efficacy and reduce side effects. Different NPs with ligands to target TrkB receptor were tested. Distribution, uptake mechanisms, trafficking, and bioefficacy of drug release of rolipram loaded NPs were evaluated. Methods: We tested lipid based nanocapsules (LNCs), Quantum Dot, silica NPs with surface modification by peptides mimicking TrkB or TrkB activating antibodies. Bioefficacy of drug release was tested with rolipram loaded LNCs to prevent cisplatin-induced apoptosis. We established different cell culture models with SH-SY-5Y and inner ear derived cell lines and used neonatal and adult mouse explants. Uptake and trafficking was evaluated with FACS and confocal as well as transmission electron microscopy. Results: Plain NPs show some selectivity in uptake related to the in vitro system properties, carrier material, and NP size. Some peptide ligands provide enhanced targeted uptake to neuronal cells but failed to show this in cell cultures. Agonistic antibodies linked to silica NPs showed TrkB activation and enhanced binding to inner ear derived cells. Rolipram loaded LNCs proved as effective carriers to prevent cisplatin-induced apoptosis. Discussion: Most NPs with targeting ligands showed limited effects to enhance uptake. NP aggregation and unspecific binding may change uptake mechanisms and impair endocytosis by an overload of NPs. This may affect survival signaling. NPs with antibodies activate survival signaling and show effective binding to TrkB positive cells but needs further optimization for specific internalization. Bioefficiacy of rolipram release confirms LNCs as encouraging vectors for drug delivery of lipophilic agents to the inner ear with ideal release characteristics independent of endocytosis

  15. JE-ADVAX vaccine protection against Japanese encephalitis virus mediated by memory B cells in the absence of CD8(+) T cells and pre-exposure neutralizing antibody.

    PubMed

    Larena, Maximilian; Prow, Natalie A; Hall, Roy A; Petrovsky, Nikolai; Lobigs, Mario

    2013-04-01

    JE-ADVAX is a new, delta inulin-adjuvanted, Japanese encephalitis (JE) candidate vaccine with a strong safety profile and potent immunogenicity that confers efficient immune protection not only against JE virus but also against related neurotropic flaviviruses such as West Nile virus. In this study, we investigated the immunological mechanism of protection by JE-ADVAX vaccine using knockout mice deficient in B cells or CD8(+) T cells and poor persistence of neutralizing antibody or by adoptive transfer of immune splenocyte subpopulations. We show that memory B cells induced by JE-ADVAX provide long-lived protection against JE even in the absence of detectable pre-exposure serum neutralizing antibodies and without the requirement of CD8(+) T cells. Upon virus encounter, these vaccine-induced memory B cells were rapidly triggered to produce neutralizing antibodies that then protected immunized mice from morbidity and mortality. The findings suggest that the extent of the B-cell memory compartment might be a better immunological correlate for clinical efficacy of JE vaccines than the currently recommended measure of serum neutralizing antibody. This may explain the paradox where JE protection is observed in some subjects even in the absence of detectable serum neutralizing antibody. Our investigation also established the suitability of a novel flavivirus challenge model (β(2)-microglobulin-knockout mice) for studies of the role of B-cell memory responses in vaccine protection.

  16. Combination of monoclonal antibodies with DST inhibits accelerated rejection mediated by memory T cells to induce long-lived heart allograft acceptance in mice.

    PubMed

    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.

  17. Antibody-mediated resistance against plant pathogens.

    PubMed

    Safarnejad, Mohammad Reza; Jouzani, Gholamreza Salehi; Tabatabaei, Meisam; Tabatabaie, Meisam; Twyman, Richard M; Schillberg, Stefan

    2011-01-01

    Plant diseases have a significant impact on the yield and quality of crops. Many strategies have been developed to combat plant diseases, including the transfer of resistance genes to crops by conventional breeding. However, resistance genes can only be introgressed from sexually-compatible species, so breeders need alternative measures to introduce resistance traits from more distant sources. In this context, genetic engineering provides an opportunity to exploit diverse and novel forms of resistance, e.g. the use of recombinant antibodies targeting plant pathogens. Native antibodies, as a part of the vertebrate adaptive immune system, can bind to foreign antigens and eliminate them from the body. The ectopic expression of antibodies in plants can also interfere with pathogen activity to confer disease resistance. With sufficient knowledge of the pathogen life cycle, it is possible to counter any disease by designing expression constructs so that pathogen-specific antibodies accumulate at high levels in appropriate sub-cellular compartments. Although first developed to tackle plant viruses and still used predominantly for this purpose, antibodies have been targeted against a diverse range of pathogens as well as proteins involved in plant-pathogen interactions. Here we comprehensively review the development and implementation of antibody-mediated disease resistance in plants.

  18. Platelets in Early Antibody-Mediated Rejection of Renal Transplants

    PubMed Central

    Kuo, Hsiao-Hsuan; Fan, Ran; Dvorina, Nina; Chiesa-Vottero, Andres

    2015-01-01

    Antibody-mediated rejection is a major complication in renal transplantation. The pathologic manifestations of acute antibody-mediated rejection that has progressed to functional impairment of a renal transplant have been defined in clinical biopsy specimens. However, the initial stages of the process are difficult to resolve with the unavoidable variables of clinical studies. We devised a model of renal transplantation to elucidate the initial stages of humoral rejection. Kidneys were orthotopically allografted to immunodeficient mice. After perioperative inflammation subsided, donor-specific alloantibodies were passively transferred to the recipient. Within 1 hour after a single transfer of antibodies, C4d was deposited diffusely on capillaries, and von Willebrand factor released from endothelial cells coated intravascular platelet aggregates. Platelet-transported inflammatory mediators platelet factor 4 and serotonin accumulated in the graft at 100- to 1000-fold higher concentrations compared with other platelet-transported chemokines. Activated platelets that expressed P-selectin attached to vascular endothelium and macrophages. These intragraft inflammatory changes were accompanied by evidence of acute endothelial injury. Repeated transfers of alloantibodies over 1 week sustained high levels of platelet factor 4 and serotonin. Platelet depletion decreased platelet mediators and altered the accumulation of macrophages. These data indicate that platelets augment early inflammation in response to donor-specific antibodies and that platelet-derived mediators may be markers of evolving alloantibody responses. PMID:25145937

  19. Antibody-mediated inhibition of ricin toxin retrograde transport.

    PubMed

    Yermakova, Anastasiya; Klokk, Tove Irene; Cole, Richard; Sandvig, Kirsten; Mantis, Nicholas J

    2014-04-08

    Ricin is a member of the ubiquitous family of plant and bacterial AB toxins that gain entry into the cytosol of host cells through receptor-mediated endocytosis and retrograde traffic through the trans-Golgi network (TGN) and endoplasmic reticulum (ER). While a few ricin toxin-specific neutralizing monoclonal antibodies (MAbs) have been identified, the mechanisms by which these antibodies prevent toxin-induced cell death are largely unknown. Using immunofluorescence confocal microscopy and a TGN-specific sulfation assay, we demonstrate that 24B11, a MAb against ricin's binding subunit (RTB), associates with ricin in solution or when prebound to cell surfaces and then markedly enhances toxin uptake into host cells. Following endocytosis, however, toxin-antibody complexes failed to reach the TGN; instead, they were shunted to Rab7-positive late endosomes and LAMP-1-positive lysosomes. Monovalent 24B11 Fab fragments also interfered with toxin retrograde transport, indicating that neither cross-linking of membrane glycoproteins/glycolipids nor the recently identified intracellular Fc receptor is required to derail ricin en route to the TGN. Identification of the mechanism(s) by which antibodies like 24B11 neutralize ricin will advance our fundamental understanding of protein trafficking in mammalian cells and may lead to the discovery of new classes of toxin inhibitors and therapeutics for biodefense and emerging infectious diseases. IMPORTANCE Ricin is the prototypic member of the AB family of medically important plant and bacterial toxins that includes cholera and Shiga toxins. Ricin is also a category B biothreat agent. Despite ongoing efforts to develop vaccines and antibody-based therapeutics against ricin, very little is known about the mechanisms by which antibodies neutralize this toxin. In general, it is thought that antibodies simply prevent toxins from attaching to cell surface receptors or promote their clearance through Fc receptor (FcR)-mediated uptake

  20. Phenotypes of antibody-mediated rejection in organ transplants.

    PubMed

    Mengel, Michael; Husain, Sufia; Hidalgo, Luis; Sis, Banu

    2012-06-01

    Antibody-mediated hyperacute rejection was the first rejection phenotype observed in human organ transplants. This devastating phenotype was eliminated by reliable crossmatch technologies. Since then, the focus was on T-cell-mediated rejection and de novo donor-specific antibodies were considered an epiphenomenon of cognate T-cell activation. The immune theory was that controlling the T-cell response would entail elimination of antibody-mediated rejection (ABMR). With modern immunosuppressive drugs, T-cell-mediated rejection is essentially treatable. However, this did not prevent ABMR from emerging as a significant phenotype in all types of organ transplants. It became obvious that both rejection types require distinct treatment and thus reliable diagnosis. This is the current challenge. ABMR, depending on stage, grade, time course, organ type or prior treatment, can present with a wide spectrum of phenotypes. This review summarizes the current diagnostic consensus for ABMR, describes unmet needs and challenges in diagnostics, and proposes new approaches for consideration. © 2012 The Authors. Transplant International © 2012 European Society for Organ Transplantation.

  1. Neuronal Surface Antibody-Mediated Autoimmune Encephalitis

    PubMed Central

    Linnoila, Jenny J.; Rosenfeld, Myrna R.; Dalmau, Josep

    2016-01-01

    In the past few years, many autoimmune encephalitides have been identified, with specific clinical syndromes and associated antibodies against neuronal surface antigens. There is compelling evidence that many of these antibodies are pathogenic and most of these encephalitides are highly responsive to immunotherapies. The clinical spectra of some of these antibody-mediated syndromes, especially those reported in only a few patients, are evolving. Others, such as anti-N-methyl-D-aspartate (NMDA) receptor encephalitis, are well characterized. Diagnosis involves recognizing the specific syndromes and identifying the antibody in a patient’s cerebrospinal fluid (CSF) and/or serum. These syndromes are associated with variable abnormalities in CSF, magnetic resonance imaging, and electroencephalography. Treatment is often multidisciplinary and should be focused upon neutralizing the effects of antibodies and eliminating their source. Overlapping disorders have been noted, with some patients having more than one neurologic autoimmune disease. In other patients, viral infections such as herpes simplex virus encephalitis trigger robust antineuronal autoimmune responses. PMID:25369441

  2. Molecular diagnosis of antibody-mediated rejection in human kidney transplants.

    PubMed

    Sellarés, J; Reeve, J; Loupy, A; Mengel, M; Sis, B; Skene, A; de Freitas, D G; Kreepala, C; Hidalgo, L G; Famulski, K S; Halloran, P F

    2013-04-01

    Antibody-mediated rejection is the major cause of kidney transplant failure, but the histology-based diagnostic system misses most cases due to its requirement for C4d positivity. We hypothesized that gene expression data could be used to test biopsies for the presence of antibody-mediated rejection. To develop a molecular test, we prospectively assigned diagnoses, including C4d-negative antibody-mediated rejection, to 403 indication biopsies from 315 patients, based on histology (microcirculation lesions) and donor-specific HLA antibody. We then used microarray data to develop classifiers that assigned antibody-mediated rejection scores to each biopsy. The transcripts distinguishing antibody-mediated rejection from other conditions were mostly expressed in endothelial cells or NK cells, or were IFNG-inducible. The scores correlated with the presence of microcirculation lesions and donor-specific antibody. Of 45 biopsies with scores>0.5, 39 had been diagnosed as antibody-mediated rejection on the basis of histology and donor-specific antibody. High scores were also associated with unanimity among pathologists that antibody-mediated rejection was present. The molecular score also strongly predicted future graft loss in Cox regression analysis. We conclude that microarray assessment of gene expression can assign a probability of ABMR to transplant biopsies without knowledge of HLA antibody status, histology, or C4d staining, and predicts future failure.

  3. NK cell-mediated antibody-dependent cellular cytotoxicity is enhanced by tamoxifen in HER2/neu non-amplified, but not HER2/neu-amplified, breast cancer cells.

    PubMed

    Richards, John O; Albers, Alex J; Smith, Thomas S; Tjoe, Judy A

    2016-11-01

    Tumor-targeting antibodies have been successful in the treatment of various types of cancers. Antibodies engage the immune system with their Fc, stimulating immune cell effector function. In the clinic, FcγRIIIa polymorphisms with higher affinity for the Fc of antibodies were shown to improve response rates and overall survival. Efforts have been made to modify the Fc to enhance affinity to Fc receptors and thereby improve effector function. An alternative for improving immune effector function may be to increase the level of tumor antigen expression. In this study, tamoxifen was used to increase HER2/neu protein level to determine whether increased tumor antigen expression could enhance NK cell-mediated antibody-dependent cytotoxicity (ADCC). Tamoxifen was found to increase HER2/neu 1.5-fold to threefold in breast cancer cell lines that were HER2/neu non-amplified. Using flow cytometry to simultaneously evaluate NK cell degranulation and tumor cell death, the increase in HER2/neu enhanced NK cell-mediated ADCC. However, in cells that had HER2/neu gene amplification and estrogen receptor expression, tamoxifen elevated HER2/neu but failed to improve NK cell function. The quantity of HER2/neu on the tumor cell surface was approximately double that of the number of Fc receptors found on NK cells. This appears to reflect a ceiling at which increasing antigen expression fails to improve NK cell effector function. This has clinical implications as trying to increase antigen expression to enhance NK cell function may be useful for patients with antigen-low tumors, but not in those whose tumors have gene amplification or high levels of antigen expression.

  4. CD147 monoclonal antibody mediated by chitosan nanoparticles loaded with α-hederin enhances antineoplastic activity and cellular uptake in liver cancer cells

    PubMed Central

    Zhu, Rong; Zhang, Chun-ge; Liu, Yang; Yuan, Zhi-qiang; Chen, Wei-liang; Yang, Shu-di; Li, Ji-zhao; Zhu, Wen-jing; Zhou, Xiao-feng; You, Ben-gang; Zhang, Xue-nong

    2015-01-01

    An antibody that specifically interacts with an antigen could be applied to an active targeting delivery system. In this study, CD147 antibody was coupled with α-hed chitosan nanoparticles (α-Hed-CS-NPs). α-Hed-CS-CD147-NPs were round and spherical in shape, with an average particle size of 148.23 ± 1.75 nm. The half-maximum inhibiting concentration (IC50) of α-Hed-CS-CD147-NPs in human liver cancer cell lines HepG2 and SMMC-7721 was lower than that of free α-Hed and α-Hed-CS-NPs. α-Hed-induced cell death was mainly triggered by apoptosis. The increase in intracellular accumulation of α-Hed-CS-CD147-NPs was also related to CD147-mediated internalization through the Caveolae-dependent pathway and lysosomal escape. The higher targeting antitumor efficacy of α-Hed-CS-CD147-NPs than that α-Hed-CS-NPs was attributed to its stronger fluorescence intensity in the tumor site in nude mice. PMID:26639052

  5. CD147 monoclonal antibody mediated by chitosan nanoparticles loaded with α-hederin enhances antineoplastic activity and cellular uptake in liver cancer cells.

    PubMed

    Zhu, Rong; Zhang, Chun-ge; Liu, Yang; Yuan, Zhi-qiang; Chen, Wei-liang; Yang, Shu-di; Li, Ji-zhao; Zhu, Wen-jing; Zhou, Xiao-feng; You, Ben-gang; Zhang, Xue-nong

    2015-12-07

    An antibody that specifically interacts with an antigen could be applied to an active targeting delivery system. In this study, CD147 antibody was coupled with α-hed chitosan nanoparticles (α-Hed-CS-NPs). α-Hed-CS-CD147-NPs were round and spherical in shape, with an average particle size of 148.23 ± 1.75 nm. The half-maximum inhibiting concentration (IC50) of α-Hed-CS-CD147-NPs in human liver cancer cell lines HepG2 and SMMC-7721 was lower than that of free α-Hed and α-Hed-CS-NPs. α-Hed-induced cell death was mainly triggered by apoptosis. The increase in intracellular accumulation of α-Hed-CS-CD147-NPs was also related to CD147-mediated internalization through the Caveolae-dependent pathway and lysosomal escape. The higher targeting antitumor efficacy of α-Hed-CS-CD147-NPs than that α-Hed-CS-NPs was attributed to its stronger fluorescence intensity in the tumor site in nude mice.

  6. The function of multiple extracellular matrix receptors in mediating cell adhesion to extracellular matrix: preparation of monoclonal antibodies to the fibronectin receptor that specifically inhibit cell adhesion to fibronectin and react with platelet glycoproteins Ic-IIa

    PubMed Central

    1988-01-01

    We have identified monoclonal antibodies that inhibit human cell adhesion to collagen (P1H5), fibronectin (P1F8 or P1D6), and collagen and fibronectin (P1B5) that react with a family of structurally similar glycoproteins referred to as extracellular matrix receptors (ECMRs) II, VI, and I, respectively. Each member of this family contains a unique alpha subunit, recognized by the antibodies, and a common beta subunit, each of approximately 140 kD. We show here that ECMR VI is identical to the fibronectin receptor (FNR), very late antigen (VLA) 5, and platelet glycoproteins Ic-IIa and shall be referred to as FNR. Monoclonal antibodies to FNR inhibit lymphocyte, fibroblast, and platelet adhesion to fibronectin-coated surfaces. ECMRs I, II, and FNR were differentially expressed in platelets, resting or activated lymphocytes, and myeloid, epithelial, endothelial, and fibroblast cell populations, suggesting a functional role for the receptors in vascular emigration and selective tissue localization. Tissue staining of human fetal skin localized ECMRs I and II to the basal epidermis primarily, while monoclonal antibodies to the FNR stained both the dermis and epidermis. Experiments carried out to investigate the functional roles of these receptors in mediating cell adhesion to complex extracellular matrix (ECM) produced by cells in culture revealed that complete inhibition of cell adhesion to ECM required antibodies to both the FNR and ECMR II, the collagen adhesion receptor. These results show that multiple ECMRs function in combination to mediate cell adhesion to complex EMC templates and predicts that variation in ECM composition and ECMR expression may direct cell localization to specific tissue domains. PMID:2846588

  7. Antibody-Mediated Rejection: A Review

    PubMed Central

    Garces, Jorge Carlos; Giusti, Sixto; Staffeld-Coit, Catherine; Bohorquez, Humberto; Cohen, Ari J.; Loss, George E.

    2017-01-01

    Background: Chronic antibody injury is a serious threat to allograft outcomes and is therefore the center of active research. In the continuum of allograft rejection, the development of antibodies plays a critical role. In recent years, an increased recognition of molecular and histologic changes has provided a better understanding of antibody-mediated rejection (AMR), as well as potential therapeutic interventions. However, several pathways are still unknown, which accounts for the lack of efficacy of some of the currently available agents that are used to treat rejection. Methods: We review the current diagnostic criteria for AMR; AMR paradigms; and desensitization, treatment, and prevention strategies. Results: Chronic antibody-mediated endothelial injury results in transplant glomerulopathy, manifested as glomerular basement membrane duplication, double contouring, or splitting. Clinical manifestations of AMR include proteinuria and a rise in serum creatinine. Current strategies for the treatment of AMR include antibody depletion with plasmapheresis (PLEX), immunoadsorption (IA), immunomodulation with intravenous immunoglobulin (IVIG), and T cell– or B cell–depleting agents. Some treatment benefits have been found in using PLEX and IA, and some small nonrandomized trials have identified some benefits in using rituximab and the proteasome inhibitor-based therapy bortezomib. More recent histologic follow-ups of patients treated with bortezomib have not shown significant benefits in terms of allograft outcomes. Furthermore, no specific treatment approaches have been approved by the US Food and Drug Administration. Other agents used for more difficult rejections include bortezomib and eculizumab (an anti-C5 monoclonal antibody). Conclusion: AMR is a fascinating field with ample opportunities for research and progress in the future. Despite the use of advanced techniques for the detection of human leukocyte antigen (HLA) or non-HLA donor-specific antibodies

  8. Maternal antibody decay and antibody-mediated immune responses in chicken pullets fed prebiotics and synbiotics.

    PubMed

    Alizadeh, M; Munyaka, P; Yitbarek, A; Echeverry, H; Rodriguez-Lecompte, J C

    2017-01-01

    Three experiments were conducted to evaluate the effect of yeast-derived carbohydrates (YDC), and a blend of probiotics and YDC (synbiotic, SNB) on serum IgG concentration, maternal-derived antibody (MDA) decay, and specific antibody-mediated immune response in chick pullets following immunization with T-cell dependent antigens. A total of 300 day-old pullet chicks were randomly assigned to 3 dietary treatments including: a basal diet (Control), and diets containing YDC, and SNB (Lactobacillus acidophilus, L. casei, Streptococcus faecium, and Bacillus subtilis, and YDC). In experiment one, on d 1 and wk 3, 4, 5, and 6, blood samples were collected and serum were analyzed by ELISA for total IgG (Y), and MDA against Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV). The second experiment examined the specific antibody against infectious bronchitis virus (IBV) in pullet chicks following vaccination against IBV at d 1. Finally, in experiment 3, on d 21 and 28 posthatch, 10 birds per treatment were immunized intramuscularly with both sheep red blood cells (SRBC) and bovine serum albumin (BSA), and 11 after immunization serum samples were analyzed by hemagglutination assay for antibody response to SRBC, and by ELISA for serum IgM and IgG response to BSA. The results demonstrated that diet containing SNB increased serum IgG at wk 3 posthatch. However, the decay rate of MDA against NDV and IBDV were not affected by dietary treatments. Birds fed YDC showed higher specific antibody response against IBV in wk 4, while both diets containing YDC and SNB decreased antibody response to IBV in wk 6. In addition, specific antibody response against SRBC and BSA was not affected by diets. In conclusion, supplementation of diet with SNB improved humoral immunity by increasing IgG concentration in serum, and modulated the adaptive antibody-mediated immune response against IBV. © 2016 Poultry Science Association Inc.

  9. Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries.

    PubMed

    Waldmeier, Lorenz; Hellmann, Ina; Gutknecht, Chantal K; Wolter, Fabian I; Cook, Skylar C; Reddy, Sai T; Grawunder, Ulf; Beerli, Roger R

    2016-01-01

    In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette. We demonstrate that stably transposed cells co-express transmembrane and secreted antibodies at levels comparable to those provided by dedicated constructs for secreted and membrane-associated IgGs. This unique feature expedites the screening and antibody characterization process by obviating the need for intermediate sequencing and re-cloning of individual antibody clones into separate expression vectors for functional screening purposes. In a series of proof-of-concept experiments, we demonstrate the seamless integration of antibody discovery with functional screening for various antibody properties, including binding affinity and suitability for preparation of antibody-drug conjugates.

  10. Transpo-mAb display: Transposition-mediated B cell display and functional screening of full-length IgG antibody libraries

    PubMed Central

    Waldmeier, Lorenz; Hellmann, Ina; Gutknecht, Chantal K.; Wolter, Fabian I.; Cook, Skylar C.; Reddy, Sai T.; Grawunder, Ulf; Beerli, Roger R.

    2016-01-01

    ABSTRACT In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette. We demonstrate that stably transposed cells co-express transmembrane and secreted antibodies at levels comparable to those provided by dedicated constructs for secreted and membrane-associated IgGs. This unique feature expedites the screening and antibody characterization process by obviating the need for intermediate sequencing and re-cloning of individual antibody clones into separate expression vectors for functional screening purposes. In a series of proof-of-concept experiments, we demonstrate the seamless integration of antibody discovery with functional screening for various antibody properties, including binding affinity and suitability for preparation of antibody-drug conjugates. PMID:26986818

  11. Silica vesicles as nanocarriers and adjuvants for generating both antibody and T-cell mediated immune resposes to Bovine Viral Diarrhoea Virus E2 protein.

    PubMed

    Mody, Karishma T; Mahony, Donna; Zhang, Jun; Cavallaro, Antonino S; Zhang, Bing; Popat, Amirali; Mahony, Timothy J; Yu, Chengzhong; Mitter, Neena

    2014-12-01

    Bovine Viral Diarrhoea Virus (BVDV) is widely distributed in cattle industries and causes significant economic losses worldwide annually. A limiting factor in the development of subunit vaccines for BVDV is the need to elicit both antibody and T-cell-mediated immunity as well as addressing the toxicity of adjuvants. In this study, we have prepared novel silica vesicles (SV) as the new generation antigen carriers and adjuvants. With small particle size of 50 nm, thin wall (~6 nm), large cavity (~40 nm) and large entrance size (5.9 nm for SV-100 and 16 nm for SV-140), the SV showed high loading capacity (∼ 250 μg/mg) and controlled release of codon-optimised E2 (oE2) protein, a major immunogenic determinant of BVDV. The in vivo functionality of the system was validated in mice immunisation trials comparing oE2 plus Quil A (50 μg of oE2 plus 10 μg of Quil A, a conventional adjuvant) to the oE2/SV-140 (50 μg of oE2 adsorbed to 250 μg of SV-140) or oE2/SV-140 together with 10 μg of Quil A. Compared to the oE2 plus Quil A, which generated BVDV specific antibody responses at a titre of 10(4), the oE2/SV-140 group induced a 10 times higher antibody response. In addition, the cell-mediated response, which is essential to recognise and eliminate the invading pathogens, was also found to be higher [1954-2628 spot forming units (SFU)/million cells] in mice immunised with oE2/SV-140 in comparison to oE2 plus Quil A (512-1369 SFU/million cells). Our study has demonstrated that SV can be used as the next-generation nanocarriers and adjuvants for enhanced veterinary vaccine delivery.

  12. Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in (51)Cr-release and CD107a assays.

    PubMed

    Mata, Mariana M; Mahmood, Fareeha; Sowell, Ryan T; Baum, Linda L

    2014-04-01

    Freshly isolated PBMC are broadly used as effector cells in functional assays that evaluate antibody-dependent cell mediated cytotoxicity (ADCC) and NK activity; however, they introduce natural-individual donor-to-donor variability. Cryopreserved PBMC provide a more consistent source of effectors than fresh cells in cytotoxicity assays. Our objective was to determine the effects of cryopreservation of effector PBMC on cell frequency, and on the magnitude and specificity of ADCC and NK activity. Fresh, frozen/overnight rested and frozen/not rested PBMC were used as effector cells in (51)Cr-release and CD107a degranulation assays. Frozen/overnight rested PBMC had higher ADCC and NK activity in both assays when compared to fresh PBMC; however, when using frozen/not rested PBMC, ADCC and NK activities were significantly lower than fresh PBMC. Background CD107a degranulation in the absence of target cell stimulation was greater in PBMC that were frozen/not rested when compared to fresh PBMC or PBMC that were frozen overnight and rested. The percentages of CD16(+)CD56(dim) NK cells and CD14(+) monocytes were lower in PBMC that were frozen and rested overnight than in fresh PBMC. CD16 expression on CD56(dim) NK cells was similar for all PBMC treatments. PBMC that were frozen and rested overnight were comparable to fresh PBMC effectors. PBMC that were frozen and used immediately when evaluating ADCC or NK activity using either a (51)Cr-release assay or a CD107a degranulation assay had the lowest activity. Clinical studies of antibodies that mediate ADCC would benefit from using effector cells that have been frozen, thawed and rested overnight prior to assay.

  13. CHO-S antibody titers >1 gram/liter using flow electroporation-mediated transient gene expression followed by rapid migration to high-yield stable cell lines.

    PubMed

    Steger, Krista; Brady, James; Wang, Weili; Duskin, Meg; Donato, Karen; Peshwa, Madhusudan

    2015-04-01

    In recent years, researchers have turned to transient gene expression (TGE) as an alternative to CHO stable cell line generation for early-stage antibody development. Despite advances in transfection methods and culture optimization, the majority of CHO-based TGE systems produce insufficient antibody titers for extensive use within biotherapeutic development pipelines. Flow electroporation using the MaxCyte STX Scalable Transfection System is a highly efficient, scalable means of CHO-based TGE for gram-level production of antibodies without the need for specialized expression vectors or genetically engineered CHO cell lines. CHO cell flow electroporation is easily scaled from milligram to multigram quantities without protocol reoptimization while maintaining transfection performance and antibody productivity. In this article, data are presented that demonstrate the reproducibility, scalability, and antibody production capabilities of CHO-based TGE using the MaxCyte STX. Data show optimization of posttransfection parameters such as cell density, media composition, and feed strategy that result in secreted antibody titers >1 g/L and production of multiple grams of antibody within 2 weeks of a single CHO-S cell transfection. In addition, data are presented to demonstrate the application of scalable electroporation for the rapid generation of high-yield stable CHO cell lines to bridge the gap between early- and late-stage antibody development activities.

  14. Blockage of CD59 Function Restores Activities of Neutralizing and Nonneutralizing Antibodies in Triggering Antibody-Dependent Complement-Mediated Lysis of HIV-1 Virions and Provirus-Activated Latently Infected Cells

    PubMed Central

    Yang, Kai; Lan, Jie; Shepherd, Nicole; Hu, Ningjie; Xing, Yanyan; Byrd, Daniel; Amet, Tohti; Jewell, Corlin; Gupta, Samir; Kounga, Carole

    2015-01-01

    ABSTRACT Both HIV-1 virions and infected cells use their surface regulators of complement activation (RCA) to resist antibody-dependent complement-mediated lysis (ADCML). Blockage of the biological function of RCA members, particularly CD59 (a key RCA member that controls formation of the membrane attack complex at the terminal stage of the complement activation cascades via all three activation pathways), has rendered both HIV-1 virions and infected cells sensitive to ADCML mediated by anti-Env antibodies (Abs) or sera/plasma from patients at different stages of viral infection. In the current study, we used the well-characterized anti-HIV-1 neutralizing Abs (nAbs), including 2G12, 2F5, and 4E10, and non-nAbs, including 2.2C, A32, N5-i5, and N12-i15, to investigate whether the enhancement of ADCML by blockage of CD59 function is mediated by nAbs, non-nAbs, or both. We found that all nAbs and two non-nAbs (N5-i5 and A32) strongly reacted to three HIV-1 laboratory strains (R5, X4, and R5/X4), six primary isolates, and provirus-activated ACH-2 cells examined. In contrast, two non-nAbs, 2.2C and N12-i15, reacted weakly and did not react to these targets, respectively. After blockage of CD59 function, the reactive Abs, regardless of their neutralizing activities, significantly enhanced specific ADCML of HIV-1 virions (both laboratory strains and primary isolates) and provirus-activated latently infected cells. The ADMCL efficacy positively correlated with the enzyme-linked immunosorbent assay-reactive intensity of those Abs with their targets. Thus, blockage of RCA function represents a novel approach to restore activities of both nAbs and non-nAbs in triggering ADCML of HIV-1 virions and provirus-activated latently infected cells. IMPORTANCE There is a renewed interest in the potential role of non-nAbs in the control of HIV-1 infection. Our data, for the first time, demonstrated that blockage of the biological function of RCA members rendered both HIV-1 virions and

  15. Blockage of CD59 Function Restores Activities of Neutralizing and Nonneutralizing Antibodies in Triggering Antibody-Dependent Complement-Mediated Lysis of HIV-1 Virions and Provirus-Activated Latently Infected Cells.

    PubMed

    Yang, Kai; Lan, Jie; Shepherd, Nicole; Hu, Ningjie; Xing, Yanyan; Byrd, Daniel; Amet, Tohti; Jewell, Corlin; Gupta, Samir; Kounga, Carole; Gao, Jimin; Yu, Qigui

    2015-09-01

    Both HIV-1 virions and infected cells use their surface regulators of complement activation (RCA) to resist antibody-dependent complement-mediated lysis (ADCML). Blockage of the biological function of RCA members, particularly CD59 (a key RCA member that controls formation of the membrane attack complex at the terminal stage of the complement activation cascades via all three activation pathways), has rendered both HIV-1 virions and infected cells sensitive to ADCML mediated by anti-Env antibodies (Abs) or sera/plasma from patients at different stages of viral infection. In the current study, we used the well-characterized anti-HIV-1 neutralizing Abs (nAbs), including 2G12, 2F5, and 4E10, and non-nAbs, including 2.2C, A32, N5-i5, and N12-i15, to investigate whether the enhancement of ADCML by blockage of CD59 function is mediated by nAbs, non-nAbs, or both. We found that all nAbs and two non-nAbs (N5-i5 and A32) strongly reacted to three HIV-1 laboratory strains (R5, X4, and R5/X4), six primary isolates, and provirus-activated ACH-2 cells examined. In contrast, two non-nAbs, 2.2C and N12-i15, reacted weakly and did not react to these targets, respectively. After blockage of CD59 function, the reactive Abs, regardless of their neutralizing activities, significantly enhanced specific ADCML of HIV-1 virions (both laboratory strains and primary isolates) and provirus-activated latently infected cells. The ADMCL efficacy positively correlated with the enzyme-linked immunosorbent assay-reactive intensity of those Abs with their targets. Thus, blockage of RCA function represents a novel approach to restore activities of both nAbs and non-nAbs in triggering ADCML of HIV-1 virions and provirus-activated latently infected cells. There is a renewed interest in the potential role of non-nAbs in the control of HIV-1 infection. Our data, for the first time, demonstrated that blockage of the biological function of RCA members rendered both HIV-1 virions and infected cells

  16. Donor antibodies to HNA-3a implicated in TRALI reactions prime neutrophils and cause PMN-mediated damage to human pulmonary microvascular endothelial cells in a two-event in vitro model.

    PubMed

    Silliman, Christopher C; Curtis, Brian R; Kopko, Patricia M; Khan, Samina Y; Kelher, Marguerite R; Schuller, Randy M; Sannoh, Baindu; Ambruso, Daniel R

    2007-02-15

    Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality. Antibodies to HNA-3a are commonly implicated in TRALI. We hypothesized that HNA-3a antibodies prime neutrophils (PMNs) and cause PMN-mediated cytotoxicity through a two-event pathogenesis. Isolated HNA-3a+ or HNA-3a- PMNs were incubated with plasma containing HNA-3a antibodies implicated in TRALI, and their ability to prime the oxidase was measured. Human pulmonary microvascular endothelial cells (HMVECs) were activated with endotoxin or buffer, HNA-3a+ or HNA-3a- PMNs were added, and the coculture was incubated with plasma+/-antibodies to HNA-3a. PMN-mediated damage was measured by counting viable HMVECs/mm2. Plasma containing HNA-3a antibodies primed the fMLP-activated respiratory burst of HNA-3a+, but not HNA-3a-, PMNs and elicited PMN-mediated damage of LPS-activated HMVECs when HNA-3a+, but not HNA-3a-, PMNs were used. Thus, antibodies to HNA-3a primed PMNs and caused PMN-mediated HMVEC cytotoxicity in a two-event model identical to biologic response modifiers implicated in TRALI.

  17. The herpes virus Fc receptor gE-gI mediates antibody bipolar bridging to clear viral antigens from the cell surface.

    PubMed

    Ndjamen, Blaise; Farley, Alexander H; Lee, Terri; Fraser, Scott E; Bjorkman, Pamela J

    2014-03-01

    The Herpes Simplex Virus 1 (HSV-1) glycoprotein gE-gI is a transmembrane Fc receptor found on the surface of infected cells and virions that binds human immunoglobulin G (hIgG). gE-gI can also participate in antibody bipolar bridging (ABB), a process by which the antigen-binding fragments (Fabs) of the IgG bind a viral antigen while the Fc binds to gE-gI. IgG Fc binds gE-gI at basic, but not acidic, pH, suggesting that IgG bound at extracellular pH by cell surface gE-gI would dissociate and be degraded in acidic endosomes/lysosomes if endocytosed. The fate of viral antigens associated with gE-gI-bound IgG had been unknown: they could remain at the cell surface or be endocytosed with IgG. Here, we developed an in vitro model system for ABB and investigated the trafficking of ABB complexes using 4-D confocal fluorescence imaging of ABB complexes with transferrin or epidermal growth factor, well-characterized intracellular trafficking markers. Our data showed that cells expressing gE-gI and the viral antigen HSV-1 gD endocytosed anti-gD IgG and gD in a gE-gI-dependent process, resulting in lysosomal localization. These results suggest that gE-gI can mediate clearance of infected cell surfaces of anti-viral host IgG and viral antigens to evade IgG-mediated responses, representing a general mechanism for viral Fc receptors in immune evasion and viral pathogenesis.

  18. The Herpes Virus Fc Receptor gE-gI Mediates Antibody Bipolar Bridging to Clear Viral Antigens from the Cell Surface

    PubMed Central

    Ndjamen, Blaise; Farley, Alexander H.; Lee, Terri; Fraser, Scott E.; Bjorkman, Pamela J.

    2014-01-01

    The Herpes Simplex Virus 1 (HSV-1) glycoprotein gE-gI is a transmembrane Fc receptor found on the surface of infected cells and virions that binds human immunoglobulin G (hIgG). gE-gI can also participate in antibody bipolar bridging (ABB), a process by which the antigen-binding fragments (Fabs) of the IgG bind a viral antigen while the Fc binds to gE-gI. IgG Fc binds gE-gI at basic, but not acidic, pH, suggesting that IgG bound at extracellular pH by cell surface gE-gI would dissociate and be degraded in acidic endosomes/lysosomes if endocytosed. The fate of viral antigens associated with gE-gI–bound IgG had been unknown: they could remain at the cell surface or be endocytosed with IgG. Here, we developed an in vitro model system for ABB and investigated the trafficking of ABB complexes using 4-D confocal fluorescence imaging of ABB complexes with transferrin or epidermal growth factor, well-characterized intracellular trafficking markers. Our data showed that cells expressing gE-gI and the viral antigen HSV-1 gD endocytosed anti-gD IgG and gD in a gE-gI–dependent process, resulting in lysosomal localization. These results suggest that gE-gI can mediate clearance of infected cell surfaces of anti-viral host IgG and viral antigens to evade IgG-mediated responses, representing a general mechanism for viral Fc receptors in immune evasion and viral pathogenesis. PMID:24604090

  19. Gamma Interferon Is Required for Optimal Antibody-Mediated Immunity against Genital Chlamydia Infection

    PubMed Central

    Naglak, Elizabeth K.; Morrison, Sandra G.

    2016-01-01

    Defining the mechanisms of immunity conferred by the combination of antibody and CD4+ T cells is fundamental to designing an efficacious chlamydial vaccine. Using the Chlamydia muridarum genital infection model of mice, which replicates many features of human C. trachomatis infection and avoids the characteristic low virulence of C. trachomatis in the mouse, we previously demonstrated a significant role for antibody in immunity to chlamydial infection. We found that antibody alone was not protective. Instead, protection appeared to be conferred through an undefined antibody-cell interaction. Using gene knockout mice and in vivo cellular depletion methods, our data suggest that antibody-mediated protection is dependent on the activation of an effector cell population in genital tract tissues by CD4+ T cells. Furthermore, the CD4+ T cell-secreted cytokine gamma interferon (IFN-γ) was found to be a key component of the protective antibody response. The protective function of IFN-γ was not related to the immunoglobulin class or to the magnitude of the Chlamydia-specific antibody response or to recruitment of an effector cell population to genital tract tissue. Rather, IFN-γ appears to be necessary for activation of the effector cell population that functions in antibody-mediated chlamydial immunity. Our results confirm the central role of antibody in immunity to chlamydia reinfection and demonstrate a key function for IFN-γ in antibody-mediated protection. PMID:27600502

  20. Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies.

    PubMed

    Fauvelle, Catherine; Felmlee, Daniel J; Crouchet, Emilie; Lee, JiYoung; Heydmann, Laura; Lefèvre, Mathieu; Magri, Andrea; Hiet, Marie-Sophie; Fofana, Isabel; Habersetzer, François; Foung, Steven K H; Milne, Ross; Patel, Arvind H; Vercauteren, Koen; Meuleman, Philip; Zeisel, Mirjam B; Bartenschlager, Ralf; Schuster, Catherine; Baumert, Thomas F

    2016-01-01

    Efforts to develop an effective vaccine against hepatitis C virus (HCV) have been hindered by the propensity of the virus to evade host immune responses. HCV particles in serum and in cell culture associate with lipoproteins, which contribute to viral entry. Lipoprotein association has also been proposed to mediate viral evasion of the humoral immune response, though the mechanisms are poorly defined. We used small interfering RNAs to reduce levels of apolipoprotein E (apoE) in cell culture-derived HCV-producing Huh7.5-derived hepatoma cells and confirmed its depletion by immunoblot analyses of purified viral particles. Before infection of naïve hepatoma cells, we exposed cell culture-derived HCV strains of different genotypes, subtypes, and variants to serum and polyclonal and monoclonal antibodies isolated from patients with chronic HCV infection. We analyzed the interaction of apoE with viral envelope glycoprotein E2 and HCV virions by immunoprecipitation. Through loss-of-function studies on patient-derived HCV variants of several genotypes and subtypes, we found that the HCV particle apoE allows the virus to avoid neutralization by patient-derived antibodies. Functional studies with human monoclonal antiviral antibodies showed that conformational epitopes of envelope glycoprotein E2 domains B and C were exposed after depletion of apoE. The level and conformation of virion-associated apoE affected the ability of the virus to escape neutralization by antibodies. In cell-infection studies, we found that HCV-associated apoE helps the virus avoid neutralization by antibodies against HCV isolated from chronically infected patients. This method of immune evasion poses a challenge for the development of HCV vaccines. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

  1. Molecular Communication Modeling of Antibody-Mediated Drug Delivery Systems.

    PubMed

    Chahibi, Youssef; Akyildiz, Ian F; Balasubramaniam, Sasitharan; Koucheryavy, Yevgeni

    2015-07-01

    Antibody-mediated Drug Delivery Systems (ADDS) are emerging as one of the most encouraging therapeutic solutions for treating several diseases such as human cancers. ADDS use small molecules (antibodies) that propagate in the body and bind selectively to their corresponding receptors (antigens) expressed at the surface of the diseased cells. In this paper, the Molecular Communication (MC) paradigm, where information is conveyed through the concentration of molecules, is advocated for the engineering of ADDS and modeling their complex behavior, to provide a realistic model without the over-complication of system biology models, and the limitations of experimental approaches. The peculiarities of antibodies, including their anisotropic transport and complex electrochemical structure, are taken into account to develop an analytical model of the ADDS transport and antigen-binding kinetics. The end-to-end response of ADDS, from the drug injection to the drug absorption, is mathematically derived based on the geometry of the antibody molecule, the electrochemical structure of the antibody-antigen complex, and the physiology of the patient. The accuracy of the MC model is validated by finite-element (COMSOL) simulations. The implications of the complex interplay between the transport and kinetics parameters on the performance of ADDS are effectively captured by the proposed MC model. The MC model of ADDS will enable the discovery and optimization of drugs in a versatile, cost-efficient, and reliable manner.

  2. A multi-subunit chlamydial vaccine induces antibody and cell-mediated immunity in immunized koalas (Phascolarctos cinereus): comparison of three different adjuvants.

    PubMed

    Carey, Alison J; Timms, Peter; Rawlinson, Galit; Brumm, Jacqui; Nilsson, Karen; Harris, Jonathon M; Beagley, Kenneth W

    2010-02-01

    Chlamydial infections represent a major threat to the survival of the koala. Infections caused by Chlamydia pecorum cause blindness, infertility, pneumonia and urinary tract infections and represent a threat to the survival of the species. Little is known about the immune response in koalas, or the safety of commonly used adjuvants for induction of protective systemic and mucosal immunity. of study In the present study, we immunized 18 healthy female koalas subcutaneously with a combination of three chlamydial antigens [major outer membrane protein (MOMP), NrdB and TC0512 (Omp85)] mixed with one of three different adjuvants [Alhydrogel, Immunostimulating Complex (ISC) and TiterMax Gold]. All adjuvants induced strong neutralizing IgG responses in plasma against the three antigens with prolonged responses lasting more than 270 days seen in Alhydrogel and ISC immunized animals. Cloacal IgG responses lasting >270 days were also induced in ISC-immunized animals. Chlamydia-specific peripheral blood mononuclear cell proliferative responses were elicited by both Alhydrogel and ISC, and these lasted >270 days in the ISC group. The data show that a multi-subunit chlamydial vaccine, given subcutaneously, can elicit Chlamydia-specific cell-mediated and antibody responses in the koala demonstrating that the development of a protective vaccine is feasible.

  3. Influence of the Envelope gp120 Phe 43 Cavity on HIV-1 Sensitivity to Antibody-Dependent Cell-Mediated Cytotoxicity Responses.

    PubMed

    Prévost, Jérémie; Zoubchenok, Daria; Richard, Jonathan; Veillette, Maxime; Pacheco, Beatriz; Coutu, Mathieu; Brassard, Nathalie; Parsons, Matthew S; Ruxrungtham, Kiat; Bunupuradah, Torsak; Tovanabutra, Sodsai; Hwang, Kwan-Ki; Moody, M Anthony; Haynes, Barton F; Bonsignori, Mattia; Sodroski, Joseph; Kaufmann, Daniel E; Shaw, George M; Chenine, Agnès L; Finzi, Andrés

    2017-04-01

    HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cellular-mediated cytotoxicity (ADCC). HIV-1 has evolved sophisticated mechanisms to avoid the exposure of Env ADCC epitopes by downregulating CD4 and by limiting the overall amount of Env on the cell surface. In HIV-1, substitution of large residues such as histidine or tryptophan for serine 375 (S375H/W) in the gp120 Phe 43 cavity, where Phe 43 of CD4 contacts gp120, results in the spontaneous sampling of an Env conformation closer to the CD4-bound state. While residue S375 is well conserved in the majority of group M HIV-1 isolates, CRF01_AE strains have a naturally occurring histidine at this position (H375). Interestingly, CRF01_AE is the predominant circulating strain in Thailand, where the RV144 trial took place. In this trial, which resulted in a modest degree of protection, ADCC responses were identified as being part of the correlate of protection. Here we investigate the influence of the Phe 43 cavity on ADCC responses. Filling this cavity with a histidine or tryptophan residue in Env with a natural serine residue at this position (S375H/W) increased the susceptibility of HIV-1-infected cells to ADCC. Conversely, the replacement of His 375 by a serine residue (H375S) within HIV-1 CRF01_AE decreased the efficiency of the ADCC response. Our results raise the intriguing possibility that the presence of His 375 in the circulating strain where the RV144 trial was held contributed to the observed vaccine efficacy.IMPORTANCE HIV-1-infected cells presenting Env in the CD4-bound conformation on their surface are preferentially targeted by ADCC mediated by HIV-positive (HIV(+)) sera. Here we show that the gp120 Phe 43 cavity modulates the propensity of Env to sample this conformation and therefore affects the susceptibility of infected cells to ADCC. CRF01_AE HIV-1 strains have an unusual Phe 43 cavity

  4. gp49B-mediated negative regulation of antibody production by memory and marginal zone B cells.

    PubMed

    Fukao, Saori; Haniuda, Kei; Nojima, Takuya; Takai, Toshiyuki; Kitamura, Daisuke

    2014-07-15

    The rapid Ab responses observed after primary and secondary immunizations are mainly derived from marginal zone (MZ) and memory B cells, respectively, but it is largely unknown how these responses are negatively regulated. Several inhibitory receptors have been identified and their roles have been studied, but mainly on follicular B cells and much less so on MZ B, and never on memory B cells. gp49B is an Ig superfamily member that contains two ITIMs in its cytoplasmic tail, and it has been shown to negatively regulate mast cell, macrophage, and NK cell responses. In this study, we demonstrate that gp49B is preferentially expressed on memory and MZ B cells. We show that gp49B(-/-) mice produce more IgM after a primary immunization and more IgM and IgG1 after a secondary immunization than gp49B(+/+) mice in T cell-dependent immune responses. Memory and MZ B cells from gp49B(-/-) mice also produce more Abs upon in vitro stimulation with CD40 than those from gp49B(+/+) mice. The in vitro IgM production by MZ B cells from gp49B(+/+), but not gp49B(-/-), mice is suppressed by interaction with a putative gp49B ligand, the integrin αvβ3 heterodimer. In addition, gp49B(-/-) mice exhibited exaggerated IgE production in the memory recall response. These results suggest that plasma cell development from memory and MZ B cells, as well as subsequent Ab production, are suppressed via gp49B. In memory B cells, this suppression also prevents excessive IgE production, thus curtailing allergic diseases. Copyright © 2014 by The American Association of Immunologists, Inc.

  5. Evaluation of antibody-dependent cell-mediated cytotoxicity activity and cetuximab response in KRAS wild-type metastatic colorectal cancer patients.

    PubMed

    Lo Nigro, Cristiana; Ricci, Vincenzo; Vivenza, Daniela; Monteverde, Martino; Strola, Giuliana; Lucio, Francesco; Tonissi, Federica; Miraglio, Emanuela; Granetto, Cristina; Fortunato, Mirella; Merlano, Marco Carlo

    2016-02-15

    To investigate the prognostic role of invariant natural killer T (iNKT) cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in wild type KRAS metastatic colorectal cancer (mCRC) patients treated with cetuximab. Forty-one KRAS wt mCRC patients, treated with cetuximab and irinotecan-based chemotherapy in II and III lines were analyzed. Genotyping of single nucleotide polymorphism (SNP)s in the FCGR2A, FCGR3A and in the 3' untranslated regions of KRAS and mutational analysis for KRAS, BRAF and NRAS genes was determined either by sequencing or allelic discrimination assays. Enriched NK cells were obtained from lymphoprep-peripheral blood mononuclear cell and iNKT cells were defined by co-expression of CD3, TCRVα24, TCRVβ11. ADCC was evaluated as ex vivo NK-dependent activity, measuring lactate dehydrogenase release. At basal, mCRC patients performing ADCC activity above the median level (71%) showed an improved overall survival (OS) compared to patients with ADCC below (median 16 vs 8 mo; P = 0.026). We did not find any significant correlation of iNKT cells with OS (P = 0.19), albeit we observed a trend to a longer survival after 10 mo in patients with iNKT above median basal level (0.382 cells/microliter). Correlation of OS and progression-free survival (PFS) with interesting SNPs involved in ADCC ability revealed not to be significant. Patients carrying alleles both with A in FCGR2A and TT in FCGR3A presented a trend of longer PFS (median 9 vs 5 mo; P = 0.064). Chemotherapy impacted both iNKT cells and ADCC activity. Their prognostic values get lost when we analysed them after 2 and 4 mo of treatment. Our results suggest a link between iNKT cells, basal ADCC activity, genotypes in FCGR2A and FCGR3A, and efficacy of cetuximab in KRAS wt mCRC patients.

  6. Antibody-mediated Hsp70 protein therapy.

    PubMed

    Hansen, James E; Sohn, William; Kim, Charles; Chang, Sophia S; Huang, Natalie C; Santos, Donaldson G; Chan, Grace; Weisbart, Richard H; Nishimura, Robert N

    2006-05-09

    Intracellular Hsp70 provides cytoprotection against a variety of stressful stimuli, and an effective means of increasing intracellular Hsp70 levels could prove beneficial in the prevention and treatment of a variety of human diseases. A novel protein transduction domain consisting of the single chain Fv fragment of an anti-DNA antibody known to penetrate into living cells and tissues, mAb 3E10, has recently been used to deliver functional proteins to cells. The ability of the single chain Fv fragment to deliver Hsp70 into living cells was tested by generating an Fv-Hsp70 fusion protein. Fv-Hsp70 was produced as a secreted protein in both COS-7 cells and the methylotropic yeast strain Pichia pastoris and was shown capable of penetrating into COS-7 cells and primary rat cortical neurons. Pre-treatment with Fv-Hsp70 protected both COS-7 cells and primary rat cortical neurons against subsequent exposure to hydrogen peroxide. These results provide the first evidence that the Fv fragment of mAb 3E10 is capable of delivering proteins to neurons and indicate its potential in the development of Hsp70 protein therapy.

  7. Generation of new peptide-Fc fusion proteins that mediate antibody-dependent cellular cytotoxicity against different types of cancer cells

    PubMed Central

    Sioud, Mouldy; Westby, Phuong; Olsen, Julie Kristine E.; Mobergslien, Anne

    2015-01-01

    Antibody-dependent cellular cytotoxicity (ADCC), a key effector function for the clinical effectiveness of monoclonal antibodies, is triggered by the engagement of the antibody Fc domain with the Fcγ receptors expressed by innate immune cells such as natural killer (NK) cells and macrophages. Here, we fused cancer cell-binding peptides to the Fc domain of human IgG1 to engineer novel peptide-Fc fusion proteins with ADCC activity. The designed fusion proteins were expressed in human embryonic kidney 293T cells, followed by purification and characterization by western blots. One of the engineered variants (WN-Fc), bound with high affinity to a wide range of solid tumor cell lines (e.g., colon, lung, prostate, skin, ovarian, and mammary tumors). Treatment of cancer cells with the engineered peptide-Fc fusions in the presence of effector NK cells potentially enhanced cytotoxicity, degranulation, and interferon-γ production by NK cells when compared to cells treated with the Fc control. The presence of competing peptides inhibited NK cell activation. Furthermore, a bispecific peptide-Fc fusion protein activated NK cells against HER-1- and/or HER-2-expressing cancer cells. Collectively, the engineered peptide-Fc fusions constitute a new promising strategy to recruit and activate NK cells against tumor cells, a primary goal of cancer immunotherapy. PMID:26605373

  8. A novel modified vaccination technique produces IgG antibodies that cause complement-mediated lysis of multiple myeloma cells carrying CD38 antigen.

    PubMed

    Barabas, Arpad Z; Cole, Chad D; Graeff, Richard M; Morcol, Tulin; Lafreniere, Rene

    2016-01-01

    Objectives were to: 1) induce a lytic IgG antibody (ab) response (via the so called `third vaccination method') against CD38 antigen (ag) residing on the extra-cellular domain of multiple myeloma (MM) cells in recipient rabbits, by combining the CD38 ag with donor-derived anti-CD38 ag lytic IgG ab into an immune complex (IC); and 2) determine whether abs produced would cause complement-mediated lysis (in vitro) of human MM cells containing CD38 ag. The vaccine was created in a two-step process. First, ab (rabbit anti-CD38 ag IgG ab) was raised in donor rabbits by injections of low molecular weight soluble CD38 ag in Freund's complete adjuvant (FCA) and aqueous solution. Second, transfer of pathogenic lytic IgG ab response into recipient rabbits was achieved by injections of ICs composed of CD38 ag and homologous anti-CD38 ag IgG ab. Consequently, recipient rabbits produced the same ab with the same specificity against the target ag as was present in the inoculum, namely agglutinating, precipitating and lytic (as demonstrated in vitro). In an in vitro study, in the presence of complement, donor and recipient rabbits' immune sera caused lysis of CD38 ag associated human MM cells. The most effective lytic ab response causing sera were those from donor rabbits injected with CD38 ag in FCA and those from rabbits injected with ICs, especially when they were administered in adjuvants. These results provided proof of concept that the third vaccination method has good potential as a stand-alone and efficacious method of controlling cancer.

  9. Therapy of B-cell malignancies by anti–HLA-DR humanized monoclonal antibody, IMMU-114, is mediated through hyperactivation of ERK and JNK MAP kinase signaling pathways

    PubMed Central

    Gupta, Pankaj; Chen, Xiaochuan; Cardillo, Thomas M.; Furman, Richard R.; Chen, Susan; Chang, Chien-Hsing; Goldenberg, David M.

    2010-01-01

    A humanized IgG4 anti–HLA-DR monoclonal antibody (IMMU-114), engineered to avoid side effects associated with complement activation, was examined for binding and cytotoxicity on leukemia, lymphoma, and multiple myeloma cell lines and chronic lymphocytic leukemia (CLL) patient specimens, followed by evaluation of the effects of IMMU-114 on extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways. HLA-DR was expressed on the majority of these cells at markedly higher levels than CD20, CD22, and CD74. IMMU-114 was toxic to mantle cell lymphoma, CLL, acute lymphoblastic leukemia, hairy cell leukemia, non-Hodgkin lymphoma (including rituximab-resistant), and multiple myeloma cell lines, and also patient CLL cells. IMMU-114 induced disease-free survival in tumor-bearing SCID mice with early-stage disease and in models that are relatively resistant to anti-CD20 monoclonal antibodies. Despite positive staining, acute myelogenous leukemic cells were not killed by IMMU-114. The ability of IMMU-114 to induce activation of ERK and JNK signaling correlated with cytotoxicity and differentiates the mechanism of action of IMMU-114 from monoclonal antibodies against CD20 and CD74. Thus, antigen expression is not sufficient for cytotoxicity; antibody-induced hyperactivation of ERK and JNK mitogen activated protein kinase signaling pathways are also required. PMID:20101022

  10. Autologous antibodies that bind neuroblastoma cells.

    PubMed

    Sun, Yujing; Sholler, Giselle S; Shukla, Girja S; Pero, Stephanie C; Carman, Chelsea L; Zhao, Ping; Krag, David N

    2015-11-01

    Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.

  11. Acute antibody-mediated rejection in kidney transplant recipients.

    PubMed

    Davis, Scott; Cooper, James E

    2017-01-01

    Antibody-mediated rejection has now been recognized as one of the most important causes of graft loss. Transplantation across HLA barriers and nonadherence can result in acute antibody-mediated rejection, which is associated with particularly worse graft outcomes. New technologies, including genomic studies and assays to detect and define donor-specific antibodies, have provided important insights into the pathophysiology and diagnosis of acute antibody-mediated rejection but have engendered many questions about the clinical application of these tests in the prognosis and prevention of this protean disease process. In this article, we review the pathophysiology of acute antibody-mediated rejection, the evolving diagnostic criteria, and specific challenges related to its prognosis, treatment, and prevention.

  12. Antibody repertoire complexity and effector cell biology determined by assays for IgE-mediated basophil and T-cell activation.

    PubMed

    Lund, Gitte; Willumsen, Nicholas; Holm, Jens; Christensen, Lars Harder; Würtzen, Peter Adler; Lund, Kaare

    2012-09-28

    Effector cell activation and T-cell activation, the latter mediated by facilitated antigen presentation, are immunological mechanisms that play crucial roles in the manifestation and maintenance of allergic disease. In addition to their relevance for the pathogenesis of allergy in-vivo, in-vitro assays based on these immunological mechanisms have been established and used for diagnostics, for monitoring the progression of disease and for the effect of specific immunotherapy as well as for basic research purposes. Here we review different parameters that affect effector cell activation and facilitated antigen uptake and presentation, including assay designs, readout parameters and critical experimental conditions. Central to the two immunological mechanisms is complex formation between allergen-specific IgE, allergen, and cell surface-anchored immunoglobulin receptor; the high affinity IgE-receptor FcεRI on basophils and mast cells, and the low affinity IgE-receptor FcεRII (CD23) on B-cells. Accordingly, the effect of IgE repertoire complexity and allergen diversity on effector cell and facilitated antigen presentation is discussed in detail.

  13. Impaired Antibody-mediated Protection and Defective IgA B-Cell Memory in Experimental Infection of Adults with Respiratory Syncytial Virus.

    PubMed

    Habibi, Maximillian S; Jozwik, Agnieszka; Makris, Spyridon; Dunning, Jake; Paras, Allan; DeVincenzo, John P; de Haan, Cornelis A M; Wrammert, Jens; Openshaw, Peter J M; Chiu, Christopher

    2015-05-01

    Despite relative antigenic stability, respiratory syncytial virus (RSV) reinfects throughout life. After more than 40 years of research, no effective human vaccine exists and correlates of protection remain poorly defined. Most current vaccine candidates seek to induce high levels of RSV-specific serum neutralizing antibodies, which are associated with reduced RSV-related hospitalization rates in observational studies but may not actually prevent infection. To characterize correlates of protection from infection and the generation of RSV-specific humoral memory to promote effective vaccine development. We inoculated 61 healthy adults with live RSV and studied protection from infection by serum and mucosal antibody. We analyzed RSV-specific peripheral blood plasmablast and memory B-cell frequencies and antibody longevity. Despite moderately high levels of preexisting serum antibody, 34 (56%) became infected, of whom 23 (68%) developed symptomatic colds. Prior RSV-specific nasal IgA correlated significantly more strongly with protection from polymerase chain reaction-confirmed infection than serum neutralizing antibody. Increases in virus-specific antibody titers were variable and transient in infected subjects but correlated with plasmablasts that peaked around Day 10. During convalescence, only IgG (and no IgA) RSV-specific memory B cells were detectable in peripheral blood. This contrasted with natural influenza infection, in which virus-specific IgA memory B cells were readily recovered. This observed specific defect in IgA memory may partly explain the ability of RSV to cause recurrent symptomatic infections. If so, vaccines able to induce durable RSV-specific IgA responses may be more protective than those generating systemic antibody alone.

  14. Impaired Antibody-mediated Protection and Defective IgA B-Cell Memory in Experimental Infection of Adults with Respiratory Syncytial Virus

    PubMed Central

    Habibi, Maximillian S.; Jozwik, Agnieszka; Makris, Spyridon; Dunning, Jake; Paras, Allan; DeVincenzo, John P.; de Haan, Cornelis A. M.; Wrammert, Jens; Openshaw, Peter J. M.

    2015-01-01

    Rationale: Despite relative antigenic stability, respiratory syncytial virus (RSV) reinfects throughout life. After more than 40 years of research, no effective human vaccine exists and correlates of protection remain poorly defined. Most current vaccine candidates seek to induce high levels of RSV-specific serum neutralizing antibodies, which are associated with reduced RSV-related hospitalization rates in observational studies but may not actually prevent infection. Objectives: To characterize correlates of protection from infection and the generation of RSV-specific humoral memory to promote effective vaccine development. Methods: We inoculated 61 healthy adults with live RSV and studied protection from infection by serum and mucosal antibody. We analyzed RSV-specific peripheral blood plasmablast and memory B-cell frequencies and antibody longevity. Measurements and Main Results: Despite moderately high levels of preexisting serum antibody, 34 (56%) became infected, of whom 23 (68%) developed symptomatic colds. Prior RSV-specific nasal IgA correlated significantly more strongly with protection from polymerase chain reaction–confirmed infection than serum neutralizing antibody. Increases in virus-specific antibody titers were variable and transient in infected subjects but correlated with plasmablasts that peaked around Day 10. During convalescence, only IgG (and no IgA) RSV-specific memory B cells were detectable in peripheral blood. This contrasted with natural influenza infection, in which virus-specific IgA memory B cells were readily recovered. Conclusions: This observed specific defect in IgA memory may partly explain the ability of RSV to cause recurrent symptomatic infections. If so, vaccines able to induce durable RSV-specific IgA responses may be more protective than those generating systemic antibody alone. PMID:25730467

  15. Targeting Tumor Cells with Anti-CD44 Antibody Triggers Macrophage-Mediated Immune Modulatory Effects in a Cancer Xenograft Model.

    PubMed

    Maisel, Daniela; Birzele, Fabian; Voss, Edgar; Nopora, Adam; Bader, Sabine; Friess, Thomas; Goller, Bernhard; Laifenfeld, Daphna; Weigand, Stefan; Runza, Valeria

    2016-01-01

    CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages) to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP) of the malignant cells by macrophages.

  16. Targeting Tumor Cells with Anti-CD44 Antibody Triggers Macrophage-Mediated Immune Modulatory Effects in a Cancer Xenograft Model

    PubMed Central

    Maisel, Daniela; Birzele, Fabian; Voss, Edgar; Nopora, Adam; Bader, Sabine; Friess, Thomas; Goller, Bernhard; Laifenfeld, Daphna; Weigand, Stefan; Runza, Valeria

    2016-01-01

    CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages) to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP) of the malignant cells by macrophages. PMID:27463372

  17. Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins.

    PubMed

    Wang, Sheng-Fan; Tseng, Sung-Pin; Yen, Chia-Hung; Yang, Jyh-Yuan; Tsao, Ching-Han; Shen, Chun-Wei; Chen, Kuan-Hsuan; Liu, Fu-Tong; Liu, Wu-Tse; Chen, Yi-Ming Arthur; Huang, Jason C

    2014-08-22

    The severe acute respiratory syndrome coronavirus (SARS-CoV) still carries the potential for reemergence, therefore efforts are being made to create a vaccine as a prophylactic strategy for control and prevention. Antibody-dependent enhancement (ADE) is a mechanism through which dengue viruses, feline coronaviruses, and HIV viruses take advantage of anti-viral humoral immune responses to infect host target cells. Here we describe our observations of SARS-CoV using ADE to enhance the infectivity of a HL-CZ human promonocyte cell line. Quantitative-PCR and immunofluorescence staining results indicate that SARS-CoV is capable of replication in HL-CZ cells, and of displaying virus-induced cytopathic effects and increased levels of TNF-α, IL-4 and IL-6 two days post-infection. According to flow cytometry data, the HL-CZ cells also expressed angiotensin converting enzyme 2 (ACE2, a SARS-CoV receptor) and higher levels of the FcγRII receptor. We found that higher concentrations of anti-sera against SARS-CoV neutralized SARS-CoV infection, while highly diluted anti-sera significantly increased SARS-CoV infection and induced higher levels of apoptosis. Results from infectivity assays indicate that SARS-CoV ADE is primarily mediated by diluted antibodies against envelope spike proteins rather than nucleocapsid proteins. We also generated monoclonal antibodies against SARS-CoV spike proteins and observed that most of them promoted SARS-CoV infection. Combined, our results suggest that antibodies against SARS-CoV spike proteins may trigger ADE effects. The data raise new questions regarding a potential SARS-CoV vaccine, while shedding light on mechanisms involved in SARS pathogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Effects of the orientation of anti-BMP2 monoclonal antibody immobilized on scaffold in antibody-mediated osseous regeneration

    PubMed Central

    Ansari, Sahar; Freire, Marcelo; Choi, Moon G; Tavari, Azadeh; Almohaimeed, Mohammad; Moshaverinia, Alireza; Zadeh, Homayoun H

    2015-01-01

    Recently, we have shown that anti-BMP2 monoclonal antibodies (mAbs) can trap endogenous osteogenic BMP ligands, which can in turn mediate osteodifferentiation of progenitor cells. The effectiveness of this strategy requires the availability of the anti-BMP-2 monoclonal antibodies antigen-binding sites for anti-BMP-2 monoclonal antibodies to bind to the scaffold through a domain that will leave its antigen-binding region exposed and available for binding to an osteogenic ligand. We examined whether antibodies bound to a scaffold by passive adsorption versus through Protein G as a linker will exhibit differences in mediating bone formation. In vitro anti-BMP-2 monoclonal antibodies was immobilized on absorbable collagen sponge (ACS) with Protein G as a linker to bind the antibody through its Fc region and implanted into rat calvarial defects. The biomechanical strength of bone regenerated by absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies immune complex was compared to ACS/anti-BMP-2 monoclonal antibodies or ACS/Protein G/isotype mAb control group. Results demonstrated higher binding of anti-BMP-2 monoclonal antibodies/BMPs to C2C12 cells, when the mAb was initially attached to recombinant Protein G or Protein G-coupled microbeads. After eight weeks, micro-CT and histomorphometric analyses revealed increased bone formation within defects implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies compared with defects implanted with absorbable collagen sponge/anti-BMP-2 monoclonal antibodies (p < 0.05). Confocal laser scanning microscopy (CLSM) confirmed increased BMP-2, -4, and -7 detection in sites implanted with absorbable collagen sponge/Protein G/anti-BMP-2 monoclonal antibodies in vivo. Biomechanical analysis revealed the regenerated bone in sites with Protein G/anti-BMP-2 monoclonal antibodies had higher mechanical strength in comparison to anti-BMP-2 monoclonal antibodies. The negative control group, Protein G

  19. SARS CoV subunit vaccine: antibody-mediated neutralisation and enhancement.

    PubMed

    Jaume, M; Yip, M S; Kam, Y W; Cheung, C Y; Kien, F; Roberts, A; Li, P H; Dutry, I; Escriou, N; Daeron, M; Bruzzone, R; Subbarao, K; Peiris, J S M; Nal, B; Altmeyer, R

    2012-02-01

    1. A SARS vaccine was produced based on recombinant native full-length Spike-protein trimers (triSpike) and efficient establishment of a vaccination procedure in rodents. 2. Antibody-mediated enhancement of SARS-CoV infection with anti-SARS-CoV Spike immune-serum was observed in vitro. 3. Antibody-mediated infection of SARS-CoV triggers entry into human haematopoietic cells via an FcγR-dependent and ACE2-, pH-, cysteine-protease-independent pathways. 4. The antibody-mediated enhancement phenomenon is not a mandatory component of the humoral immune response elicited by SARS vaccines, as pure neutralising antibody only could be obtained. 5. Occurrence of immune-mediated enhancement of SARS-CoV infection raises safety concerns regarding the use of SARS-CoV vaccine in humans and enables new ways to investigate SARS pathogenesis (tropism and immune response deregulation).

  20. Short communication: Prevalence of digital dermatitis in Canadian dairy cattle classified as high, average, or low antibody- and cell-mediated immune responders.

    PubMed

    Cartwright, S L; Malchiodi, F; Thompson-Crispi, K; Miglior, F; Mallard, B A

    2017-10-01

    Lameness is a major animal welfare issue affecting Canadian dairy producers, and it can lead to production, reproduction, and health problems in dairy cattle herds. Although several different lesions affect dairy cattle hooves, studies show that digital dermatitis is the most common lesion identified in Canadian dairy herds. It has also been shown that dairy cattle classified as having high immune response (IR) have lower incidence of disease compared with those animals with average and low IR; therefore, it has been hypothesized that IR plays a role in preventing infectious hoof lesions. The objective of this study was to compare the prevalence of digital dermatitis in Canadian dairy cattle that were classified for antibody-mediated (AMIR) and cell-mediated (CMIR) immune response. Cattle (n = 329) from 5 commercial dairy farms in Ontario were evaluated for IR using a patented test protocol that captures both AMIR and CMIR. Individuals were classified as high, average, or low responders based on standardized residuals for AMIR and CMIR. Residuals were calculated using a general linear model that included the effects of herd, parity, stage of lactation, and stage of pregnancy. Hoof health data were collected from 2011 to 2013 by the farm's hoof trimmer using Hoof Supervisor software (KS Dairy Consulting Inc., Dresser, WI). All trim events were included for each animal, and lesions were assessed as a binary trait at each trim event. Hoof health data were analyzed using a mixed model that included the effects of herd, stage of lactation (at trim date), parity (at trim date), IR category (high, average, and low), and the random effect of animal. All data were presented as prevalence within IR category. Results showed that cows with high AMIR had significantly lower prevalence of digital dermatitis than cattle with average and low AMIR. No significant difference in prevalence of digital dermatitis was observed between high, average, and low CMIR cows. These results

  1. Not All Antibodies Are Created Equal: Factors That Influence Antibody Mediated Rejection

    PubMed Central

    Butler, Carrie L.; Valenzuela, Nicole M.; Thomas, Kimberly A.

    2017-01-01

    Consistent with Dr. Paul Terasaki's “humoral theory of rejection” numerous studies have shown that HLA antibodies can cause acute and chronic antibody mediated rejection (AMR) and decreased graft survival. New evidence also supports a role for antibodies to non-HLA antigens in AMR and allograft injury. Despite the remarkable efforts by leaders in the field who pioneered single antigen bead technology for detection of donor specific antibodies, a considerable amount of work is still needed to better define the antibody attributes that are associated with AMR pathology. This review highlights what is currently known about the clinical context of pre and posttransplant antibodies, antibody characteristics that influence AMR, and the paths after donor specific antibody production (no rejection, subclinical rejection, and clinical dysfunction with AMR). PMID:28373996

  2. Development of a novel mammalian cell surface antibody display platform.

    PubMed

    Zhou, Chen; Jacobsen, Frederick W; Cai, Ling; Chen, Qing; Shen, Weyen David

    2010-01-01

    Antibody display systems have been successfully applied to screen, select and characterize antibody fragments. These systems typically use prokaryotic organisms such as phage and bacteria or lower eukaryotic organisms, such as yeast. These organisms possess either no or different post-translational modification functions from mammalian cells and prefer to display small antibody fragments instead of full-length IgGs. We report here a novel mammalian cell-based antibody display platform that displays full-length functional antibodies on the surface of mammalian cells. Through recombinase-mediated DNA integration, each host cell contains one copy of the gene of interest in the genome. Utilizing a hot-spot integration site, the expression levels of the gene of interest are high and comparable between clones, ensuring a high signal to noise ratio. Coupled with fluorescence-activated cell sorting (FACS) technology, our platform is high throughput and can distinguish antibodies with very high antigen binding affinities directly on the cell surface. Single-round FACS can enrich high affinity antibodies by more than 500 fold. Antibodies with significantly improved neutralizing activity have been identified from a randomly mutagenized library, demonstrating the power of this platform in screening and selecting antibody therapeutics.

  3. Postoperative rebound of antiblood type antibodies and antibody-mediated rejection after ABO-incompatible living-related kidney transplantation.

    PubMed

    Ishida, Hideki; Kondo, Tsunenori; Shimizu, Tomokazu; Nozaki, Taiji; Tanabe, Kazunari

    2015-03-01

    The purpose of this study is to examine whether postoperative antiblood type antibody rebound is attributed to kidney allograft rejection in ABO blood type-incompatible (ABO-I) living-related kidney transplantation (KTx). A total of 191 ABO-I recipients who received ABO-I living-related KTx between 2001 and 2013 were divided into two groups: Group 1 consisted of low rebound [(≦1:32), N = 170] and Group 2 consisted of high rebound [(≧1:64), N = 21], according to the levels of the rebounded antiblood type antibodies within 1 year after transplantation. No prophylactic treatment for rejection was administered for elevated antiblood type antibodies, regardless of the levels of the rebounded antibodies. Within 1 year after transplantation, T-cell-mediated rejection was observed in 13 of 170 recipients (13/170, 8%) in Group 1 and in 2 of 21 recipients (2/21, 10%) in Group 2 (Groups 1 vs. 2, P = 0.432). Antibody-mediated rejection was observed in 15 of 170 recipients (15/170, 9%) and 2 of 21 recipients (2/21, 10%) in Groups 1 and 2, respectively (P = 0.898). In this study, we found no correlation between the postoperative antiblood type antibody rebound and the incidence of acute rejection. We concluded that no treatment is necessary for rebounded antiblood type antibodies.

  4. Cell-targeting antibodies in immunity to Ebola

    PubMed Central

    Schmaljohn, Alan; Lewis, George K.

    2016-01-01

    As the 2014–15 Ebola virus epidemic in West Africa evolved from emergency to lesson, developers of both vaccines and therapeutic antibodies were left with the puzzlement of what kinds of anti-Ebola antibodies are predictably desirable in treating the afflicted, and what antibodies might account for the specific and lasting protection elicited by the more effective vaccines. The facile answer in virology is that neutralizing antibody (NAb) is desired and required. However, with Ebola and other filoviruses (as with many prior viral examples), there are multiple discordances in which neutralizing antibodies fail to protect animals, and others in which antibody-mediated protection is observed in the absence of measured virus neutralization. Explanation presumably resides in the protective role of antibodies that bind and functionally ‘target’ virus-infected cells, here called ‘cell-targeting antibody’, or CTAb. To be clear, many NAbs are also CTAbs, and in the case of Ebola the great majority of NAbs are likely CTAbs. Isotype, glycosylation, and other features of CTAbs are likely crucial in their capacity to mediate protection. Overall, results and analysis invite an increasingly complex view of antibody-mediated immunity to enveloped viruses. PMID:27005312

  5. Infection of CD4{sup +} T lymphocytes by the human T cell leukemia virus type 1 is mediated by the glucose transporter GLUT-1: Evidence using antibodies specific to the receptor's large extracellular domain

    SciTech Connect

    Jin, Qingwen; Agrawal, Lokesh; VanHorn-Ali, Zainab; Alkhatib, Ghalib . E-mail: galkhati@iupui.edu

    2006-05-25

    To analyze HTLV-1 cytotropism, we developed a highly sensitive vaccinia virus-based assay measuring activation of a reporter gene upon fusion of two distinct cell populations. We used this system in a functional cDNA screening to isolate and confirm that the glucose transporter protein 1 (GLUT-1) is a receptor for HTLV-1. GLUT-1 is a ubiquitously expressed plasma membrane glycoprotein with 12 transmembrane domains and 6 extracellular loops (ECL). We demonstrate for the first time that peptide antibodies (GLUT-IgY) raised in chicken to the large extracellular loop (ECL1) detect GLUT-1 at the cell surface and inhibit envelope (Env)-mediated fusion and infection. Efficient GLUT-IgY staining was detected with peripheral blood CD4{sup +} lymphocytes purified by positive selection. Further, GLUT-IgY caused efficient inhibition of Env-mediated fusion and infection of CD4{sup +} T and significantly lower inhibition of CD8{sup +} T lymphocytes. The specificity of GLUT-IgY antibodies to GLUT-1 was demonstrated by ECL1 peptide competition studies. Grafting ECL1 of GLUT-1 onto the receptor-negative GLUT-3 conferred significant receptor activity. In contrast, grafting ECL1 of GLUT-3 onto GLUT-1 resulted in a significant loss of the receptor activity. The ECL1-mediated receptor activity was efficiently blocked with four different human monoclonal antibody (HMab) to HTLV-1 Env. The ECL1-derived peptide blocked HTLV-1 Env-mediated fusion with several nonhuman mammalian cell lines. The results demonstrate the utilization of cell surface GLUT-1 in HTLV-1 infection of CD4{sup +} T lymphocytes and implicate a critical role for the ECL1 region in viral tropism.

  6. Identifying Subphenotypes of Antibody-Mediated Rejection in Kidney Transplants.

    PubMed

    Halloran, P F; Merino Lopez, M; Barreto Pereira, A

    2016-03-01

    The key lesions in antibody-mediated kidney transplant rejection (ABMR) are microcirculation inflammation (peritubular capillaritis and/or glomerulitis lesions, abbreviated "pg") and glomerular double contours (cg lesions). We used these features to explore subphenotypes in 164 indication biopsies with ABMR-related diagnoses: 137 ABMR (109 pure and 28 mixed with T cell-mediated rejection [TCMR]) and 27 transplant glomerulopathy (TG), identified from prospective multicenter studies. The lesions indicated three ABMR subphenotypes: pgABMR, cgABMR, and pgcgABMR. Principal component analysis confirmed these subphenotypes and showed that TG can be reclassified as pgcgABMR (n = 17) or cgABMR (n = 10). ABMR-related biopsies included 45 pgABMR, 90 pgcgABMR, and 25 cgABMR, with four unclassifiable. Dominating all time intervals was the subphenotype pgcgABMR. The pgABMR subphenotype presented earliest (median <2 years), frequently mixed with TCMR, and was most associated with nonadherence. The cgABMR subphenotype presented late (median 9 years). Subphenotypes differed in their molecular changes, with pgABMR having the most histologic-molecular discrepancies (i.e. potential errors). Donor-specific antibody (DSA) was not identified in 29% of pgcgABMR and 46% of cgABMR, but failure rates and molecular findings were similar to cases where DSA was known to be positive. Thus, ABMR presents distinct subphenotypes, early pg-dominant, late cg-dominant, and combined pgcg phenotype, differing in time, molecular features, accompanying TCMR, HLA antibody, and probability of nonadherence.

  7. Antibodies as Mediators of Brain Pathology

    PubMed Central

    Brimberg, Lior; Mader, Simone; Fujieda, Yuichiro; Arinuma, Yoshiyuki; Kowal, Czeslawa; Volpe, Bruce T.; Diamond, Betty

    2016-01-01

    The brain is normally sequestered from antibody exposure by the blood brain barrier. However, antibodies can access the brain during fetal development before the barrier achieves full integrity, and in disease states when barrier integrity is compromised. Recent studies suggest that antibodies contribute to brain pathology associated with autoimmune diseases such as systemic lupus erythematosus and neuromyelitis optica, and can lead to transient or permanent behavioral or cognitive abnormalities. We review these findings here and examine the circumstances associated with antibody entry into the brain, the routes of access and the mechanisms that then effect pathology. Understanding these processes and the nature and specificity of neuronal autoantibodies may reveal therapeutic strategies toward alleviating or preventing the neurological pathologies and behavioral abnormalities associated with autoimmune disease. PMID:26494046

  8. Vector-mediated antibody gene transfer for infectious diseases.

    PubMed

    Schnepp, Bruce C; Johnson, Philip R

    2015-01-01

    This chapter discusses the emerging field of vector-mediated antibody gene transfer as an alternative vaccine for infectious disease, with a specific focus on HIV. However, this methodology need not be confined to HIV-1; the general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets like hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis. This approach is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. With vector-mediated gene transfer, the antibody gene is delivered to the host, via a recombinant adeno-associated virus (rAAV) vector; this in turn results in long-term endogenous antibody expression from the injected muscle that confers protective immunity. Vector-mediated antibody gene transfer can rapidly move existing, potent broadly cross-neutralizing HIV-1-specific antibodies into the clinic. The gene transfer products demonstrate a potency and breadth identical to the original product. This strategy eliminates the need for immunogen design and interaction with the adaptive immune system to generate protection, a strategy that so far has shown limited promise.

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

    PubMed Central

    2012-01-01

    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 < 0.05) and CD4+CD25+CD127-IFNγ+ PBL (anti-CD178, anti-CD152, anti-CD279, anti-CD95: p < 0.05). Effector cell proliferation increased with increasing antibody concentrations in culture medium (anti-CD178 and anti-CD279: p < 0.05). Conversely, high concentrations of recombinant proteins induced formation of CD4+CD25+Foxp3+IFNγ+ PBL (rCD152 and rCD95: p < 0.05) and decreased cell proliferation dose-dependently (rCD178 and rCD95: p < 0.05). Our data suggest an inverse association of iTreg induction with effector cell proliferation in cell culture which is dependent on the concentration of monoclonal antibodies against iTreg surface determinants. 3-day co-cultures of polyclonally stimulated PBL with separated CD4+CD25+CD127-IFNγ+ PBL showed lower cell proliferation than co-cultures with CD4+CD25+CD127-IFNγ- PBL (p < 0.05). Cell proliferation increased strongly in CD4+CD25+CD127-IFNγ- PBL-containing co-cultures in the presence of monoclonal antibody (anti-CD28, anti-CD152, anti-CD279: p

  10. Decreased complement mediated binding of antibody//sup 3/-dsDNA immune complexes to the red blood cells of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies

    SciTech Connect

    Taylor, R.P.; Horgan, C.; Buschbacher, R.; Brunner, C.M.; Hess, C.E.; O'Brien, W.M.; Wanebo, H.J.

    1983-06-01

    The complement mediated binding of prepared antibody//sup 3/H-dsDNA immune complexes to the red blood cells obtained from a number of patient populations has been investigated. Patients with solid tumors have binding activity similar to that seen in a normal group of individuals. However, a significant fraction of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies have lowered binding activity compared with normal subjects. Quantitative studies indicate the lowered activity probably arises due to a decrease in complement receptors on the respective red blood cells. The potential importance and implications of these findings are briefly discussed.

  11. Antibody-independent, interleukin-17A-mediated, cross-serotype immunity to pneumococci in mice immunized intranasally with the cell wall polysaccharide.

    PubMed

    Malley, Richard; Srivastava, Amit; Lipsitch, Marc; Thompson, Claudette M; Watkins, Claire; Tzianabos, Arthur; Anderson, Porter W

    2006-04-01

    Serotype-specific immunity to Streptococcus pneumoniae is conferred by antibodies to the capsular polysaccharides, which define the 90 known serotypes. Whether antibody to the species-common cell wall polysaccharide (C-Ps) is protective has been a matter of controversy. Here we show that C-Ps given intranasally with mucosal adjuvant increased the resistance of mice to experimental nasopharyngeal colonization by capsulated S. pneumoniae of serotype 6B. This immunity could be induced in mice congenitally lacking immunoglobulin but was dependent upon CD4+ T cells. Elimination of the charged amino group on the polymer backbone by N acetylation of C-Ps reduced the immunity, as did treatment of the mice with antibody to the cytokine interleukin-17A at the time of challenge, both consistent with the hypothesis of T-cell activation due to the zwitterionic motif of the polymer. C-Ps also protected in a model of fatal aspiration pneumonia by heavily capsulated serotype 3. These findings suggest a novel immunization strategy against S. pneumoniae.

  12. Radiosensitization and growth inhibition of cancer cells mediated by an scFv antibody gene against DNA-PKcs in vitro and in vivo

    PubMed Central

    2010-01-01

    Background Overexpression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is commonly occurred in cancers and causes radioresistance and poor prognosis. In present study, the single-chain variable antibody fragments (scFv) targeting DNA-PKcs was developed for the application of radiosensitization in vitro and in vivo. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody. Methods DNA-PKcs epitopes were predicted and cloned. A humanized semisynthetic scFv library and the phage-display antibodies technology were employed to screen DNA-PKcs scFv antibody. DNA damage repair was analyzed by comet assay and immunofluorescence detection of γH2AX foci. The radiosensitization in vivo was determined on Balb/c athymic mice transplanted tumours of HeLa cells. Results Four epitopes of DNA-PKcs have been predicted and expressed as the antigens, and a specific human anti-DNA-PKcs scFv antibody gene, anti-DPK3-scFv, was obtained by screening the phage antibody library using the DNA-PKcs peptide DPK3. The specificity of anti-DPK3-scFv was verified, in vitro. Transfection of HeLa cells with the anti-DPK3-scFv gene resulted in an increased sensitivity to IR, decreased repair capability of DNA double-strand breaks (DSB) detected by comet assay and immunofluorescence detection of γH2AX foci. Moreover, the kinase activity of DNA-PKcs was inhibited by anti-DPK3-scFv, which was displayed by the decreased phosphorylation levels of its target Akt/S473 and the autophosphorylation of DNA-PKcs on S2056 induced by radiation. Measurement of the growth and apoptosis rates showed that anti-DPK3-scFv enhanced the sensitivity of tumours transplanted in Balb/c athymic mice to radiation therapy. Conclusion The antiproliferation and radiosensitizing effects of anti-DPK3-scFv via targeting DNA-PKcs make it very appealing for the development as a novel biological radiosensitizer for cancer therapeutic potential

  13. Fatal immune-mediated pancytopenia and a TRALI-like syndrome associated with high titers of recipient-type antibodies against donor-derived peripheral blood cells after allogeneic bone marrow transplantation following dose reduced conditioning.

    PubMed

    Knop, Stefan; Bux, Juergen; Kroeber, Stefan M; Bader, Peter; Hebart, Holger; Kanz, Lothar; Einsele, Hermann

    2004-05-01

    Pancytopenia occurring after bone marrow transplantation is a rare complication. A 47 year old patient with progression of multiple myeloma after standard therapy received an allogeneic marrow graft from a matched unrelated donor. The non-myeloablative conditioning regimen consisted of fludarabine, cyclophosphamide, rabbit anti-thymocyte globulin and total body irradiation. GVHD prophylaxis consisted of cyclosporine. Neutrophil engraftment was as expected and the patient was discharged without signs of acute GvHD. On day +34 the patient presented with clinical and laboratory findings consistent with severe pancytopenia. Antibodies against red cells, platelets, lymphocytes and granulocytes were detected in extremely high titers. Immune-mediated pancytopenia was refractory on multiple immunosuppressive treatment strategies. Proliferation of polyclonal plasma cells of recipient-type that was documented postmortem, was most likely responsible for excessive antibody formation.

  14. Nonabsorbable Rabbit Anti-Salmonella typhimurium Antibody as Detected by the Complement-Mediated Bactericidal Reaction

    PubMed Central

    Herzberg, Mendel; Kenny, Kathryn V.; Robbins, John B.

    1966-01-01

    Herzberg, Mendel (University of Florida, Gainesville), Kathryn V. Kenny, and John B. Robbins. Nonabsorbable rabbit anti-Salmonella typhimurium antibody as detected by the complement-mediated bactericidal reaction. J. Bacteriol. 91:1548–1555. 1966.—A portion of antibody active in the complement-mediated bactericidal reaction against Salmonella typhimurium from hyperimmune rabbit serum has been shown to be nonabsorbable by repeated serial absorptions with whole heat-killed or living bacteria. The first two absorptions remove 90 to 95% of the activity, but 1 to 5% cannot be removed by subsequent absorptions. The nonabsorbable antibody appears to be a macroglobulin by density-gradient centrifugation and by comparison of activity and absorbability of purified γ-M and γ-G immune globulins. Alternative hypotheses involving low avidity antibody or antibody to minor cell antigenic components are offered in explanation of the phenomenon. PMID:4160234

  15. Antibodies to Phospholipids and Liposomes: Binding of Antibodies to Cells

    DTIC Science & Technology

    1987-01-01

    LIPOSOMES: BINDING OF ANTIBODIES TO CELLS 12. PERSONAL AUTHOR(S) W.E. FOGLER , G. M. SWARTZ, AND C.R. ALVING 13a TYPE OF REPORT 13b. TIME COVERED 14. DATE...Elsevier BBA 73693 Antibodies to phospholipids and liposomes: binding of antibodies to cells William E. Fogler *, Glenn M. Swartz, Jr. and Carl R. Alving...Immunol. 21. Research Associateship from the U.S. National 12863-86812Hall. T. and Esser, K. (1984) 3. Immunol. 132. 2059-2063 Research Council. 13 Fogler

  16. Monoclonal antibodies to endogenous galactose-specific tumor cell lectins.

    PubMed Central

    Raz, A; Meromsky, L; Carmi, P; Karakash, R; Lotan, D; Lotan, R

    1984-01-01

    A monoclonal antibody, 5D7, was obtained after immunization of syngeneic mice with B16 melanoma cell extracts enriched for endogenous lectin activity and screening for inhibition of lectin-mediated hemagglutination. Binding of this antibody to affinity-purified B16 melanoma galactoside-specific lectin was revealed by solid-phase radioimmunoassay and binding to the surface of viable B16 cells was demonstrated by indirect immunofluorescence. Inhibition of lectin activity and cell surface labeling by 5D7 antibody were also found with several types of cultured human and murine cells including melanoma, sarcoma and carcinoma. This monoclonal antibody should be useful for evaluating the role of tumor cell surface lectins in intercellular interactions and metastasis. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6084591

  17. Selection of Antibodies Interfering with Cell Surface Receptor Signaling Using Embryonic Stem Cell Differentiation.

    PubMed

    Melidoni, Anna N; Dyson, Michael R; McCafferty, John

    2016-01-01

    Antibodies able to bind and modify the function of cell surface signaling components in vivo are increasingly being used as therapeutic drugs. The identification of such "functional" antibodies from within large antibody pools is, therefore, the subject of intense research. Here we describe a novel cell-based expression and reporting system for the identification of functional antibodies from antigen-binding populations preselected with phage display. The system involves inducible expression of the antibody gene population from the Rosa-26 locus of embryonic stem (ES) cells, followed by secretion of the antibodies during ES cell differentiation. Target antigens are cell-surface signaling components (receptors or ligands) with a known effect on the direction of cell differentiation (FGFR1 mediating ES cell exit from self renewal in this particular protocol). Therefore, inhibition or activation of these components by functional antibodies in a few elite clones causes a shift in the differentiation outcomes of these clones, leading to their phenotypic selection. Functional antibody genes are then recovered from positive clones and used to produce the purified antibodies, which can be tested for their ability to affect cell fates exogenously. Identified functional antibody genes can be further introduced in different stem cell types. Inducible expression of functional antibodies has a temporally controlled protein-knockdown capability, which can be used to study the unknown role of the signaling pathway in different developmental contexts. Moreover, it provides a means for control of stem cell differentiation with potential in vivo applications.

  18. Concise Review: Stem Cells As an Emerging Platform for Antibody Therapy of Cancer

    PubMed Central

    Frank, Richard T; Najbauer, Joseph; Aboody, Karen S

    2010-01-01

    Monoclonal antibodies are important tools for cancer therapy, however, three factors limit their effectiveness: toxicity, poor tumor penetration, and inability to cross the blood-brain barrier. This review discusses the emerging field of stem cell-mediated antibody delivery and how this approach may improve antibody therapy of cancer by overcoming these obstacles. STEM CELLS 2010;28:2084–2087 PMID:21089119

  19. A Nanoparticle Platform To Evaluate Bioconjugation and Receptor-Mediated Cell Uptake Using Cross-Linked Polyion Complex Micelles Bearing Antibody Fragments.

    PubMed

    Florinas, Stelios; Liu, Marc; Fleming, Ryan; Van Vlerken-Ysla, Lilian; Ayriss, Joanne; Gilbreth, Ryan; Dimasi, Nazzareno; Gao, Changshou; Wu, Herren; Xu, Ze-Qi; Chen, Shaoyi; Dirisala, Anjaneyulu; Kataoka, Kazunori; Cabral, Horacio; Christie, R James

    2016-05-09

    Targeted nanomedicines are a promising technology for treatment of disease; however, preparation and characterization of well-defined protein-nanoparticle systems remain challenging. Here, we describe a platform technology to prepare antibody binding fragment (Fab)-bearing nanoparticles and an accompanying real-time cell-based assay to determine their cellular uptake compared to monoclonal antibodies (mAbs) and Fabs. The nanoparticle platform was composed of core-cross-linked polyion complex (PIC) micelles prepared from azide-functionalized PEG-b-poly(amino acids), that is, azido-PEG-b-poly(l-lysine) [N3-PEG-b-PLL] and azido-PEG-b-poly(aspartic acid) [N3-PEG-b-PAsp]. These PIC micelles were 30 nm in size and contained approximately 10 polymers per construct. Fabs were derived from an antibody binding the EphA2 receptor expressed on cancer cells and further engineered to contain a reactive cysteine for site-specific attachment and a cleavable His tag for purification from cell culture expression systems. Azide-functionalized micelles and thiol-containing Fab were linked using a heterobifunctional cross-linker (FPM-PEG4-DBCO) that contained a fluorophenyl-maleimide for stable conjugation to Fabs thiols and a strained alkyne (DBCO) group for coupling to micelle azide groups. Analysis of Fab-PIC micelle conjugates by fluorescence correlation spectroscopy, size exclusion chromatography, and UV-vis absorbance determined that each nanoparticle contained 2-3 Fabs. Evaluation of cellular uptake in receptor positive cancer cells by real-time fluorescence microscopy revealed that targeted Fab-PIC micelles achieved higher cell uptake than mAbs and Fabs, demonstrating the utility of this approach to identify targeted nanoparticle constructs with unique cellular internalization properties.

  20. Procedures for Sxs antigen detection by antibody-mediated cytotoxicity tests. A comparative analysis.

    PubMed

    Sánchez, A; Jiménez, R; Burgos, M; Díaz de la Guardia, R

    1994-11-01

    Biological reagents used in the serological detection of Sxs antigen by antibody-mediated cytotoxicity tests were compared in order to optimize the method. Our analyses showed that: (a) red cell-free spleen cells are the best target cells, (b) rabbit serum used as the complement source should be obtained from females, and absorbed with female spleen cells before use, (c) antiserum obtained by immunizing females with repeated injections of syngenic male spleen cells provides the highest anti-Sxs antibody titer, and (d) of the different biological fluids investigated, testis supernatant has highest concentration of Sxs antigen.

  1. The role of complement in antibody-mediated rejection in kidney transplantation.

    PubMed

    Stegall, Mark D; Chedid, Marcio F; Cornell, Lynn D

    2012-11-01

    Over the past decade, several studies have suggested that the complement system has an active role in both acute and chronic allograft rejection. These studies have been facilitated by improved techniques to detect antibody-mediated organ rejection, including immunohistological staining for C4d deposition in the allograft and solid-phase assays that identify donor-specific alloantibodies (DSAs) in the serum of transplant recipients. Studies with eculizumab, a humanized monoclonal antibody directed against complement component C5, have shown that activation of the terminal complement pathway is necessary for the development of acute antibody-mediated rejection in recipients of living-donor kidney allografts who have high levels of DSAs. The extent to which complement activation drives chronic antibody-mediated injury leading to organ rejection is less clear. In chronic antibody-mediated injury, early complement activation might facilitate chemotaxis of inflammatory cells into the allograft in a process that later becomes somewhat independent of DSA levels and complement factors. In this Review, we discuss the different roles that the complement system might have in antibody-mediated allograft rejection, with specific emphasis on renal transplantation.

  2. Antibodies to CD40 prevent Epstein-Barr virus-mediated human B-cell lymphomagenesis in severe combined immune deficient mice given human peripheral blood lymphocytes.

    PubMed

    Murphy, W J; Funakoshi, S; Beckwith, M; Rushing, S E; Conley, D K; Armitage, R J; Fanslow, W C; Rager, H C; Taub, D D; Ruscetti, F W

    1995-09-01

    CD40 is expressed on both normal and neoplastic B lymphocytes. Signal transduction through CD40 in vitro has been shown to exert stimulatory effects on normal B cells and inhibitory effects on Epstein-Barr virus (EBV)-induced B-cell lymphoma lines and some other cell lines derived from patients with aggressive histology lymphoma. The transfer of normal human peripheral blood lymphocytes (huPBL) from EBV-seropositive donors into severe combined immune deficient (SCID) mice has been previously shown to result in the generation of human B-cell lymphomas. These tumors are similar to the highly aggressive EBV-induced lymphomas that can arise clinically after transplantation or in the setting of immunodeficiency. Treatment of huPBL-SCID chimeric mice with anti-CD40 or anti-CD20 monoclonal antibodies (MoAb) significantly delayed the development of EBV-induced B-cell lymphoma. However, the effects of the two MoAb were mechanistically distinct. Anti-CD40 treatment prevented lymphoma generation, while still allowing for functional human B-cell engraftment in the huPBL-SCID mice compared with mice receiving no treatment, all of which succumbed to lymphoma. By contrast, treatment with anti-CD20 significantly inhibited total human B-cell engraftment in the SCID recipients, which accounted for the absence of lymphomas. In vitro assays examining the transformation of human B cells by EBV also indicated that anti-CD40 could directly inhibit EBV-transformation, whereas anti-CD20 antibodies had no effect. Thus, anti-CD40 exerts selective effects to allow for the engraftment of normal human B cells and prevent the emergence of EBV lymphomas. Stimulation of CD40 by antibodies or its physiologic ligand may, therefore, be of significant clinical use in the prevention of EBV-induced B lymphomas that may arise when EBV-seropositive individuals receive immunosuppressive regimens after transplantation or in immune deficiency states, such as acquired immune deficiency syndrome.

  3. Antibody-mediated cofactor-driven reactions

    DOEpatents

    Schultz, Peter G.

    1993-01-01

    Chemical reactions capable of being rate-enhanced by auxiliary species which interact with the reactants but do not become chemically bound to them in the formation of the final product are performed in the presence of antibodies which promote the reactions. The antibodies contain regions within their antigen binding sites which recognize the auxiliary species in a conformation which promotes the reaction. The antigen binding site frequently recognizes a particular transition state complex or other high energy complex along the reaction coordinate, thereby promoting the progress of the reaction along the desired route as opposed to other less favorable routes. Various classes of reaction together with appropriate antigen binding site specificities tailored for each are disclosed.

  4. Acute antibody-mediated rejection of cardiac transplants.

    PubMed

    Reed, Elaine F; Demetris, Anthony J; Hammond, Elizabeth; Itescu, Silviu; Kobashigawa, Jon A; Reinsmoen, Nancy L; Rodriguez, E Rene; Rose, Marlene; Stewart, Susan; Suciu-Foca, Nicole; Zeevi, Adriana; Fishbein, Michael C

    2006-02-01

    Under the direction of the International Society for Heart and Lung Transplantation, a multidisciplinary review of the cardiac biopsy grading system was undertaken in 2004, with task forces examining the areas of histopathology of rejection, clinical issues, and research. An important new area addressed by the Immunopathology Task Force sub-committee was the clinical and diagnostic criteria for antibody-mediated rejection. This article is a companion paper to the revised working formulation for the standardization of nomenclature in the diagnosis of heart rejection and reviews the published literature documenting the serologic and morphologic evidence that antibody-mediated rejection is clinically significant and associated with graft loss, accelerated transplant-associated coronary artery disease, and death. This article also provides a more in-depth analysis of antibody-mediated rejection developed by the Immunopathology Task Force for revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection.

  5. Antibody-Mediated Clearance of Alphavirus Infection from Neurons

    NASA Astrophysics Data System (ADS)

    Levine, Beth; Hardwick, J. Marie; Trapp, Bruce D.; Crawford, Thomas O.; Bollinger, Robert C.; Griffin, Diane E.

    1991-11-01

    Humoral immunity is important for protection against viral infection and neutralization of extracellular virus, but clearance of virus from infected tissues is thought to be mediated solely by cellular immunity. However, in a SCID mouse model of persistent alphavirus encephalomyelitis, adoptive transfer of hyperimmune serum resulted in clearance of infectious virus and viral RNA from the nervous system, whereas adoptive transfer of sensitized T lymphocytes had no effect on viral replication. Three monoclonal antibodies to two different epitopes on the E2 envelope glycoprotein mediated viral clearance. Treatment of alphavirus-infected primary cultured rat neurons with these monoclonal antibodies to E2 resulted in decreased viral protein synthesis, followed by gradual termination of mature infectious virion production. Thus, antibody can mediate clearance of alphavirus infection from neurons by restricting viral gene expression.

  6. Antibody-mediated neutralization of flaviviruses: A reductionist view

    PubMed Central

    Dowd, Kimberly A.; Pierson, Theodore C.

    2011-01-01

    Flaviviruses are a group of ~70 small RNA viruses responsible for significant morbidity and mortality across the globe. Efforts to develop effective vaccines for several clinically important flaviviruses are underway. Antibodies are a significant component of the host’s protective response against flavivirus infection with the potential to contribute to immunity via several distinct mechanisms, including an ability to directly neutralize virus infection. Conversely, virus-reactive antibodies have been implicated in the increased risk of severe clinical manifestations following secondary dengue virus infection. In this review, we will discuss recent progress toward understanding the molecular basis of antibody-mediated neutralization of flaviviruses. Neutralization requires engagement of the virion with a stoichiometry that exceeds a required threshold. From this perspective, we will discuss viral and host factors that impact the number of antibody molecules bound to the virus particle and significantly modulate the potency of neutralizing antibodies. PMID:21255816

  7. The alpha-receptor for platelet-derived growth factor as a target for antibody-mediated inhibition of skeletal metastases from prostate cancer cells.

    PubMed

    Russell, M R; Jamieson, W L; Dolloff, N G; Fatatis, A

    2009-01-22

    Bone resorption by osteoclasts is thought to promote the proliferation of prostate cancer cells disseminated to the skeleton (Mundy, 2002). Using a mouse model of experimental metastasis, we found that although late-stage metastatic tumors were indeed surrounded by osteoclasts, these cells were spatially unrelated to the small foci of cancer cells in early-stage metastases. This is the first evidence that survival and growth of disseminated prostate cancer cells immediately after their extravasation may not depend on osteoclast involvement. Interestingly, prostate cancer cells expressing the alpha-receptor for platelet-derived growth factor (PDGFRalpha) progress during early-stages of skeletal dissemination, whereas cells expressing lower levels or lacking this receptor fail to survive after extravasation in the bone marrow. However, non-metastatic cells acquire bone-metastatic potential upon ectopic overexpression of PDGFRalpha. Finally, functional blockade of human PDGFRalpha on prostate cancer cells utilizing a novel humanized monoclonal antibody -- soon to undergo phase-II clinical trials -- significantly impairs the establishment of early skeletal metastases. In conclusion, our results strongly implicate PDGFRalpha in prostate cancer bone tropism through its promotion of survival and progression of early-metastatic foci, providing ground for therapeutic strategies aimed at preventing or containing the initial progression of skeletal metastases in patients affected by prostate adenocarcinoma.

  8. Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21)

    PubMed Central

    McEwan, William A.; Bidgood, Susanna R.; Towers, Greg J.; Johnson, Chris M.; James, Leo C.

    2010-01-01

    Antibodies provide effective antiviral immunity despite the fact that viruses escape into cells when they infect. Here we show that antibodies remain attached to viruses after cell infection and mediate an intracellular immune response that disables virions in the cytosol. We have discovered that cells possess a cytosolic IgG receptor, tripartite motif-containing 21 (TRIM21), which binds to antibodies with a higher affinity than any other IgG receptor in the human body. TRIM21 rapidly recruits to incoming antibody-bound virus and targets it to the proteasome via its E3 ubiquitin ligase activity. Proteasomal targeting leads to rapid degradation of virions in the cytosol before translation of virally encoded genes. Infection experiments demonstrate that at physiological antibody concentrations TRIM21 neutralizes viral infection. These results reveal an intracellular arm of adaptive immunity in which the protection mediated by antibodies does not end at the cell membrane but continues inside the cell to provide a last line of defense against infection. PMID:21045130

  9. Antibody Against Integrin Lymphocyte Function-Associated Antigen 1 Inhibits HIV Type 1 Infection in Primary Cells Through Caspase-8-Mediated Apoptosis

    PubMed Central

    Walker, Tiffany N.; Cimakasky, Lisa M.; Coleman, Ebony M.; Madison, M. Nia

    2013-01-01

    Abstract HIV-1 infection induces formation of a virological synapse wherein CD4, chemokine receptors, and cell-adhesion molecules such as lymphocyte function-associated antigen 1 (LFA-1) form localized domains on the cell surface. Studies show that LFA-1 on the surface of HIV-1 particles retains its adhesion function and enhances virus attachment to susceptible cells by binding its counterreceptor intercellular adhesion molecule 1 (ICAM-1). This virus–cell interaction augments virus infectivity by facilitating binding and entry events. In this study, we demonstrate that inhibition of the LFA-1/ICAM-1 interaction by a monoclonal antibody leads to decreased virus production and spread in association with increased apoptosis of HIV-infected primary T cells. The data indicate that the LFA-1/ICAM-1 interaction may limit apoptosis in HIV-1-infected T cells. This phenomenon appears similar to anoikis wherein epithelial cells are protected from apoptosis conferred by ligand-bound integrins. These results have implications for further understanding HIV pathogenesis and replication in peripheral compartments and lymphoid organs. PMID:22697794

  10. Antibody against integrin lymphocyte function-associated antigen 1 inhibits HIV type 1 infection in primary cells through caspase-8-mediated apoptosis.

    PubMed

    Walker, Tiffany N; Cimakasky, Lisa M; Coleman, Ebony M; Madison, M Nia; Hildreth, James E K

    2013-02-01

    HIV-1 infection induces formation of a virological synapse wherein CD4, chemokine receptors, and cell-adhesion molecules such as lymphocyte function-associated antigen 1 (LFA-1) form localized domains on the cell surface. Studies show that LFA-1 on the surface of HIV-1 particles retains its adhesion function and enhances virus attachment to susceptible cells by binding its counterreceptor intercellular adhesion molecule 1 (ICAM-1). This virus-cell interaction augments virus infectivity by facilitating binding and entry events. In this study, we demonstrate that inhibition of the LFA-1/ICAM-1 interaction by a monoclonal antibody leads to decreased virus production and spread in association with increased apoptosis of HIV-infected primary T cells. The data indicate that the LFA-1/ICAM-1 interaction may limit apoptosis in HIV-1-infected T cells. This phenomenon appears similar to anoikis wherein epithelial cells are protected from apoptosis conferred by ligand-bound integrins. These results have implications for further understanding HIV pathogenesis and replication in peripheral compartments and lymphoid organs.

  11. Controlled Human Malaria Infection (CHMI) differentially affects cell-mediated and antibody responses to CSP and AMA1 induced by adenovirus vaccines with and without DNA-priming.

    PubMed

    Sedegah, Martha; Hollingdale, Michael R; Farooq, Fouzia; Ganeshan, Harini; Belmonte, Maria; Huang, Jun; Abot, Esteban; Limbach, Keith; Chuang, Ilin; Tamminga, Cindy; Epstein, Judith E; Villasante, Eileen

    2015-01-01

    We have previously shown that a DNA-prime followed by an adenovirus-5 boost vaccine containing CSP and AMA1 (DNA/Ad) successfully protected 4 of 15 subjects to controlled human malaria infection (CHMI). However, the adenovirus-5 vaccine alone (AdCA) failed to induce protection despite eliciting cellular responses that were often higher than those induced by DNA/Ad. Here we determined the effect of CHMI on pre-CHMI cellular and antibody responses against CSP and AMA1 expressed as fold-changes in activities. Generally, in the DNA/Ad trial, CHMI caused pre-CHMI ELISpot IFN-γ and CD8+ T cell IFN-γ responses of the protected subjects to fall but among non-protected subjects, CHMI caused rises of pre-CHMI ELISpot IFN-γ but falls of CD8+ T cell IFN-γ responses. In contrast in the AdCA trial, CHMI caused both pre-CHMI ELISpot IFN-γ and CD8+ T cell IFN-γ responses of the AdCA subjects to fall. We suggest that the falls in activities are due to migration of peripheral CD8+ T cells to the liver in response to developing liver stage parasites, and this fall, in the DNA/Ad trial, is masked in ELISpot responses of the non-protected subjects by rises in other immune cell types. In addition, CHMI caused falls in antibody activities of protected subjects, but rises in non-protected subjects in both trials to CSP, and dramatically in the AdCA trial to AMA1, reaching 380 μg/ml that is probably due to boosting by transient blood stage infection before chloroquine treatment. Taken together, these results further define differences in cellular responses between DNA/Ad and AdCA trials, and suggest that natural transmission may boost responses induced by these malaria vaccines especially when protection is not achieved.

  12. Controlled Human Malaria Infection (CHMI) differentially affects cell-mediated and antibody responses to CSP and AMA1 induced by adenovirus vaccines with and without DNA-priming

    PubMed Central

    Sedegah, Martha; Hollingdale, Michael R; Farooq, Fouzia; Ganeshan, Harini; Belmonte, Maria; Huang, Jun; Abot, Esteban; Limbach, Keith; Chuang, Ilin; Tamminga, Cindy; Epstein, Judith E; Villasante, Eileen

    2015-01-01

    We have previously shown that a DNA-prime followed by an adenovirus-5 boost vaccine containing CSP and AMA1 (DNA/Ad) successfully protected 4 of 15 subjects to controlled human malaria infection (CHMI). However, the adenovirus-5 vaccine alone (AdCA) failed to induce protection despite eliciting cellular responses that were often higher than those induced by DNA/Ad. Here we determined the effect of CHMI on pre-CHMI cellular and antibody responses against CSP and AMA1 expressed as fold-changes in activities. Generally, in the DNA/Ad trial, CHMI caused pre-CHMI ELISpot IFN-γ and CD8+ T cell IFN-γ responses of the protected subjects to fall but among non-protected subjects, CHMI caused rises of pre-CHMI ELISpot IFN-γ but falls of CD8+ T cell IFN-γ responses. In contrast in the AdCA trial, CHMI caused both pre-CHMI ELISpot IFN-γ and CD8+ T cell IFN-γ responses of the AdCA subjects to fall. We suggest that the falls in activities are due to migration of peripheral CD8+ T cells to the liver in response to developing liver stage parasites, and this fall, in the DNA/Ad trial, is masked in ELISpot responses of the non-protected subjects by rises in other immune cell types. In addition, CHMI caused falls in antibody activities of protected subjects, but rises in non-protected subjects in both trials to CSP, and dramatically in the AdCA trial to AMA1, reaching 380 μg/ml that is probably due to boosting by transient blood stage infection before chloroquine treatment. Taken together, these results further define differences in cellular responses between DNA/Ad and AdCA trials, and suggest that natural transmission may boost responses induced by these malaria vaccines especially when protection is not achieved. PMID:26292027

  13. Varicella-zoster virus-specific, cell-mediated immunity with interferon-gamma release assay after vaccination of college students with no or intermediate IgG antibody response.

    PubMed

    Terada, Kihei; Itoh, Yuri; Fujii, Akihide; Kitagawa, Seiko; Ogita, Satoko; Ouchi, Kazunobu

    2015-02-01

    This study measured Varicella-zoster virus (VZV) specific cell-mediated immunity (CMI) and antibodies to clarify immune response after vaccination in 68 college students with negative or intermediate IgG antibody status. The enrolled numbers of negative, intermediate, and positive VZV-IgG antibody were 27, 41, and 28 students, respectively. The positive rates of CMI were 3.7% (1/27), 41.5% (17/41), and 96.4% (27/28) before vaccination, respectively. After vaccination, the IgG antibody titers became significantly higher in the intermediate IgG group compared to those in the negative IgG group (P < 0.01), but CMI did not differ significantly between the two groups. Ninety-three percent (38/41) of the intermediate IgG antibody group and 41% (11/27) of the negative IgG antibody group became positive for the IgG antibody after vaccination (P < 0.0001). When subjects were divided into negative, intermediate, and positive CMI by interferon-gamma values before vaccination, the IgG antibody and interferon-gamma values increased significantly in the positive CMI group compared to the negative CMI group after vaccination (P < 0.01 and P < 0.01, respectively). All (17/17) of positive CMI group and 61% (27/44) of negative CMI group became positive for the IgG antibody after vaccination (P < 0.01). Ninety-four percent (16/17) of positive CMI group and 59% (28/44) of negative CMI group became positive for CMI after vaccination (P < 0.05). Ninety-six percent (22/23) of the subjects with a history of vaccination became IgG seropositive after a second dose of vaccination, but 22% (5/23) of them remained negative for CMI. CMI is valuable information to identify potential non-responders to vaccination and to predict risk of clinical VZV infection.

  14. HIV-1 Replication in Langerhans and Interstitial Dendritic Cells Is Inhibited by Neutralizing and Fc-Mediated Inhibitory Antibodies ▿ †

    PubMed Central

    Peressin, M.; Holl, V.; Schmidt, S.; Decoville, T.; Mirisky, D.; Lederle, A.; Delaporte, M.; Xu, K.; Aubertin, A. M.; Moog, C.

    2011-01-01

    Langerhans cells (LCs) and interstitial dendritic cells (IDCs) may be among the first human immunodeficiency virus type 1 (HIV-1) targets after sexual transmission. We generated cells of these types by differentiation of purified CD34+ cord blood cells. After in vitro infection with R5-tropic strains, we obtained similar percentages of infected cells for both dendritic cell (DC) subsets. Moreover, LC infection was not increased by blockage of langerin by antilangerin. These results indicate that, under our experimental conditions, there was no evidence of any preference of HIV replication in LCs versus IDCs. The inhibitory activity of HIV-1-specific IgAs and IgGs against HIV-1 replication in LCs and IDCs was analyzed. We found that neutralizing antibodies inhibit HIV-1 infection of both DC subsets. Interestingly, HIV-1 was inhibited more efficiently by the IgGs than the corresponding IgA, due to an Fcγ receptor-dependent mechanism. Moreover, nonneutralizing inhibitory IgGs were able to inhibit infection of both LCs and IDCs. These results underline the importance of HIV-1 inhibition by the binding of the Fc part of IgGs to Fcγ receptors and suggest that the induction of neutralizing and nonneutralizing inhibitory IgGs in addition to neutralizing IgAs at mucosal sites may contribute to protection against sexual transmission of HIV-1. PMID:21084491

  15. Selective suppression of Th2 cell-mediated lung eosinophilic inflammation by anti-major facilitator super family domain containing 10 monoclonal antibody.

    PubMed

    Nishimura, Tomoe; Saeki, Mayumi; Motoi, Yuji; Kitamura, Noriko; Mori, Akio; Kaminuma, Osamu; Hiroi, Takachika

    2014-05-01

    The eosinophil is deeply associated with the pathogenesis of bronchial asthma and other allergic diseases. We recently identified a novel eosinophil-specific cell surface molecule, major facilitator super family domain containing 10 (Mfsd10). A monoclonal antibody (mAb) against Mfsd10 (M2) showed selective binding and neutralizing activities for eosinophils. However, the relative potency of the blockage of Mfsd10 and other eosinophil-specific molecules for the treatment of allergic diseases has not been evaluated. Therefore, in this study, the effects of M2 and an anti-Siglec-F mAb on antigen-immunized and antigen-specific Th2 cell-transferred murine eosinophilic inflammation models were comparatively investigated. Ovalbumin (OVA)-specific Th2 cells were differentiated from naïve CD4+ T cells of DO11.10/RAG-2-/- mice in vitro and cytokine producing activity of the Th2 cells was examined. OVA-immunized and Th2 cell-transferred BALB/c mice were treated with M2 or anti-Siglec-F and challenged with OVA. Then the number of inflammatory cells and the concentration of IL-5 in the bronchoalveolar lavage fluid (BALF) were determined. Antigen-specific Th2 cells produced large amounts of IL-4, IL-5 and IL-13 but not IL-17A or IFN-γ. Administration of M2 significantly suppressed antigen-induced lung eosinophil infiltration both in OVA-immunized and Th2 cell-transferred mice. The potency as well as selectivity of M2 for down-regulating eosinophils was quite similar to that of anti-Siglec-F. Both mAbs did not affect antigen-induced IL-5 production in the lungs. Mfsd10 as well as Siglec-F could be an effective target to treat eosinophil-related disorders including bronchial asthma.

  16. Complement-fixing antibodies against denatured HLA and MICA antigens are associated with antibody mediated rejection.

    PubMed

    Cai, Junchao; Terasaki, Paul I; Zhu, Dong; Lachmann, Nils; Schönemann, Constanze; Everly, Matthew J; Qing, Xin

    2016-02-01

    We have found antibodies against denatured HLA class I antigens in the serum of allograft recipients which were not significantly associated with graft failure. It is unknown whether transplant recipients also have denatured HLA class II and MICA antibodies. The effects of denatured HLA class I, class II, and MICA antibodies on long-term graft outcome were further investigated based on their ability to fix complement c1q. In this 4-year retrospective cohort study, post-transplant sera from 975 kidney transplant recipients were tested for antibodies against denatured HLA/MICA antigens and these antibodies were further classified based on their ability to fix c1q. Thirty percent of patients had antibodies against denatured HLA class I, II, or MICA antigens. Among them, 8.5% and 21.5% of all patients had c1q-fixing and non c1q-fixing antibodies respectively. There was no significant difference on graft survival between patients with or without antibodies against denatured HLA/MICA. However, when these antibodies were further classified according to their ability to fix c1q, patients with c1q-fixing antibodies had a significantly lower graft survival rate than patients without antibodies or patients with non c1q-fixing antibodies (p=0.008). In 169 patients who lost renal grafts, 44% of them had c1q-fixing antibodies against denatured HLA/MICA antigens, which was significantly higher than that in patients with functioning renal transplants (25%, p<0.0001). C1q-fixing antibodies were more significantly associated with graft failure caused by AMR (72.73%) or mixed AMR/CMR (61.9%) as compared to failure due to CMR (35.3%) or other causes (39.2%) (p=0.026). Transplant recipients had antibodies against denatured HLA class I, II, and MICA antigens. However, only c1q-fixing antibodies were associated with graft failure which was related to antibody mediated rejection. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Targeted Fcγ Receptor (FcγR)-mediated Clearance by a Biparatopic Bispecific Antibody*

    PubMed Central

    Kasturirangan, Srinath; Rainey, G. Jonah; Xu, Linda; Wang, Xinwei; Portnoff, Alyse; Chen, Tracy; Fazenbaker, Christine; Zhong, Helen; Bee, Jared; Zeng, Zhutian; Jenne, Craig; Wu, Herren; Gao, Changshou

    2017-01-01

    Soluble ligands have commonly been targeted by antibody therapeutics for cancers and other diseases. Although monoclonal antibodies targeting such ligands can block their interactions with their cognate receptors, they can also significantly increase the half-life of their ligands by FcRn-mediated antibody recycling, thereby evading ligand renal clearance and requiring increasingly high antibody doses to neutralize the increasing pool of target. To overcome this issue, we generated a bispecific/biparatopic antibody (BiSAb) that targets two different epitopes on IL-6 to block IL-6-mediated signaling. The BiSAb formed large immune complexes with IL-6 that can bind Fcγ receptors on phagocytic cells and are rapidly internalized. In addition, rapid clearance of the BiSAb·IL-6 complex was observed in mice while the parental antibodies prolonged the serum half-life of IL-6. Intravital imaging of the liver in mice confirmed that the rapid clearance of these large immune complexes was associated with Fcγ receptor-dependent binding to Kupffer cells in the liver. The approach described here provides a general strategy for therapeutic antibodies with the ability to not only neutralize but also actively drive clearance of their soluble antigens. PMID:28100773

  18. Targeted Fcγ Receptor (FcγR)-mediated Clearance by a Biparatopic Bispecific Antibody.

    PubMed

    Kasturirangan, Srinath; Rainey, G Jonah; Xu, Linda; Wang, Xinwei; Portnoff, Alyse; Chen, Tracy; Fazenbaker, Christine; Zhong, Helen; Bee, Jared; Zeng, Zhutian; Jenne, Craig; Wu, Herren; Gao, Changshou

    2017-03-10

    Soluble ligands have commonly been targeted by antibody therapeutics for cancers and other diseases. Although monoclonal antibodies targeting such ligands can block their interactions with their cognate receptors, they can also significantly increase the half-life of their ligands by FcRn-mediated antibody recycling, thereby evading ligand renal clearance and requiring increasingly high antibody doses to neutralize the increasing pool of target. To overcome this issue, we generated a bispecific/biparatopic antibody (BiSAb) that targets two different epitopes on IL-6 to block IL-6-mediated signaling. The BiSAb formed large immune complexes with IL-6 that can bind Fcγ receptors on phagocytic cells and are rapidly internalized. In addition, rapid clearance of the BiSAb·IL-6 complex was observed in mice while the parental antibodies prolonged the serum half-life of IL-6. Intravital imaging of the liver in mice confirmed that the rapid clearance of these large immune complexes was associated with Fcγ receptor-dependent binding to Kupffer cells in the liver. The approach described here provides a general strategy for therapeutic antibodies with the ability to not only neutralize but also actively drive clearance of their soluble antigens.

  19. Antibody-dependent cellular cytotoxicity-mediating antibodies from an HIV-1 vaccine efficacy trial target multiple epitopes and preferentially use the VH1 gene family.

    PubMed

    Bonsignori, Mattia; Pollara, Justin; Moody, M Anthony; Alpert, Michael D; Chen, Xi; Hwang, Kwan-Ki; Gilbert, Peter B; Huang, Ying; Gurley, Thaddeus C; Kozink, Daniel M; Marshall, Dawn J; Whitesides, John F; Tsao, Chun-Yen; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Rerks-Ngarm, Supachai; Kim, Jerome H; Michael, Nelson L; Tomaras, Georgia D; Montefiori, David C; Lewis, George K; DeVico, Anthony; Evans, David T; Ferrari, Guido; Liao, Hua-Xin; Haynes, Barton F

    2012-11-01

    The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n = 19), a non-A32-blockable conformational epitope (n = 1), and the gp120 Env variable loops (n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs.

  20. Antibody-Mediated Phosphatidylserine Blockade Enhances the Antitumor Responses to CTLA-4 and PD-1 Antibodies in Melanoma.

    PubMed

    Freimark, Bruce D; Gong, Jian; Ye, Dan; Gray, Michael J; Nguyen, Van; Yin, Shen; Hatch, Michaela M S; Hughes, Christopher C W; Schroit, Alan J; Hutchins, Jeff T; Brekken, Rolf A; Huang, Xianming

    2016-06-01

    In tumor-bearing animals, the membrane phospholipid phosphatidylserine (PS) suppresses immune responses, suggesting that PS signaling could counteract the antitumor effect of antibody-driven immune checkpoint blockade. Here, we show that treating melanoma-bearing mice with a PS-targeting antibody enhances the antitumor activity of downstream checkpoint inhibition. Combining PS-targeting antibodies with CTLA-4 or PD-1 blockade resulted in significantly greater inhibition of tumor growth than did single-agent therapy. Moreover, combination therapy enhanced CD4(+) and CD8(+) tumor-infiltrating lymphocyte numbers; elevated the fraction of cells expressing the proinflammatory cytokines IL2, IFNγ, and TNFα; and increased the ratio of CD8 T cells to myeloid-derived suppressor cells and regulatory T cells in tumors. Similar changes in immune cell profiles were observed in splenocytes. Taken together, these data show that antibody-mediated PS blockade enhances the antitumor efficacy of immune checkpoint inhibition. Cancer Immunol Res; 4(6); 531-40. ©2016 AACR.

  1. Oral administration of Aloe vera gel, anti-microbial and anti-inflammatory herbal remedy, stimulates cell-mediated immunity and antibody production in a mouse model

    PubMed Central

    Bałan, Barbara Joanna; Niemcewicz, Marcin; Kocik, Janusz; Jung, Leszek; Skopiński, Piotr

    2014-01-01

    Introduction Aloe vera (L.) Burm. f. (Aloe barbadensis Mill) Liliaceae, succulent plant native to northern Africa, is presently cultivated in many regions of the world. Traditionally, its inner part of parenchyma, which contains aloe gel, was used for the treatment of minor wounds, inflammatory skin disorders, thermal and radiation burns and to alleviate chronic osteoarthritis pain. It also possesses some antimicrobial activity. Now, aloe gel is also increasingly consumed as a dietary supplement. Some data suggest its immunomodulatory properties. The aim of the study The aim of the study was to evaluate the influence of orally administered aloe gel on some parameters of cellular and humoral immunity viz. mitogen-induced proliferation of splenic lymphocytes and their chemokinetic activity, and anti-sheep red blood cells (SRBC) antibody production in Balb/c mice. Results Daily treatment of mice for 14 and 21 days with 50 µl or 150 µl of aloe gel dose resulted in enhanced chemokinetic activity and stronger response of their splenic lymphocytes to mitogen PHA and enhancement of anti-SRBC antibody production. PMID:26155113

  2. Epitope distance to the target cell membrane and antigen size determine the potency of T cell-mediated lysis by BiTE antibodies specific for a large melanoma surface antigen.

    PubMed

    Bluemel, Claudia; Hausmann, Susanne; Fluhr, Petra; Sriskandarajah, Mirnalini; Stallcup, William B; Baeuerle, Patrick A; Kufer, Peter

    2010-08-01

    Melanoma chondroitin sulfate proteoglycan (MCSP; also called CSPG4, NG2, HMW-MAA, MSK16, MCSPG, MEL-CSPG, or gp240) is a surface antigen frequently expressed on human melanoma cells, which is involved in cell adhesion, invasion and spreading, angiogenesis, complement inhibition, and signaling. MCSP has therefore been frequently selected as target antigen for development of antibody- and vaccine-based therapeutic approaches. We have here used a large panel of monoclonal antibodies against human MCSP for generation of single-chain MCSP/CD3-bispecific antibodies of the BiTE (for bispecific T cell engager) class. Despite similar binding affinity to MCSP, respective BiTE antibodies greatly differed in their potency of redirected lysis of CHO cells stably transfected with full-length human MCSP, or with various MCSP deletion mutants and fusion proteins. BiTE antibodies binding to the membrane proximal domain D3 of MCSP were more potent than those binding to more distal domains. This epitope distance effect was corroborated with EpCAM/CD3-bispecific BiTE antibody MT110 by testing various fusion proteins between MCSP and EpCAM as surface antigens. CHO cells expressing small surface target antigens were generally better lysed than those expressing larger target antigens, indicating that antigen size was also an important determinant for the potency of BiTE antibody. The present study for the first time relates the positioning of binding domains and size of surface antigens to the potency of target cell lysis by BiTE-redirected cytotoxic T cells. In case of the MCSP antigen, this provides the basis for selection of a maximally potent BiTE antibody candidate for development of a novel melanoma therapy.

  3. Similar Idiotypes in Antibody-Forming Cells and in Cells Synthesizing Immunoglobulins Without Detectable Antibody Function

    PubMed Central

    Cazenave, P. -A.; Ternynck, T.; Avrameas, S.

    1974-01-01

    The occurrence of immunoglobulins with and without antibody specificity and with and without idiotypic specificity was studied, by use of enzyme-labeled antigen and antibodies, in lymph node cells of rabbits immunized with horse-radish peroxidase and hen ovalbumin. Some cells, containing immunoglobulins without detectable antibody function, were shown to contain idiotypes similar to those found in antibody-producing cells. PMID:4140504

  4. Monoclonal antibodies reacting with murine teratocarcinoma cells.

    PubMed Central

    Goodfellow, P N; Levinson, J R; Williams, V E; McDevitt, H O

    1979-01-01

    Monoclonal antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a rat immunized with the C3H mouse teratocarcinoma C86-S1. After the fusion two clones were chosen for further analysis. The first clone, 3C4-10, produced an antibody recognizing an antigen with a distribution restricted to teratocarcinoma cell lines, an endoderm cell line, and a neuroblastoma. The second clone, 4A1-9, produced an antibody that reacted with all cultured murine cells tested and adult brain. Neither antibody reacted with preimplantation embryos. The 3C4-10 antibody recognized an antigen associated with proteins. The apparent molecular weight of the 3C4-10 antigen was greater than 100,000. PMID:284353

  5. Soluble Envelope Glycoprotein Trimers from a CD4-Independent HIV-1 Elicit Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies in Guinea Pigs

    PubMed Central

    Murray, Marta K.; Teran, Victor A.; Chapleau, Jean-Philippe; Wang, Baomin; Kim, Su Hyon; LaBranche, Celia C.; Richard, Jonathan; Montefiori, David C.

    2015-01-01

    CD4-independent HIV-1 variants can infect coreceptor-expressing cells lacking CD4. The envelope (Env) glycoproteins on these HIV-1 variants expose a coreceptor binding site that overlaps some CD4-induced (CD4i) epitopes. Reports have demonstrated that CD4i antibodies mediate antibody-dependent cellular cytotoxicity (ADCC). Here we investigated the immunogenicity of soluble Env trimers (sgp140) from a CD4-independent HIV-1 in guinea pigs and found that the sgp140 elicited ADCC-mediating antibodies. Therefore, these sgp140 might be useful in vaccine regimens aimed at eliciting ADCC responses. PMID:26246571

  6. Consensus opinion from the antibody working group on the diagnosis, reporting, and risk assessment for antibody-mediated rejection and desensitization protocols.

    PubMed

    Montgomery, Robert A; Hardy, Mark A; Jordan, Stanley C; Racusen, Lorraine C; Ratner, Lloyd E; Tyan, Dolly B; Zachary, Andrea A

    2004-07-27

    During the past few decades, much of the experimental and clinical effort in solid-organ transplantation has been directed toward ameliorating or abrogating T-cell-mediated responses. As a result, universally understood and accepted nomenclature and diagnostic criteria have evolved. Humoral immunity in transplantation has yet to undergo a similar renaissance. Readers of transplant journals regularly find it difficult and often impossible to interpret data on the diagnosis and management of antibody-mediated rejection. The Antibody Working Group was assembled in an attempt to provide guidelines for the standardization of nomenclature, diagnostic criteria, reporting, antibody profiling, and risk assessment.

  7. Patient-Derived Antibody Targets Tumor Cells

    Cancer.gov

    An NCI Cancer Currents blog on an antibody derived from patients that killed tumor cells in cell lines of several cancer types and slowed tumor growth in mouse models of brain and lung cancer without evidence of side effects.

  8. Montanide™ ISA 71 VG adjuvant enhances antibody and cell-mediated immune responses to profilin subunit antigen vaccination and promotes protection against Eimeria acervulina and Eimeria tenella.

    PubMed

    Jang, Seung I; Lillehoj, Hyun S; Lee, Sung Hyen; Lee, Kyung Woo; Lillehoj, Erik P; Bertrand, François; Dupuis, Laurent; Deville, Sébastien

    2011-01-01

    The present study was conducted to investigate the immunoenhancing effects of Montanide™ ISA 71 VG adjuvant on profilin subunit antigen vaccination. Broiler chickens were immunized subcutaneously with a purified Eimeria acervulina recombinant profilin protein, either alone or mixed with ISA 71 VG, and host immune responses were evaluated. After secondary immunization, antigen-specific antibody and T-cell responses were higher in the group which received profilin plus ISA 71 VG compared with the other groups. Furthermore, body weight gains and fecal oocyst shedding were evaluated following oral challenge infection with live E. acervulina or Eimeria tenella oocysts. Vaccination with profilin plus ISA 71 VG reduced oocyst shedding compared with animals immunized with profilin alone. These results demonstrate that the recombinant profilin subunit vaccine, when given in combination with Montanide™ ISA 71 VG, augments protective immunity against E. acervulina and E. tenella. Published by Elsevier Inc.

  9. Are matrix-immobilized neoglycoproteins, plant and human lectins and carbohydrate--binding antibodies from human serum mediators of adhesion in vitro for carcinoma and lymphosarcoma cells?

    PubMed

    Wawotzny, R; André, S; Dong, X; Joshi, S S; Gabius, H J

    1995-01-01

    Mediation of cell adhesion by defined molecules can be studied by their immobilization onto a nitrocellulose matrix and incubation with cells. In order to infer the capacity of deliberately selected protein-carbohydrate interactions to establish sugar-inhibitable cell adhesion, a panel of immobilized neoglycoproteins was employed for the murine lymphosarcoma lines RAW-117 with low (P) and high (H10) metastatic capacity, a human mammary carcinoma line (DU4475) and three human colon carcinoma lines (C205, SW480, SW620). Exhibiting an otherwise rather similar behavior relative to the line with low metastatic potential, the murine line RAW117-H10 bound strongly to the matrix with carboxyl group-bearing N-acetylneuraminic acid and glucuronic acid as well as rhamnose. Whereas the analysis of carbohydrate-mediated adhesion yielded comparable results for the three colon carcinoma lines, a markedly reduced number of adherent cells was counted for matrix-attached alpha- and beta-galactosyl, alpha-mannosyl and alpha-glucosyl moieties in the case of the mammary carcinoma line, raising evidence for cell lineage-dependent alterations of this property. From the carbohydrate-binding proteins, the plant lectin, concanavalin A and Viscum album agglutinin almost invariably served well as cell adhesion molecules. Appropriate cell surface sugar receptors, probed with neoglycoproteins, and glycoconjugates, probed with lectins, thus can contribute to adhesion in this model system. The immobilized human beta-galactoside-binding lectin (Mr 14kDa) caused adhesion of the murine lines and one colon carcinoma line (SW480). Neither C-reactive protein under conditions that induce its activity as lectin nor serum amyloid P component nor a lactose-binding immunoglobulin G fraction from human serum were reactive. However, cell adhesion to the alpha-galactoside-binding immunoglobulin G fraction of human serum was seen with the murine line of low metastatic capacity and the mammary carcinoma line

  10. Designing a HER2/neu promoter to drive α1,3galactosyltransferase expression for targeted anti-αGal antibody-mediated tumor cell killing

    PubMed Central

    Lanteri, Marion; Ollier, Laurence; Giordanengo, Valérie; Lefebvre, Jean-Claude

    2005-01-01

    Introduction Our goal was to specifically render tumor cells susceptible to natural cytolytic anti-αGal antibodies by using a murine α1,3galactosyltransferase (mαGalT) transgene driven by a designed form of HER2/neu promoter (pNeu), the transcription of which is frequently observed to be above basal in breast tumors. Indeed, the αGalT activity that promotes Galα1,3Galβ1,4GlcNAc-R (αGal) epitope expression has been mutationally disrupted during the course of evolution, starting from Old World primates, and this has led to the counter-production of large amounts of cytotoxic anti-αGal antibodies in recent primates, including man. Method Expression of the endogenous c-erbB-2 gene was investigated in various cell lines by northern blotting. A mαGalT cDNA was constructed into pcDNA3 vector downstream of the original CMV promoter (pCMV/mαGalT) and various forms of pNeu were prepared by PCR amplification and inserted in the pCMV/mαGalT construct upstream of the mαGalT cDNA, in the place of the CMV promoter. These constructs were transferred into HEK-293 control and breast tumor cell lines. Stably transfected cells were analyzed by northern blotting for their expression of αGalT and c-erbB-2, and by flow cytometry for their binding with fluorescein isothiocyanate-conjugated Griffonia simplicifolia/isolectin B4. Results We show that expression of the mαGalT was up- or down-modulated according to the level of endogenous pNeu activity and the particular form of constructed pNeu. Among several constructs, two particular forms of the promoter, pNeu250 containing the CCAAT box and the PEA3 motif adjacent to the TATAA box, and pNeu664, which has three additional PEA3 motifs upstream of the CCAAT box, were found to promote differential αGalT expression. Conclusion Our results strengthen current concepts about the crucial role played by the proximal PEA3 motif of pNeu, and may represent a novel therapeutic approach for the development of targeted transgene expression

  11. Specific antibody-mediated effect on the immune response

    PubMed Central

    Murgita, R. A.; Vas, S. I.

    1972-01-01

    The effect of prior administration of two classes of 7S mouse anti-sheep red blood cell (SRBC) antibodies on the primary immune response to SRBC was studied. Mouse γG1 and γG2 immunoglobulins were isolated from serum by zone electrophoresis and density gradient isolectric focusing. The immunoglobulins were defined by qualitative and quantitative studies, and their effects on the primary response were determined by administering doses ranging from 0.5 to 0.001 mg of immunoglobulin 2 hours before injection of antigen. Gamma G1 antibody suppressed the response to SRBC at all concentrations. High doses of γG2 antibody partially suppressed 19S plaque-forming cells (PFC), but had no significant effect on serum haemagglutinin (HA) levels. Low doses of γG2 antibody specifically augmented both the 19S PFC and serum HA levels. It is concluded that the specific suppressing and augmenting influences of antibodies on the primary response are a function of class. In addition, it is proposed that γG1 and γG2 antibodies can act as specific regulatory elements during the primary response by exerting these two competing biological effects on antibody synthesis. ImagesFIG. 2 PMID:4554743

  12. Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity

    PubMed Central

    Calow, Jenny; Bockau, Ulrike; Struwe, Weston B.; Nowaczyk, Marc M.; Loser, Karin; Crispin, Max

    2016-01-01

    ABSTRACT Antibody glycosylation is a key parameter in the optimization of antibody therapeutics. Here, we describe the production of the anti-cancer monoclonal antibody rituximab in the unicellular ciliate, Tetrahymena thermophila. The resulting antibody demonstrated enhanced antibody-dependent cell-mediated cytotoxicity, which we attribute to unusual N-linked glycosylation. Detailed chromatographic and mass spectrometric analysis revealed afucosylated, oligomannose-type glycans, which, as a whole, displayed isomeric structures that deviate from the typical human counterparts, but whose branches were equivalent to fragments of metabolic intermediates observed in human glycoproteins. From the analysis of deposited crystal structures, we predict that the ciliate glycans adopt protein-carbohydrate interactions with the Fc domain that closely mimic those of native complex-type glycans. In addition, terminal glucose structures were identified that match biosynthetic precursors of human glycosylation. Our results suggest that ciliate-based expression systems offer a route to large-scale production of monoclonal antibodies exhibiting glycosylation that imparts enhanced cell killing activity. PMID:27594301

  13. Peritoneal lavage cells of Indonesian thin-tail sheep mediate antibody-dependent superoxide radical cytotoxicity in vitro against newly excysted juvenile Fasciola gigantica but not juvenile Fasciola hepatica.

    PubMed

    Piedrafita, David; Estuningsih, Endah; Pleasance, Jill; Prowse, Rhoda; Raadsma, Herman W; Meeusen, Els N T; Spithill, Terry W

    2007-04-01

    Indonesian thin-tail (ITT) sheep resist infection by Fasciola gigantica by an immunological mechanism within 2 to 4 weeks of infection yet are susceptible to F. hepatica infection. Studies of ITT sheep show that little liver damage occurs following F. gigantica infection, suggesting that the invading parasites are killed within the peritoneum or shortly after reaching the liver. We investigated whether cells isolated from the peritoneums of ITT sheep could kill newly excysted juvenile F. gigantica in vitro and act as a potential mechanism of resistance against F. gigantica infection. Peritoneal cells from F. gigantica-infected sheep, rich in macrophages and eosinophils, mediated antibody-dependent cytotoxicity against juvenile F. gigantica in vitro. Cytotoxicity was dependent on contact between the parasite and effector cells. Isolated mammary gland eosinophils of F. gigantica-infected sheep, or resident peritoneal monocytes/macrophages from uninfected sheep, also killed the juvenile parasites in vitro. By using inhibitors, we show that the molecular mechanism of killing in these assays was dependent on the production of superoxide radicals by macrophages and eosinophils. In contrast, this cytotoxic mechanism was ineffective against juvenile F. hepatica parasites in vitro. Analysis of superoxide dismutase activity and mRNA levels showed that activity and gene expression were higher in F. hepatica than in F. gigantica, suggesting a possible role for this enzyme in the resistance of F. hepatica to superoxide-mediated killing. We suggest that ovine macrophages and eosinophils, acting in concert with a specific antibody, may be important effector cells involved in the resistance of ITT sheep to F. gigantica.

  14. Peritoneal Lavage Cells of Indonesian Thin-Tail Sheep Mediate Antibody-Dependent Superoxide Radical Cytotoxicity In Vitro against Newly Excysted Juvenile Fasciola gigantica but Not Juvenile Fasciola hepatica▿

    PubMed Central

    Piedrafita, David; Estuningsih, Endah; Pleasance, Jill; Prowse, Rhoda; Raadsma, Herman W.; Meeusen, Els N. T.; Spithill, Terry W.

    2007-01-01

    Indonesian thin-tail (ITT) sheep resist infection by Fasciola gigantica by an immunological mechanism within 2 to 4 weeks of infection yet are susceptible to F. hepatica infection. Studies of ITT sheep show that little liver damage occurs following F. gigantica infection, suggesting that the invading parasites are killed within the peritoneum or shortly after reaching the liver. We investigated whether cells isolated from the peritoneums of ITT sheep could kill newly excysted juvenile F. gigantica in vitro and act as a potential mechanism of resistance against F. gigantica infection. Peritoneal cells from F. gigantica-infected sheep, rich in macrophages and eosinophils, mediated antibody-dependent cytotoxicity against juvenile F. gigantica in vitro. Cytotoxicity was dependent on contact between the parasite and effector cells. Isolated mammary gland eosinophils of F. gigantica-infected sheep, or resident peritoneal monocytes/macrophages from uninfected sheep, also killed the juvenile parasites in vitro. By using inhibitors, we show that the molecular mechanism of killing in these assays was dependent on the production of superoxide radicals by macrophages and eosinophils. In contrast, this cytotoxic mechanism was ineffective against juvenile F. hepatica parasites in vitro. Analysis of superoxide dismutase activity and mRNA levels showed that activity and gene expression were higher in F. hepatica than in F. gigantica, suggesting a possible role for this enzyme in the resistance of F. hepatica to superoxide-mediated killing. We suggest that ovine macrophages and eosinophils, acting in concert with a specific antibody, may be important effector cells involved in the resistance of ITT sheep to F. gigantica. PMID:17210661

  15. Quantitative model of antibody- and soluble CD4-mediated neutralization of primary isolates and T-cell line-adapted strains of human immunodeficiency virus type 1.

    PubMed Central

    Klasse, P J; Moore, J P

    1996-01-01

    Primary isolates (PI) of human immunodeficiency virus type 1 (HIV-1) are considerably less sensitive than T-cell line-adapted strains to neutralization by soluble CD4 and by most cross-reactive monoclonal antibodies to the viral envelope (Env) glycoprotein, as well as by postinfection and postvaccination sera (J. P. Moore and D. D. Ho, AIDS 9 [suppl. A]:5117-5136, 1995). We developed a quantitative model to explain the neutralization resistance of PI. The factors incorporated into the model are the dissociation constants for the binding of the neutralizing agent to native Env oligomers, the number of outer Env molecules on the viral surface (which decreases by shedding), and the minimum number of Env molecules required for attachment and fusion. We conclude that modest differences in all these factors can, when combined, explain a relative neutralization resistance of PI versus T-cell line-adapted strains that sometimes amounts to several orders of magnitude. The hypothesis that neutralization of HIV is due to the reduction below a minimum number of the Env molecules on a virion available for attachment and fusion is at odds with single- and few-hit neutralization theories. Our analysis of these ideas favors the hypothesis that neutralization of HIV is instead a competitive blocking of interactions with cellular factors, including adsorption receptors. PMID:8648701

  16. Early eradication of persistent Salmonella infection primes antibody-mediated protective immunity to recurrent infection.

    PubMed

    Johanns, Tanner M; Law, Calvin Y; Kalekar, Lokeshchandra A; O'Donnell, Hope; Ertelt, James M; Rowe, Jared H; Way, Sing Sing

    2011-04-01

    Typhoid fever is a systemic, persistent infection caused by host-specific strains of Salmonella. Although the use of antibiotics has reduced the complications associated with primary infection, recurrent infection remains an important cause of ongoing human morbidity and mortality. Herein, we investigated the impacts of antibiotic eradication of primary infection on protection against secondary recurrent infection. Using a murine model of persistent Salmonella infection, we demonstrate protection against recurrent infection is sustained despite early eradication of primary infection. In this model, protection is not mediated by CD4(+) or CD8(+) T cells because depletion of these cells either alone or in combination prior to rechallenge does not abrogate protection. Instead, infection followed by antibiotic-mediated clearance primes robust levels of Salmonella-specific antibody that can adoptively transfer protection to naïve mice. Thus, eradication of persistent Salmonella infection primes antibody-mediated protective immunity to recurrent infection.

  17. Toxin-Mediated Paracellular Transport of Antitoxin Antibodies Facilitates Protection against Clostridium difficile Infection

    PubMed Central

    Zhang, Z.; Chen, X.; Hernandez, L. D.; Lipari, P.; Flattery, A.; Chen, S.-C.; Kramer, S.; Polishook, J. D.; Racine, F.; Cape, H.; Kelly, C. P.

    2014-01-01

    The exotoxins TcdA and TcdB are the major virulence factors of Clostridium difficile. Circulating neutralizing antitoxin antibodies are protective in C. difficile infection (CDI), as demonstrated, in part, by the protective effects of actoxumab and bezlotoxumab, which bind to and neutralize TcdA and TcdB, respectively. The question of how systemic IgG antibodies neutralize toxins in the gut lumen remains unresolved, although it has been suggested that the Fc receptor FcRn may be involved in active antibody transport across the gut epithelium. In this study, we demonstrated that genetic ablation of FcRn and excess irrelevant human IgG have no impact on actoxumab-bezlotoxumab-mediated protection in murine and hamster models of CDI, suggesting that Fc-dependent transport of antibodies across the gut wall is not required for efficacy. Tissue distribution studies in hamsters suggest, rather, that the transport of antibodies depends on toxin-induced damage to the gut lining. In an in vitro two-dimensional culture system that mimics the architecture of the intestinal mucosal epithelium, toxins on the apical side of epithelial cell monolayers are neutralized by basolateral antibodies, and antibody transport across the cell layer is dramatically increased upon addition of toxin to the apical side. Similar data were obtained with F(ab′)2 fragments, which lack an Fc domain, consistent with FcRn-independent paracellular, rather than transcellular, transport of antibodies. Kinetic studies show that initial damage caused by apical toxin is required for efficient neutralization by basolateral antibodies. These data may represent a general mechanism of humoral response-mediated protection against enteric pathogens. PMID:25385797

  18. B cell Rab7 mediates induction of activation-induced cytidine deaminase expression and class-switching in T-dependent and T-independent antibody responses.

    PubMed

    Pone, Egest J; Lam, Tonika; Lou, Zheng; Wang, Rui; Chen, Yuhui; Liu, Dongfang; Edinger, Aimee L; Xu, Zhenming; Casali, Paolo

    2015-04-01

    Class switch DNA recombination (CSR) is central to the maturation of the Ab response because it diversifies Ab effector functions. Like somatic hypermutation, CSR requires activation-induced cytidine deaminase (AID), whose expression is restricted to B cells, as induced by CD40 engagement or dual TLR-BCR engagement (primary CSR-inducing stimuli). By constructing conditional knockout Igh(+/C)γ(1-cre)Rab7(fl/fl) mice, we identified a B cell-intrinsic role for Rab7, a small GTPase involved in intracellular membrane functions, in mediating AID induction and CSR. Igh(+/C)γ(1-cre)Rab7(fl/fl) mice displayed normal B and T cell development and were deficient in Rab7 only in B cells undergoing Igh(C)γ(1-cre) Iγ1-Sγ1-Cγ1-cre transcription, as induced--like Igh germline Iγ1-Sγ1-Cγ1 and Iε-Sε-Cε transcription--by IL-4 in conjunction with a primary CSR-inducing stimulus. These mice could not mount T-independent or T-dependent class-switched IgG1 or IgE responses while maintaining normal IgM levels. Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells showed, in vivo and in vitro, normal proliferation and survival, normal Blimp-1 expression and plasma cell differentiation, as well as intact activation of the noncanonical NF-κB, p38 kinase, and ERK1/2 kinase pathways. They, however, were defective in AID expression and CSR in vivo and in vitro, as induced by CD40 engagement or dual TLR1/2-, TLR4-, TLR7-, or TLR9-BCR engagement. In Igh(+/C)γ(1-cre)Rab7(fl/fl) B cells, CSR was rescued by enforced AID expression. These findings, together with our demonstration that Rab7-mediated canonical NF-κB activation, as critical to AID induction, outline a novel role of Rab7 in signaling pathways that lead to AID expression and CSR, likely by promoting assembly of signaling complexes along intracellular membranes.

  19. Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on antibody-mediated immune response and gene expression of pattern recognition receptors and cytokines in broiler chickens immunized with T-cell dependent antigens.

    PubMed

    Alizadeh, M; Rodriguez-Lecompte, J C; Echeverry, H; Crow, G H; Slominski, B A

    2016-04-01

    This study evaluated the effect of yeast-derived products on innate and antibody mediated immune response in broiler chickens following immunization with sheep red blood cells (SRBC) and bovine serum albumin (BSA). One-day-old male broiler chickens (Ross-308) were randomly assigned to 6 dietary treatments of 9 replicate cages of 5 birds each per treatment. Dietary treatments consisted of a Control diet without antibiotic, and diets containing 11 mg/kg of virginiamycin, 0.25% of yeast cell wall (YCW), 0.2% of a commercial product Maxi-Gen Plus containing processed yeast and nucleotides, 0.05% of nucleotides, or a diet containing 10% of DDGS. On days 21 and 28 post-hatching, 5 birds per treatment were immunized intramuscularly with both SRBC and BSA. One week after each immunization, blood samples were collected. Serum samples were analyzed by hemagglutination test for antibody response to SRBC, and by ELISA for serum IgM and IgG response to BSA. On d 35, 5 birds per treatment were euthanized and the tissue samples from the cecal tonsils were collected to assess the gene expression of toll-like receptors TLR2b, TLR4, and TLR21, monocyte mannose receptor (MMR), and cytokines IL-10, IL-13, IL-4, IL-12p35, and IFN-γ. The results for gene expression analysis demonstrated that the diet supplemented with YCW increased the expression of TLR2b and T-helper type 2 cytokines IL-10, IL-4, and IL-13 relative to the Control; and the expression of TLR4 and IL-13 was upregulated in the nucleotide-containing diet. However, the diets containing antibiotics or Maxi-Gen Plus downregulated the expression of IFN-γ compared to the control. The primary antibody response to SRBC was not affected by diets. However, the diet containing YCW increased the secondary antibody response to SRBC compared to the antibiotic treatment. Neither primary nor secondary IgG and IgM response against BSA were affected by diets. In conclusion, supplementation of the diet with YCW stimulated Th2 cell-mediated

  20. The antibody approach of labeling blood cells

    SciTech Connect

    Srivastava, S.C.

    1992-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated.

  1. The antibody approach of labeling blood cells

    SciTech Connect

    Srivastava, S.C.

    1991-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  2. The antibody approach of labeling blood cells

    SciTech Connect

    Srivastava, S.C.

    1991-01-01

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  3. Antibody-Mediated Bacteriorhodopsin Orientation for Molecular Device Architectures

    NASA Astrophysics Data System (ADS)

    Koyama, Koichi; Yamaguchi, Naoto; Miyasaka, Tsutomu

    1994-08-01

    A rational method for constructing highly oriented films of purple membrane (PM) has been established by using two kinds of bispecific antibodies with different antigen-binding sites, one binding to a specific side of bacteriorhodopsin and the other to a phospholipid hapten. A hapten monolayer deposited on a metal electrode was treated with a bispecific antibody solution and incubated with a PM suspension to produce a highly oriented PM film, as confirmed by electron microscopy in which an immunogold labeling technique was used. This antibody-mediated PM monolayer was then used in the construction of a light-sensing photoelectric device. A comparison of the two incorporated PM monolayers showed that highly efficient photocurrents were produced by the PM monolayer whose unidirectionally oriented cytoplasmic surface faces the electrode.

  4. How antibodies alter the cell entry pathway of dengue virus particles in macrophages

    PubMed Central

    Ayala-Nunez, Nilda V.; Hoornweg, Tabitha E.; van de Pol, Denise P.I.; Sjollema, Klaas A.; Flipse, Jacky; van der Schaar, Hilde M.; Smit, Jolanda M.

    2016-01-01

    Antibody-dependent enhancement of dengue virus (DENV) infection plays an important role in the exacerbation of DENV-induced disease. To understand how antibodies influence the fate of DENV particles, we explored the cell entry pathway of DENV in the absence and presence of antibodies in macrophage-like P388D1 cells. Recent studies unraveled that both mature and immature DENV particles contribute to ADE, hence, both particles were studied. We observed that antibody-opsonized DENV enters P388D1 cells through a different pathway than non-opsonized DENV. Antibody-mediated DENV entry was dependent on FcγRs, pH, Eps15, dynamin, actin, PI3K, Rab5, and Rab7. In the absence of antibodies, DENV cell entry was FcγR, PI3K, and Rab5-independent. Live-cell imaging of fluorescently-labeled particles revealed that actin-mediated membrane protrusions facilitate virus uptake. In fact, actin protrusions were found to actively search and capture antibody-bound virus particles distantly located from the cell body, a phenomenon that is not observed in the absence of antibodies. Overall, similar results were seen for antibody-opsonized standard and antibody-bound immature DENV preparations, indicating that the maturation status of the virus does not control the entry pathway. Collectively, our findings suggest that antibodies alter the cell entry pathway of DENV and trigger a novel mechanism of initial virus-cell contact. PMID:27385443

  5. Antibody-Mediated Protection Against SHIV Challenge Includes Systemic Clearance of Distal Virus

    PubMed Central

    Liu, Jinyan; Ghneim, Khader; Sok, Devin; Bosche, William J.; Li, Yuan; Chipriano, Elizabeth; Berkemeier, Brian; Oswald, Kelli; Borducchi, Erica; Cabral, Crystal; Peter, Lauren; Brinkman, Amanda; Shetty, Mayuri; Jimenez, Jessica; Mondesir, Jade; Lee, Benjamin; Giglio, Patricia; Chandrashekar, Abishek; Abbink, Peter; Colantonio, Arnaud; Gittens, Courtney; Baker, Chantelle; Wagner, Wendeline; Lewis, Mark G.; Li, Wenjun; Sekaly, Rafick-Pierre; Lifson, Jeffrey D.; Burton, Dennis R.; Barouch, Dan H.

    2017-01-01

    HIV-1-specific broadly neutralizing antibodies (bNAbs) can protect rhesus monkeys against simian-human immunodeficiency virus (SHIV) challenge. However, the site of antibody interception of virus and the mechanism of antibody-mediated protection remain unclear. We administered a fully protective dose of the bNAb PGT121 to rhesus monkeys and challenged them intravaginally with SHIV-SF162P3. In PGT121 treated animals, we detected low levels of viral RNA and viral DNA in distal tissues for several days following challenge. Viral RNA positive tissues showed transcriptomic changes indicative of innate immune activation, and cells from these tissues initiated infection following adoptive transfer into naïve hosts. These data demonstrate that bNAb mediated protection against a mucosal virus challenge can involve clearance of infectious virus in distal tissues. PMID:27540005

  6. Antibody-mediated rejection in kidney transplantation: a review of pathophysiology, diagnosis, and treatment options.

    PubMed

    Kim, Miae; Martin, Spencer T; Townsend, Keri R; Gabardi, Steven

    2014-07-01

    Antibody-mediated rejection (AMR), also known as B-cell-mediated or humoral rejection, is a significant complication after kidney transplantation that carries a poor prognosis. Although fewer than 10% of kidney transplant patients experience AMR, as many as 30% of these patients experience graft loss as a consequence. Although AMR is mediated by antibodies against an allograft and results in histologic changes in allograft vasculature that differ from cellular rejection, it has not been recognized as a separate disease process until recently. With an improved understanding about the importance of the development of antibodies against allografts as well as complement activation, significant advances have occurred in the treatment of AMR. The standard of care for AMR includes plasmapheresis and intravenous immunoglobulin that remove and neutralize antibodies, respectively. Agents targeting B cells (rituximab and alemtuzumab), plasma cells (bortezomib), and the complement system (eculizumab) have also been used successfully to treat AMR in kidney transplant recipients. However, the high cost of these medications, their use for unlabeled indications, and a lack of prospective studies evaluating their efficacy and safety limit the routine use of these agents in the treatment of AMR in kidney transplant recipients.

  7. Blocking by anti-idiotypic antibodies of monoclonal antibody-mediated protection against lethal Semliki Forest virus in mice.

    PubMed

    Oosterlaken, T A; Harmsen, M; Kraaijeveld, C A; Snippe, H

    1990-02-01

    Semliki Forest virus-(SEV) neutralizing monoclonal antibodies (MoAbs), produced after fusion of spleen cells from BALB/c mice and myeloma cell line P3-X63-AG8. 653 or SP2/0, were used for anti-idiotypic immunization of female BALB/c mice. Two intracutaneous immunizations (2 x 40 micrograms per animal), 3 weeks apart, with keyhole limpet haemocyanin-conjugated MoAbs mixed with the saponin Quil A were sufficient to induce high levels of anti-idiotypic antibodies in the circulation of these mice with the capacity to block specifically in vitro MoAb-mediated virus neutralization. Anti-idiotypic antibodies against SFV-neutralizing MoAbs, either passively transferred or actively acquired by immunization, are also able to abrogate (specifically) passive immunity, mediated by critical protective doses of MoAb, in mice against infection with a lethal strain of SFV. Furthermore we confirmed by intervention with anti-idiotypic serum in vivo that an SFV-neutralizing MoAb exerts its greatest protective effect during the first 2 days of infection.

  8. Alemtuzumab induction and antibody-mediated rejection in kidney transplantation.

    PubMed

    Noureldeen, T; Albekioni, Z; Machado, L; Muddana, N; Marcus, R J; Hussain, S M; Sureshkumar, K K

    2014-12-01

    Induction therapy improves graft outcomes in kidney transplant recipients (KTRs). We aimed to compare the incidences of antibody-mediated rejection (AMR) and acute cellular rejection (ACR) as well as graft and patient outcomes in KTRs who underwent induction with alemtuzumab versus rabbit-antithymocyte globulin (r-ATG). This was a single-center retrospective study involving patients who underwent kidney transplantation between January 2009 and December 2011 after receiving induction therapy with either alemtuzumab or r-ATG. Maintenance immunosuppression included tacrolimus and mycophenolate mofetil with early steroid withdrawal. Acute rejection was diagnosed using allograft biopsy. Among the 108 study patients, 68 received alemtuzumab and 40 got r-ATG. There was a significantly higher incidence of AMR (15% vs 2.5%; P = .008) and similar incidence of ACR (4.4% vs 10%; P = .69) for alemtuzumab versus r-ATG groups. One-year serum creatinine levels (l.68 ± 0.8 mg/dL vs 1.79 ± 1.8 mg/dL; P = .66) as well as graft (91.1 ± 3.5% vs 94.5 ± 3.8%; P = .48) and patient (93.8 ± 3.0% vs 96.4 ± 3.5%; P = .92) survivals were similar for the alemtuzumab versus the r-ATG groups. Our study showed a higher incidence of AMR and similar incidence of ACR in KTRs who underwent induction with alemtuzumab compared with those who received r-ATG and were maintained on tacrolimus and MMF. This was despite a lower HLA mismatch in the alemtuzumab group. One-year graft survival, patient survival, and allograft function were similar. Inadequate B-cell suppression by alemtuzumab as well as altered phenotypic and functional properties of repopulating B cells could be contributing to heightened risk of AMR in these patients. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Antibody-Mediated Rejection in Kidney Transplantation Without Evidence of Anti-HLA Antibodies?

    PubMed

    Irure, J; López-Hoyos, M; Rodrigo, E; Gómez-Román, J; Ruiz, J C; Arias, M; San Segundo, D

    2016-11-01

    The definition of antibody-mediated rejection (AMR) is based on serologic (presence and/or development of donor-specific anti-HLA antibodies [DSAs]) and histologic (C4d deposition and endothelial damage) criteria. However, several cases of AMR have been described without C4d deposition, and other cases of histologic AMR without DSAs, which could be driven by other non-HLA alloantibodies such as anti-MICA or anti-angiotensin II type I receptor (AT1R). Here we studied clinical and histologic humoral rejection in kidney transplant recipients without evidence of anti-HLA antibodies. Fifteen kidney transplant recipients with AMR defined as C4d(+) and/or histologic g+ptc without anti-HLA antibodies in screening test were studied. Sera at the moment of biopsy and 2 months earlier were studied for anti-HLA antibodies by Luminex, in neat, diluted 1/160, and sera after treatment with dithiothreitol (DTT) and confirmed by single-antigen test. The anti-AT1R was measured by enzyme-linked immunosorbent assay. A lack of anti-HLA and MICA antibodies was confirmed after anti-HLA screening test in all conditions (neat, diluted, and DTT-treated) and de novo development of AT1R antibodies was ruled out. Nevertheless, after single-antigen test, 3 patients were identified with a weak reaction against class I antigen and another 4 patients against class II antigen. Due to the lack of locus-C typing in the donors, the DSA assignment cannot be confirmed, whereas anti-HLA class II antigens were DSA. A low sensitivity in the screening of anti-HLA antibody testing was observed. Our results suggest performing single-antigen test in seronegative patients with clinical humoral rejection after screening to confirm the presence of DSA. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Effective therapy for acute antibody-mediated rejection with mild chronic changes: case report and review of the literature.

    PubMed

    Gheith, Osama; Al-Otaibi, Torki; Nampoory, Narayanan; Halim, Medhat; Nair, Prasad; Saied, Tarek; Al-Waheeb, Salah; Muzeirei, Ibraheem; Ibraheim, Mona

    2012-08-01

    To reduce the long-term toxicities of immunosuppressant drugs, corticosteroid-sparing and calcineurin-inhibitor-sparing immunosuppression protocols have become increasingly popular in managing kidney transplant recipients. The most vexing clinical condition caused by antibodies in organ transplants is antibody-mediated rejection. Limitations of the current antibody-mediated rejection therapies include (1) antibody-mediated rejection reversal tends to be gradual rather than prompt, (2) expense, (3) rejection reversal rates below 80%, (4) common appearance of chronic rejection after antibody-mediated rejection treatment, and (5) long-term persistence of donor specific antibodies after therapy. Because these limitations may be due to a lack of effects on mature plasma cells, the effects of bortezomib on mature plasma cells may represent a quantum advance in antihumoral therapy. Our experiences represent the first clinical use of bortezomib as an antihumoral agent in renal allograft recipients in Kuwait. We present 2 cases with resistant-acute antibody-mediated rejection to the standard therapies that were managed successfully with bortezomib.

  11. HIV-specific CD4-induced Antibodies Mediate Broad and Potent Antibody-dependent Cellular Cytotoxicity Activity and are Commonly Detected in Plasma from HIV-infected Humans

    PubMed Central

    Williams, Katherine L.; Cortez, Valerie; Dingens, Adam S.; Gach, Johannes S.; Rainwater, Stephanie; Weis, Julie F.; Chen, Xuemin; Spearman, Paul; Forthal, Donald N.; Overbaugh, Julie

    2015-01-01

    HIV-specific antibodies (Abs) can reduce viral burden by blocking new rounds of infection or by destroying infected cells via activation of effector cells through Fc–FcR interaction. This latter process, referred to as antibody-dependent cellular cytotoxicity (ADCC), has been associated with viral control and improved clinical outcome following both HIV and SIV infections. Here we describe an HIV viral-like particle (VLP)-based sorting strategy that led to identification of HIV-specific memory B cells encoding Abs that mediate ADCC from a subtype A-infected Kenyan woman at 914 days post-infection. Using this strategy, 12 HIV-envelope-specific monoclonal antibodies (mAbs) were isolated and three mediated potent ADCC activity when compared to well-characterized ADCC mAbs. The ADCC-mediating Abs also mediated antibody-dependent cell-mediated virus inhibition (ADCVI), which provides a net measure of Fc receptor-triggered effects against replicating virus. Two of the three ADCC-mediating Abs targeted a CD4-induced (CD4i) epitope also bound by the mAb C11; the third antibody targeted the N-terminus of V3. Both CD4i Abs identified here demonstrated strong cross-clade breadth with activity against 10 of 11 envelopes tested, including those from clades A, B, C, A/D and C/D, whereas the V3-specific antibody showed more limited breadth. Variants of these CD4i, C11-like mAbs engineered to interrupt binding to FcγRs inhibited a measurable percentage of the donor's ADCC activity starting as early as 189 days post-infection. C11-like antibodies also accounted for between 18–78% of ADCC activity in 9 chronically infected individuals from the same cohort study. Further, the two CD4i Abs originated from unique B cells, suggesting that antibodies targeting this epitope can be commonly produced. Taken together, these data provide strong evidence that CD4i, C11-like antibodies develop within the first 6 months of infection and they can arise from unique B-cell lineages in the

  12. Role of monoclonal antibodies in the treatment of immune-mediated glomerular diseases.

    PubMed

    Manrique, Joaquín; Cravedi, Paolo

    2014-05-21

    Non-specific immunosuppressants have represented for decades the only therapies for patients with immune-mediated glomerular diseases. These treatments, however, are associated with high rates of no-response and are burdened by toxicities that frequently offset the benefits of proteinuria reduction. Monoclonal antibodies targeting selective cell populations or mediators implicated in the pathophysiology of glomerular diseases have recently become available. Rituximab, a chimeric monoclonal antibody against the CD20 antigen on B cells, safely reduced proteinuria in patients with nephrotic syndrome secondary to membranous nephropathy, minimal change disease, or focal segmental glomerulosclerosis. Its ability to reduce auto-antibody formation has been instrumental to treat also ANCA-associated vasculitis, lupus nephritis, and mixed cryoglobulinemia. Many reports have also documented the efficacy of the anti-C5 humanized monoclonal antibody Eculizumab to treat atypical hemolytic uremic syndrome, C3 nephropathy, and membranoproliferative glomerulonephritis. Thanks to these encouraging findings, monoclonals are becoming very helpful tools to treat patients with glomerular diseases. Moreover, thanks to their specific mechanism of action, these and other monoclonal antibodies are important in improving our understanding of the pathophysiology of glomerular diseases. Their still high costs, however, might represent a major hurdle for their widespread implementation for all patients in need.

  13. Release of platelet activating factor in rabbits with antibody-mediated injury of the lung: the role of leukocytes and of pulmonary endothelial cells.

    PubMed

    Camussi, G; Pawlowski, I; Bussolino, F; Caldwell, P R; Brentjens, J; Andres, G

    1983-10-01

    This paper describes the release of platelet-activating factor (PAF) into the circulation of rabbits with acute pulmonary injury induced by antibody reacting with pulmonary endothelium. Eight rabbits were injected i.v. with 2 mg/kg of body weight of goat anti-rabbit lung angiotensin-converting enzyme gamma-globulin (GtARbACE). All animals developed acute pneumonitis, characterized by severe endothelial damage, accumulation of polymorphonuclear leukocytes (PMN) and platelets (Plt) in the lumina of alveolar capillaries, and deposits of goat IgG and rabbit C3 along alveolar capillary walls. Six of the rabbits died from acute pulmonary edema. PAF was detected in the plasma of all animals within 5 min after injection of GtARbACE. Five other rabbits were depleted of leukocytes by nitrogen mustard and then injected with 2 mg/kg of body weight of GtARbACE. In three of these rabbits release of PAF was demonstrated, though in amounts smaller than in non-leukocyte-depleted rabbits; all three animals died from pulmonary edema. After injection of 0.03 mg/kg of body weight of GtARbACE in six additional rabbits, three of them leukocyte-depleted, small amounts of PAF were detected in the circulation. None of these six rabbits died of pulmonary edema. PAF release was not observed in ten rabbits injected i.v. with 2 or 0.03 mg/kg of body weight of normal goat gamma-globulin. In separate experiments in vitro, incubation of isolated lung or thoracic aorta with GtARbACE resulted in deposits of goat IgG along endothelia and significant release of PAF. PAF was also released from endothelial cells removed from thoracic aorta by cellulose acetate paper and then incubated with GtARbACE. When segments of thoracic aorta were stripped of endothelium and then incubated with GtARbACE, PAF release could not be shown. The data obtained are consistent with the interpretation that PAF released into the circulation after binding of GtARbACE to the endothelia of lung and aorta originates from

  14. Resistance to respiratory syncytial virus (RSV) challenge induced by infection with a vaccinia virus recombinant expressing the RSV M2 protein (Vac-M2) is mediated by CD8+ T cells, while that induced by Vac-F or Vac-G recombinants is mediated by antibodies.

    PubMed Central

    Connors, M; Kulkarni, A B; Collins, P L; Firestone, C Y; Holmes, K L; Morse, H C; Murphy, B R

    1992-01-01

    It was previously demonstrated that the vaccinia virus recombinants expressing the respiratory syncytial virus (RSV) F, G, or M2 (also designated as 22K) protein (Vac-F, Vac-G, or Vac-M2, respectively) induced almost complete resistance to RSV challenge in BALB/c mice. In the present study, we sought to identify the humoral and/or cellular mediators of this resistance. Mice were immunized by infection with a single recombinant vaccinia virus and were subsequently given a monoclonal antibody directed against CD4+ or CD8+ T cells or gamma interferon (IFN-gamma) to cause depletion of effector T cells or IFN-gamma, respectively, at the time of RSV challenge (10 days after immunization). Mice immunized with Vac-F or Vac-G were completely or almost completely resistant to RSV challenge after depletion of both CD4+ and CD8+ T cells prior to challenge, indicating that these cells were not required at the time of virus challenge for expression of resistance to RSV infection induced by the recombinants. In contrast, the high level of protection of mice immunized with Vac-M2 was completely abrogated by depletion of CD8+ T cells, whereas depletion of CD4+ T cells or IFN-gamma resulted in intermediate levels of resistance. These results demonstrate that antibodies are sufficient to mediate the resistance to RSV induced by the F and G proteins, whereas the resistance induced by the M2 protein is mediated primarily by CD8+ T cells, with CD4+ T cells and IFN-gamma also contributing to resistance. PMID:1731105

  15. Aggregation of hapten-bearing liposomes mediated by specific antibodies.

    PubMed

    Lee, K D; Kantor, A B; Nir, S; Owicki, J C

    1993-03-01

    We studied specific membrane-membrane interactions mediated by ligand-receptor binding in a model system, which consisted of (a) FG3P, the fluorescein hapten attached to a phospholipid by a peptidyl spacer as described previously (Petrossian, A., A.B. Kantor, and J.C. Owicki. 1985. J. Lipid Res. 26:767-773), (b) antifluorescein monoclonal antibodies (MAbs), and (c) phospholipid vesicles (liposomes) into which the FG3P was incorporated. The aggregation of the hapten-bearing liposomes by four MAbs was studied by differential centrifugation. The ability of the MAbs to induce vesicle aggregation varied considerably and correlated inversely with affinity. Aggregation by one of the MAbs was studied in more detail by turbidimetry and freeze-fracture electron microscopy of samples frozen throughout the course of the aggregation. Rapid freezing was achieved with a double propane-jet apparatus. The aggregate morphologies and the time evolution of the aggregate size distribution were obtained from the two-dimensional fracture views with a stereological correction. The aggregation kinetics were simulated by considering dynamical aggregation according to a mass-action model with two parameters, the rate constants for antibody-mediated vesicle aggregation and disaggregation. Both rate constants were orders of magnitude lower than the rate constants for the corresponding interactions of antibodies with haptens either in solution or on vesicles under nonaggregating conditions.

  16. TLR5-Mediated Sensing of Gut Microbiota Is Necessary for Antibody Responses to Seasonal Influenza Vaccination

    PubMed Central

    Oh, Jason Z.; Ravindran, Rajesh; Chassaing, Benoit; Carvalho, Frederic A.; Maddur, Mohan S.; Bower, Maureen; Hakimpour, Paul; Gill, Kiran P.; Nakaya, Helder I.; Yarovinsky, Felix; Sartor, R. Balfour; Gewirtz, Andrew T.; Pulendran, Bali

    2014-01-01

    SUMMARY Systems biological analysis of immunity to the trivalent inactivated influenza vaccine (TIV) in humans revealed a correlation between early expression of TLR5 and the magnitude of the antibody response. Vaccination of Trl5−/− mice resulted in reduced antibody titers and lower frequencies of plasma cells, demonstrating a role for TLR5 in immunity to TIV. This was due to a failure to sense host microbiota. Thus, antibody responses in germ-free or antibiotic-treated mice were impaired, but restored by oral reconstitution with a flagellated, but not aflagellated, strain of E. coli. TLR5-mediated sensing of flagellin promoted plasma cell differentiation, directly, and by stimulating lymph node macrophages to produce plasma cell growth factors. Finally, TLR5-mediated sensing of the microbiota also impacted antibody responses to the inactivated polio vaccine, but not to adjuvanted vaccines or the live-attenuated yellow fever vaccine. These results reveal an unappreciated role for gut microbiota in promoting immunity to vaccination. PMID:25220212

  17. Cell-mediated cytotoxicity. Characterization of the effector cells.

    PubMed Central

    De Bracco, M M; Isturiz, M A; Manni, J A

    1976-01-01

    Isolated human mononuclear cells were fractionated according to their membrane characteristics or physical properties. Adherent cells were depleted by filtration through glass columns; phagocytic cells were removed by iron treatment and cell subpopulations capable of forming rosettes with sheep erythrocytes (E), erythrocyte-antibody-complement (EAC) and chicken erythrocyte-antibody complexes (CEA) were separated by centrifugation of Ficoll-Hypaque gradients. The functional activity of the cell subpopulations obtained was assayed by testing PHA-induced cytoxicity (PIC), antibody-dependent cytoxicity (ADCC) and blast transformation by PHA. The results of this study demonstrate that: (1) cells reacting in PIC and ADCC assays are different, adherent and phagocytic cells being necessary for full expression of PIC and not for ADCC; (2) PHA induces direct blast transformation of purified E-RFC in the absence of PIC cytotoxic cells; (3) cell populations specifically enriched in E or EAC rosette-forming cells are not cytotoxic neither in the PHA nor in antibody mediated cytotoxic assays; (4) cells participating in ADCC can be selectively purified by centrifugation of CEA rosettes. PMID:1254320

  18. Monoclonal antibodies specific for sickle cell hemoglobin

    SciTech Connect

    Jensen, R.H.; Vanderlaan, M.; Grabske, R.J.; Branscomb, E.W.; Bigbee, W.L.; Stanker, L.H.

    1985-01-01

    Two mouse hybridoma cell lines were isolated which produce monoclonal antibodies that bind hemoglobin S. The mice were immunized with peptide-protein conjugates to stimulate a response to the amino terminal peptide of the beta chain of hemoglobin S, where the single amino acid difference between A and S occurs. Immunocharacterization of the antibodies shows that they bind specifically to the immunogen peptide and to hemoglobin S. The specificity for S is high enough that one AS cell in a mixture with a million AA cells is labeled by antibody, and such cells can be analyzed by flow cytometry. Immunoblotting of electrophoretic gels allows definitive identification of hemoglobin S as compared with other hemoglobins with similar electrophoretic mobility. 12 references, 4 figures.

  19. Evidence for antimuscarinic acetylcholine receptor antibody-mediated secretory dysfunction in nod mice.

    PubMed

    Nguyen, K H; Brayer, J; Cha, S; Diggs, S; Yasunari, U; Hilal, G; Peck, A B; Humphreys-Beher, M G

    2000-10-01

    Antibodies directed against general and specific target-organ autoantigens are present in the sera of human patients and animal models with autoimmune disease. The relevance of these autoantibodies to the disease process remains ambiguous in most cases. In autoimmune exocrinopathy (Sjögren's syndrome), autoantibodies to the intracellular nuclear proteins SSA/Ro and SSB/La, as well as the cell surface muscarinic cholinergic receptor (M3) are observed. To evaluate the potential role of these factors in the loss of secretory function of exocrine tissues, a panel of monoclonal and polyclonal antibodies was developed for passive transfer into the NOD animal model. Monoclonal antibodies to mouse SSB/La, rat M3 receptor, and a rabbit polyclonal antiparotid secretory protein antibody were obtained for this study. These antibody reagents were subsequently infused into NOD-scid mice. Saliva flow rates were subsequently monitored over a 72-hour period. Submandibular gland lysates were examined by Western blotting for alteration of the distribution of the water channel protein aquaporin (AQP). Evaluation of the secretory response indicated that only antibodies directed toward the extracellular domains of the M3 receptor were capable of mediating the exocrine dysfunction aspect of the clinical pathology of the autoimmune disease. In vitro stimulation with a muscarinic agonist of submandibular gland cells isolated from mice treated with anti-M3 antibody, but not saline or the isotype control, failed to translocate AQP to the plasma membrane. These findings define a clear role for the humoral immune response and the targeting of the cell surface M3 signal transduction receptor as primary events in the development of clinical symptoms of autoimmune exocrinopathy. Furthermore, the anti-M3 receptor activity may negatively affect the secretory response through perturbation of normal signal transduction events, leading to translocation of the epithelial cell water channel.

  20. Antibody-Dependent Cell-Mediated Cytotoxicity Epitopes on the Hemagglutinin Head Region of Pandemic H1N1 Influenza Virus Play Detrimental Roles in H1N1-Infected Mice

    PubMed Central

    Ye, Zi-Wei; Yuan, Shuofeng; Poon, Kwok-Man; Wen, Lei; Yang, Dong; Sun, Zehua; Li, Cun; Hu, Meng; Shuai, Huiping; Zhou, Jie; Zhang, Mei-Yun; Zheng, Bo-Jian; Chu, Hin; Yuen, Kwok-Yung

    2017-01-01

    Engaging the antibody-dependent cell-mediated cytotoxicity (ADCC) for killing of virus-infected cells and secretion of antiviral cytokines and chemokines was incorporated as one of the important features in the design of universal influenza vaccines. However, investigation of the ADCC epitopes on the highly immunogenic influenza hemagglutinin (HA) head region has been rarely reported. In this study, we determined the ADCC and antiviral activities of two putative ADCC epitopes, designated E1 and E2, on the HA head of a pandemic H1N1 influenza virus in vitro and in a lethal mouse model. Our data demonstrated that sera from the E1-vaccinated mice could induce high ADCC activities. Importantly, the induction of ADCC response modestly decreased viral load in the lungs of H1N1-infected mice. However, the elevated ADCC significantly increased mouse alveolar damage and mortality than that of the PBS-vaccinated group (P < 0.0001). The phenotype was potentially due to an exaggerated inflammatory cell infiltration triggered by ADCC, as an upregulated release of cytotoxic granules (perforin) was observed in the lung tissue of E1-vaccinated mice after H1N1 influenza virus challenge. Overall, our data suggested that ADCC elicited by certain domains of HA head region might have a detrimental rather than protective effect during influenza virus infection. Thus, future design of universal influenza vaccine shall strike a balance between the induction of protective immunity and potential side effects of ADCC. PMID:28377769

  1. Antibody-dependent cell cytotoxicity synapses form in mice during tumor-specific antibody immunotherapy.

    PubMed

    Hubert, Pascale; Heitzmann, Adèle; Viel, Sophie; Nicolas, André; Sastre-Garau, Xavier; Oppezzo, Pablo; Pritsch, Otto; Osinaga, Eduardo; Amigorena, Sebastian

    2011-08-01

    Antibody-dependent cell cytotoxicity (ADCC) plays a critical role in monoclonal antibody (mAb)-mediated cancer therapy. ADCC, however, has not been directly shown in vivo but inferred from the requirement for IgG Fc receptors (FcγR) in tumor rejection in mice. Here, we investigated the mechanism of action of a Tn antigen-specific chimeric mAb (Chi-Tn), which binds selectively to a wide variety of carcinomas, but not to normal tissues, in both humans and mice. Chi-Tn mAb showed no direct toxicity against carcinomas cell lines in vitro but induced the rejection of a murine breast tumor in 80% to 100% of immunocompetent mice, when associated with cyclophosphamide. Tumor rejection was abolished in Fc receptors-associated γ chain (FcR-γ)-deficient mice, suggesting a role for ADCC. Indeed, tumor cells formed stable conjugates in vivo with FcR-γ chain-expressing macrophages and neutrophils in Chi-Tn mAb-treated but not in control mAb-treated mice. The contact zone between tumor cells and ADCC effectors accumulated actin, FcγR and phospho-tyrosines. The in vivo formed ADCC synapses were organized in multifocal supra-molecular activation clusters. These results show that in vivo ADCC mediated by macrophages and neutrophils during tumor rejection by Chi-Tn mAb involves a novel type of multifocal immune synapse between effectors of innate immunity and tumor cells.

  2. Acute and chronic antibody-mediated rejection in pediatric kidney transplantation.

    PubMed

    Pape, Lars; Becker, Jan U; Immenschuh, Stephan; Ahlenstiel, Thurid

    2015-03-01

    Acute antibody-mediated rejection is a diagnostic challenge in renal transplantation medicine. However, it is an important diagnosis to make, since chronic antibody-mediated rejection (CAMR) is the main cause of long-term graft loss. Antibody-mediated rejection is diagnosed by detecting donor-specific antibodies (DSAs) in the blood in combination with observing typical histomorphological signs in kidney biopsy, as described in the Banff classification. Therapy is based on the removal of DSAs by administering intravenous immunoglobulins (IVIGs), plasmapheresis, or immunoadsorption. Reoccurrence of antibodies is diminished by the use of rituximab, increased immunosuppression, and in some cases additional experimental substances. A combination of these techniques has been shown to be successful in the majority of cases of acute and chronic antibody-mediated rejection. Routine DSA monitoring is warranted for early detection of antibody-mediated rejection.

  3. A requirement for FcγR in antibody-mediated bacterial toxin neutralization

    PubMed Central

    Abboud, Nareen; Chow, Siu-Kei; Saylor, Carolyn; Janda, Alena; Ravetch, Jeffery V.; Scharff, Matthew D.

    2010-01-01

    One important function of humoral immunity is toxin neutralization. The current view posits that neutralization results from antibody-mediated interference with the binding of toxins to their targets, a phenomenon viewed as dependent only on antibody specificity. To investigate the role of antibody constant region function in toxin neutralization, we generated IgG2a and IgG2b variants of the Bacillus anthracis protective antigen–binding IgG1 monoclonal antibody (mAb) 19D9. These antibodies express identical variable regions and display the same specificity. The efficacy of antibody-mediated neutralization was IgG2a > IgG2b > IgG1, and neutralization activity required competent Fcγ receptor (FcγR). The IgG2a mAb prevented lethal toxin cell killing and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase cleavage more efficiently than the IgG1 mAb. Passive immunization with IgG1 and IgG2a mAb protected wild-type mice, but not FcγR-deficient mice, against B. anthracis infection. These results establish that constant region isotype influences toxin neutralization efficacy of certain antibodies through a mechanism that requires engagement of FcγR. These findings highlight a new parameter for evaluating vaccine responses and the possibility of harnessing optimal FcγR interactions in the design of passive immunization strategies. PMID:20921285

  4. Antibody-mediated immunity in CFW mice infected with Mycobacterium lepraemurium. Humoral immune response in murine leprosy.

    PubMed Central

    Rojas-Espinosa, O; Casoluengo-Méndez, M; Díaz, G V

    1976-01-01

    A depression in antibody-mediated immunity (AMI) measured both in terms of circulating antibody and plaque-forming cells in the spleen was observed in CFW mice infected with M. lepraemurium when sheep red blood cells (SRBC) and human gammaglobulin (HGG) were used as antigens. The impairment in AMI was evident only after 75 days of infection thereafter the antibody response to SRBC antigen progressively decreased until the last day of experimentation (135 days). Within the first 60 days of infection no alteration in AMI was observed with the HGG antigen while the response to the SRBC antigen was significantly higher in the infected animals than in uninfected controls. PMID:795574

  5. Fully Human Antagonistic Antibodies against CCR4 Potently Inhibit Cell Signaling and Chemotaxis

    PubMed Central

    Géraudie, Solène; Scheffler, Ulrike; Griep, Remko A.; Reiersen, Herald; Duncan, Alexander R.; Kiprijanov, Sergej M.

    2014-01-01

    Background CC chemokine receptor 4 (CCR4) represents a potentially important target for cancer immunotherapy due to its expression on tumor infiltrating immune cells including regulatory T cells (Tregs) and on tumor cells in several cancer types and its role in metastasis. Methodology Using phage display, human antibody library, affinity maturation and a cell-based antibody selection strategy, the antibody variants against human CCR4 were generated. These antibodies effectively competed with ligand binding, were able to block ligand-induced signaling and cell migration, and demonstrated efficient killing of CCR4-positive tumor cells via ADCC and phagocytosis. In a mouse model of human T-cell lymphoma, significant survival benefit was demonstrated for animals treated with the newly selected anti-CCR4 antibodies. Significance For the first time, successful generation of anti- G-protein coupled chemokine receptor (GPCR) antibodies using human non-immune library and phage display on GPCR-expressing cells was demonstrated. The generated anti-CCR4 antibodies possess a dual mode of action (inhibition of ligand-induced signaling and antibody-directed tumor cell killing). The data demonstrate that the anti-tumor activity in vivo is mediated, at least in part, through Fc-receptor dependent effector mechanisms, such as ADCC and phagocytosis. Anti-CC chemokine receptor 4 antibodies inhibiting receptor signaling have potential as immunomodulatory antibodies for cancer. PMID:25080123

  6. Antibody persistence and T-cell balance: Two key factors confronting HIV vaccine development

    PubMed Central

    Lewis, George K.; DeVico, Anthony L.; Gallo, Robert C.

    2014-01-01

    The quest for a prophylactic AIDS vaccine is ongoing, but it is now clear that the successful vaccine must elicit protective antibody responses. Accordingly, intense efforts are underway to identify immunogens that elicit these responses. Regardless of the mechanism of antibody-mediated protection, be it neutralization, Fc-mediated effector function, or both, antibody persistence and appropriate T-cell help are significant problems confronting the development of a successful AIDS vaccine. Here, we discuss the evidence illustrating the poor persistence of antibody responses to Env, the envelope glycoprotein of HIV-1, and the related problem of CD4+ T-cell responses that compromise vaccine efficacy by creating excess cellular targets of HIV-1 infection. Finally, we propose solutions to both problems that are applicable to all Env-based AIDS vaccines regardless of the mechanism of antibody-mediated protection. PMID:25349379

  7. Antibody-Mediated Rejection of Human Orthotopic Liver Allografts

    PubMed Central

    Demetris, A. Jake; Jaffe, Ron; Tzakis, A.; Ramsey, Glenn; Todo, S.; Belle, Steven; Esquivel, Carlos; Shapiro, Ron; Markus, Bernd; Mroczek, Elizabeth; Van Thiel, D. H.; Sysyn, Greg; Gordon, Robert; Makowka, Leonard; Starzl, Tom

    1988-01-01

    A clinicopathologic analysis of liver transplantation across major ABO blood group barriers was carried out 1) to determine if antibody-mediated (humoral) rejection was a cause of graft failure and if humoral rejection can be identified, 2) to propose criteria for establishing the diagnosis, and 3) to describe the clinical and pathologic features of humoral rejection. A total of 51 (24 primary) ABO-incompatible (ABO-I) liver grafts were transplanted into 49 recipients. There was a 46% graft failure rate during the first 30 days for primary ABO-I grafts compared with an 11% graft failure rate for primary ABO compatible (ABO-C), crossmatch negative, age, sex and priority-matched control patients (P < 0.02). A similarly high early graft failure rate (60%) was seen for nonprimary ABO-I grafts during the first 30 days. Clinically, the patients experienced a relentless rise in serum transaminases, hepatic failure, and coagulopathy during the first weeks after transplant. Pathologic examination of ABO-I grafts that failed early demonstrated widespread areas of geographic hemorrhagic necrosis with diffuse intraorgan coagulation. Prominent arterial deposition of antibody and complement components was demonstrated by immunoflourescent staining. Elution studies confirmed the presence of tissue-bound, donor-specific isoagglutinins within the grafts. No such deposition was seen in control cases. These studies confirm that antibody mediated rejection of the liver occurs and allows for the development of criteria for establishing the diagnosis. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6 PMID:3046369

  8. Universal Bispecific Antibody for Targeting Tumor Cells for Destruction by Cytotoxic T Cells

    NASA Astrophysics Data System (ADS)

    Gilliland, Lisa K.; Clark, Michael R.; Waldmann, Herman

    1988-10-01

    Previous studies have demonstrated that bispecific hybrid antibodies produced by cell-cell fusion or chemically conjugated heteroaggregates can direct cytotoxic T lymphocytes to kill target cells for which they have no intrinsic specificity, a phenomenon we call effector cell retargeting (ECR). These studies used bispecific reagents with one specificity directed to CD3 or Ti on the effector cell and the other directed to a target cell antigen. To avoid the need to create different hybrid hybridomas for each target antigen we have developed a universal means to elicit ECR through the use of an antiglobulin step. We have constructed a bispecific hybrid antibody with dual specificity for CD3 and a rat immunoglobulin light chain allotype. This bispecific antibody could mediate ECR to a range of target cells, each coated with distinct surface-binding rat monoclonal antibodies. A particular advantage of targeting to surface-bound monoclonal antibodies is that all other available effector systems may also attack the same antibody-coated target cell.

  9. Antibody-mediated immune suppression is improved when blends of anti-RBC monoclonal antibodies are used in mice.

    PubMed

    Bernardo, Lidice; Amash, Alaa; Marjoram, Danielle; Lazarus, Alan H

    2016-08-25

    Although the prevention of hemolytic disease of the fetus and newborn is highly effective using polyclonal anti-D, a recombinant alternative is long overdue. Unfortunately, anti-D monoclonal antibodies have been, at best, disappointing. To determine the primary attribute defining an optimal antibody, we assessed suppression of murine red blood cell (RBC) immunization by single-monoclonal antibodies vs defined blends of subtype-matched antibodies. Allogeneic RBCs expressing the HOD antigen (hen egg lysozyme [HEL]-ovalbumin-human transmembrane Duffy(b)) were transfused into naïve mice alone or together with selected combinations of HEL-specific antibodies, and the resulting suppressive effect was assessed by evaluating the antibody response. Polyclonal HEL antibodies dramatically inhibited the antibody response to the HOD antigen, whereas single-monoclonal HEL antibodies were less effective despite the use of saturating doses. A blend of monoclonal HEL-specific antibodies reactive with different HEL epitopes significantly increased the suppressive effect, whereas a blend of monoclonal antibodies that block each other's binding to the HEL protein did not increase suppression. In conclusion, these data show that polyclonal antibodies are superior to monoclonal antibodies at suppressing the immune response to the HOD cells, a feature that can be completely recapitulated using monoclonal antibodies to different epitopes.

  10. Culture temperature modulates aggregation of recombinant antibody in cho cells.

    PubMed

    Gomez, Natalia; Subramanian, Jayashree; Ouyang, Jun; Nguyen, Mary D H; Hutchinson, Matthew; Sharma, Vikas K; Lin, Andy A; Yuk, Inn H

    2012-01-01

    During production of therapeutic monoclonal antibodies (mAb), it is highly desirable to remove and control antibody aggregates in the manufacturing process to minimize the potential risk of immunogenicity to patients. During process development for the production of a recombinant IgG in a CHO cell line, we observed atypical high variability from 1 to 20% mAb aggregates formed during cell culture that negatively impacted antibody purification. Analytical characterization revealed the IgG aggregates were mediated by hydrophobic interactions likely caused by misfolded antibody during intracellular processing. Strikingly, data analysis showed an inverse correlation of lower cell culture temperature producing higher aggregate levels. All cultures at 37°C exhibited ≤ 5% aggregates at harvest. Aggregate levels increased 4-12-fold in 33°C cultures when compared to 37°C, with a corresponding 2-4-fold increase in heavy chain (HC) and light chain (LC) mRNA. Additionally, 37°C cases showed a greater excess of LC to HC mRNA levels. Endoplasmic reticulum (ER) chaperone expression and ER size also increased 25-75% at 33°C versus 37°C but to a lesser extent than LC and HC mRNA, consistent with a potential limiting ER folding capacity at 33°C for this cell line. Finally, we identified a 2-5-fold increase in mAb aggregate formation at 33°C compared to 37°C cultures for three additional CHO cell lines. Taken together, our observations indicate that low culture temperature can increase antibody aggregate formation in CHO cells by increasing LC and HC transcripts coupled with limited ER machinery.

  11. Targeting breast cancer stem cells with HER2-specific antibodies and natural killer cells.

    PubMed

    Diessner, Joachim; Bruttel, Valentin; Becker, Kathrin; Pawlik, Miriam; Stein, Roland; Häusler, Sebastian; Dietl, Johannes; Wischhusen, Jörg; Hönig, Arnd

    2013-01-01

    Breast cancer is the most common cancer among women worldwide. Every year, nearly 1.4 million new cases of breast cancer are diagnosed, and about 450.000 women die of the disease. Approximately 15-25% of breast cancer cases exhibit increased quantities of the trans-membrane receptor tyrosine kinase human epidermal growth factor receptor 2 (HER2) on the tumor cell surface. Previous studies showed that blockade of this HER2 proto-oncogene with the antibody trastuzumab substantially improved the overall survival of patients with this aggressive type of breast cancer. Recruitment of natural killer (NK) cells and subsequent induction of antibody-dependent cell-mediated cytotoxicity (ADCC) contributed to this beneficial effect. We hypothesized that antibody binding to HER2-positive breast cancer cells and thus ADCC might be further improved by synergistically applying two different HER2-specific antibodies, trastuzumab and pertuzumab. We found that tumor cell killing via ADCC was increased when the combination of trastuzumab, pertuzumab, and NK cells was applied to HER2-positive breast cancer cells, as compared to the extent of ADCC induced by a single antibody. Furthermore, a subset of CD44(high)CD24(low)HER2(low) cells, which possessed characteristics of cancer stem cells, could be targeted more efficiently by the combination of two HER2-specific antibodies compared to the efficiency of one antibody. These in vitro results demonstrated the immunotherapeutic benefit achieved by the combined application of trastuzumab and pertuzumab. These findings are consistent with the positive results of the clinical studies, CLEOPATRA and NEOSPHERE, conducted with patients that had HER2-positive breast cancer. Compared to a single antibody treatment, the combined application of trastuzumab and pertuzumab showed a stronger ADCC effect and improved the targeting of breast cancer stem cells.

  12. A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus infection

    SciTech Connect

    Pierson, Theodore C. . E-mail: piersontc@mail.nih.gov; Sanchez, Melissa D.; Puffer, Bridget A.; Ahmed, Asim A.; Geiss, Brian J.; Valentine, Laura E.; Altamura, Louis A.; Diamond, Michael S.; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu

    2006-03-01

    West Nile virus (WNV) is a neurotropic flavivirus within the Japanese encephalitis antigenic complex that is responsible for causing West Nile encephalitis in humans. The surface of WNV virions is covered by a highly ordered icosahedral array of envelope proteins that is responsible for mediating attachment and fusion with target cells. These envelope proteins are also primary targets for the generation of neutralizing antibodies in vivo. In this study, we describe a novel approach for measuring antibody-mediated neutralization of WNV infection using virus-like particles that measure infection as a function of reporter gene expression. These reporter virus particles (RVPs) are produced by complementation of a sub-genomic replicon with WNV structural proteins provided in trans using conventional DNA expression vectors. The precision and accuracy of this approach stem from an ability to measure the outcome of the interaction between antibody and viral antigens under conditions that satisfy the assumptions of the law of mass action as applied to virus neutralization. In addition to its quantitative strengths, this approach allows the production of WNV RVPs bearing the prM-E proteins of different WNV strains and mutants, offering considerable flexibility for the study of the humoral immune response to WNV in vitro. WNV RVPs are capable of only a single round of infection, can be used under BSL-2 conditions, and offer a rapid and quantitative approach for detecting virus entry and its inhibition by neutralizing antibody.

  13. Challenges inherent to the diagnosis of antibody-mediated rejection in lung transplantation

    PubMed Central

    Chin, Nicholas; Westall, Glen; Paraskeva, Miranda; Ciciulla, John; Cantwell, Linda; Snell, Greg

    2015-01-01

    A bilateral sequential lung transplant was performed on a young female with cystic fibrosis-related bronchiectasis. She had negative prospective T- and B-cell crossmatch, and no known donor-specific antibodies. Post-transplantation, she developed bilateral pulmonary infiltrates of uncertain etiology, compounded by persistent tachycardia and questionable medication adherence. Despite aggressive intervention for suspected cellular rejection with high-dose intravenous corticosteroid, immunoglobulin, and anti-thymocyte globulin, her condition deteriorated to ultimately require ventilatory support. The eventual discovery of eplet donor-recipient mismatches on related DQB1 alleles raised the diagnosis of antibody-mediated rejection. Before plasmapheresis could be instituted, the patient rapidly succumbed to respiratory failure. Postmortem examination confirmed features of atypical allograft rejection, without evidence of classic acute cellular rejection. This is an unconventional case of antibody-mediated lung allograft rejection – an entity that is currently a difficult diagnostic and therapeutic challenge. Prevention of donor-specific antibodies by correct donor-recipient matching, and optimizing adherence post-transplantation are most important. PMID:25802749

  14. Antibody Titer Threshold Predicts Anti-Candidal Vaccine Efficacy Even though the Mechanism of Protection Is Induction of Cell-Mediated Immunity

    PubMed Central

    Spellberg, Brad; Ibrahim, Ashraf S.; Lin, Lin; Avanesian, Valentina; Fu, Yue; Lipke, Peter; Otoo, Henry; Ho, Tiffany; Edwards, John E.

    2009-01-01

    We previously reported that vaccination with Freund’s adjuvant plus the recombinant N-terminus of the candidal adhesin, Als3p (rAls3p-N), protects mice from disseminated candidiasis. Here we report that the rAls3p-N vaccine is effective when combined with aluminum hydroxide adjuvant. Antibody titers of ≥1:6400 accurately predicted protection from infection. Nevertheless, neither B lymphocytes nor serum from immunized animals transferred protection to vaccine-naive animals. In contrast, CD3+, CD4+, or CD8+ T lymphocytes from immunized animals transferred protection, and the vaccine was efficacious in IL-4–deficient mice but not in IFN-γ–deficient mice. These data have significant implications for the development and interpretation of vaccine surrogate markers. PMID:18419471

  15. Treatment Options and Strategies for Antibody Mediated Rejection after Renal Transplantation

    PubMed Central

    Levine, Matthew H.

    2011-01-01

    Antibody mediated rejection is a significant clinical problem encountered in a subset of renal transplant recipients. This type of rejection has a variable pathogenesis from the presence of donor specific antibodies with no overt disease to immediate hyperacute rejection and many variations between. Antibody mediated rejection is more common in human leukocyte antigen sensitized patients. In general, transplant graft survival after antibody mediated rejection is jeopardized, with less than 50% graft survival 5 years after this diagnosis. A variety of agents have been utilized singly and in combinations to treat antibody mediated rejection with differing results and significant research efforts are being placed on developing new targets for intervention. These same agents have been used in desensitization protocols with some success. In this review, we describe the biology of antibody mediated rejection, review the available agents to treat this form of rejection, and highlight areas of ongoing and future research into this difficult clinical problem. PMID:21940179

  16. Antibody-Dependent Cellular Cytotoxicity against Reactivated HIV-1-Infected Cells.

    PubMed

    Lee, Wen Shi; Richard, Jonathan; Lichtfuss, Marit; Smith, Amos B; Park, Jongwoo; Courter, Joel R; Melillo, Bruno N; Sodroski, Joseph G; Kaufmann, Daniel E; Finzi, Andrés; Parsons, Matthew S; Kent, Stephen J

    2015-12-09

    Lifelong antiretroviral therapy (ART) for HIV-1 does not diminish the established latent reservoir. A possible cure approach is to reactivate the quiescent genome from latency and utilize immune responses to eliminate cells harboring reactivated HIV-1. It is not known whether antibodies within HIV-1-infected individuals can recognize and eliminate cells reactivated from latency through antibody-dependent cellular cytotoxicity (ADCC). We found that reactivation of HIV-1 expression in the latently infected ACH-2 cell line elicited antibody-mediated NK cell activation but did not result in antibody-mediated killing. The lack of CD4 expression on these HIV-1 envelope (Env)-expressing cells likely resulted in poor recognition of CD4-induced antibody epitopes on Env. To examine this further, cultured primary CD4(+) T cells from HIV-1(+) subjects were used as targets for ADCC. These ex vivo-expanded primary cells were modestly susceptible to ADCC mediated by autologous or heterologous HIV-1(+) serum antibodies. Importantly, ADCC mediated against these primary cells could be enhanced following incubation with a CD4-mimetic compound (JP-III-48) that exposes CD4-induced antibody epitopes on Env. Our studies suggest that with sufficient reactivation and expression of appropriate Env epitopes, primary HIV-1-infected cells can be targets for ADCC mediated by autologous serum antibodies and innate effector cells. The results of this study suggest that further investigation into the potential of ADCC to eliminate reactivated latently infected cells is warranted. An HIV-1 cure remains elusive due to the persistence of long-lived latently infected cells. An HIV-1 cure strategy, termed "shock and kill," aims to reactivate HIV-1 expression in latently infected cells and subsequently eliminate the reactivated cells through immune-mediated killing. While recent research efforts have focused on reversing HIV-1 latency, it remains unclear whether preexisting immune responses within HIV-1

  17. Antibody-Dependent Cellular Cytotoxicity against Reactivated HIV-1-Infected Cells

    PubMed Central

    Lee, Wen Shi; Richard, Jonathan; Lichtfuss, Marit; Smith, Amos B.; Park, Jongwoo; Courter, Joel R.; Melillo, Bruno N.; Sodroski, Joseph G.; Kaufmann, Daniel E.; Parsons, Matthew S.

    2015-01-01

    ABSTRACT Lifelong antiretroviral therapy (ART) for HIV-1 does not diminish the established latent reservoir. A possible cure approach is to reactivate the quiescent genome from latency and utilize immune responses to eliminate cells harboring reactivated HIV-1. It is not known whether antibodies within HIV-1-infected individuals can recognize and eliminate cells reactivated from latency through antibody-dependent cellular cytotoxicity (ADCC). We found that reactivation of HIV-1 expression in the latently infected ACH-2 cell line elicited antibody-mediated NK cell activation but did not result in antibody-mediated killing. The lack of CD4 expression on these HIV-1 envelope (Env)-expressing cells likely resulted in poor recognition of CD4-induced antibody epitopes on Env. To examine this further, cultured primary CD4+ T cells from HIV-1+ subjects were used as targets for ADCC. These ex vivo-expanded primary cells were modestly susceptible to ADCC mediated by autologous or heterologous HIV-1+ serum antibodies. Importantly, ADCC mediated against these primary cells could be enhanced following incubation with a CD4-mimetic compound (JP-III-48) that exposes CD4-induced antibody epitopes on Env. Our studies suggest that with sufficient reactivation and expression of appropriate Env epitopes, primary HIV-1-infected cells can be targets for ADCC mediated by autologous serum antibodies and innate effector cells. The results of this study suggest that further investigation into the potential of ADCC to eliminate reactivated latently infected cells is warranted. IMPORTANCE An HIV-1 cure remains elusive due to the persistence of long-lived latently infected cells. An HIV-1 cure strategy, termed “shock and kill,” aims to reactivate HIV-1 expression in latently infected cells and subsequently eliminate the reactivated cells through immune-mediated killing. While recent research efforts have focused on reversing HIV-1 latency, it remains unclear whether preexisting immune

  18. Monoclonal antibodies with specificity for hairy cell leukemia cells.

    PubMed Central

    Posnett, D N; Chiorazzi, N; Kunkel, H G

    1982-01-01

    Hairy cell leukemia is a well described clinical entity, but the cell of origin for this leukemic cell and its function are still unknown. There are no totally specific markers for this cell, although tartrate-resistant acid phosphatase staining has been used extensively as a diagnostic test. This study describes three monoclonal murine antibodies with variable specificity for hairy cells. Antibody 1 was highly specific for hairy cells and was not found to react with normal or leukemic cells in this limited study. It did not react with the cells of all patients. It also did not react with all of the hairy cells of some of the positive cases. Antibodies 2 and 3 reacted with virtually all hairy cells but not with normal peripheral blood cells. However, reactions were obtained with certain leukemic myelomonoblasts and some activated B cells. The most obvious use for these three antibodies is for diagnostic purposes. They should also be helpful reagents to investigate the origin of the leukemic hairy cell. The possibility that antibody 1 detects a tumor-specific antigen is discussed. Images PMID:7047565

  19. Antibody-mediated phagocytosis contributes to the anti-tumor activity of the therapeutic antibody daratumumab in lymphoma and multiple myeloma.

    PubMed

    Overdijk, Marije B; Verploegen, Sandra; Bögels, Marijn; van Egmond, Marjolein; Lammerts van Bueren, Jeroen J; Mutis, Tuna; Groen, Richard W J; Breij, Esther; Martens, Anton C M; Bleeker, Wim K; Parren, Paul W H I

    2015-01-01

    Daratumumab (DARA) is a human CD38-specific IgG1 antibody that is in clinical development for the treatment of multiple myeloma (MM). The potential for IgG1 antibodies to induce macrophage-mediated phagocytosis, in combination with the known presence of macrophages in the tumor microenvironment in MM and other hematological tumors, led us to investigate the contribution of antibody-dependent, macrophage-mediated phagocytosis to DARA's mechanism of action. Live cell imaging revealed that DARA efficiently induced macrophage-mediated phagocytosis, in which individual macrophages rapidly and sequentially engulfed multiple tumor cells. DARA-dependent phagocytosis by mouse and human macrophages was also observed in an in vitro flow cytometry assay, using a range of MM and Burkitt's lymphoma cell lines. Phagocytosis contributed to DARA's anti-tumor activity in vivo, in both a subcutaneous and an intravenous leukemic xenograft mouse model. Finally, DARA was shown to induce macrophage-mediated phagocytosis of MM cells isolated from 11 of 12 MM patients that showed variable levels of CD38 expression. In summary, we demonstrate that phagocytosis is a fast, potent and clinically relevant mechanism of action that may contribute to the therapeutic activity of DARA in multiple myeloma and potentially other hematological tumors.

  20. Neutralizing antibodies are unable to inhibit direct viral cell-to-cell spread of human cytomegalovirus.

    PubMed

    Jacob, Christian L; Lamorte, Louie; Sepulveda, Eliud; Lorenz, Ivo C; Gauthier, Annick; Franti, Michael

    2013-09-01

    Infection with human cytomegalovirus (CMV) during pregnancy is the most common cause of congenital disorders, and can lead to severe life-long disabilities with associated high cost of care. Since there is no vaccine or effective treatment, current efforts are focused on identifying potent neutralizing antibodies. A panel of CMV monoclonal antibodies identified from patent applications, was synthesized and expressed in order to reproduce data from the literature showing that anti-glycoprotein B antibodies neutralized virus entry into all cell types and that anti-pentameric complex antibodies are highly potent in preventing virus entry into epithelial cells. It had not been established whether antibodies could prevent subsequent rounds of infection that are mediated primarily by direct cell-to-cell transmission. A thorough validation of a plaque reduction assay to monitor cell-to-cell spread led to the conclusion that neutralizing antibodies do not significantly inhibit plaque formation or reduce plaque size when they are added post-infection. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Role of C1q complement fixing antibody assay in therapeutic plasma exchange management of pediatric cardiac antibody mediated rejection.

    PubMed

    Onwuemene, Oluwatoyosi A; Heath, Deneen M; Hartman, Carol; Wong, Edward C C

    2017-08-01

    Pediatric cardiac transplant patients with antibody-mediated rejection (AMR) often undergo therapeutic plasma exchange (TPE) to remove pathologic donor specific antibodies (DSA). In cases where DSA persist, it is unclear how long TPE should be continued. We report a case of a 17-year-old cardiac transplant patient with AMR where use of a C1q complement fixing antibody assay helped guide TPE cessation. This report adds to the existing literature that highlights the potential clinical significance of C1q antibodies in AMR management. © 2016 Wiley Periodicals, Inc.

  2. Acute antibody-mediated rejection after intestinal transplantation

    PubMed Central

    Wu, Guo-Sheng; Cruz Jr, Ruy J; Cai, Jun-Chao

    2016-01-01

    AIM To investigate the incidence, risk factors and clinical outcomes of acute antibody-mediated rejection (ABMR) after intestinal transplantation (ITx). METHODS A retrospective single-center analysis was performed to identify cases of acute ABMR after ITx, based on the presence of donor-specific antibody (DSA), acute tissue damage, C4d deposition, and allograft dysfunction. RESULTS Acute ABMR was identified in 18 (10.3%) out of 175 intestinal allografts with an average occurrence of 10 d (range, 4-162) after ITx. All acute ABMR cases were presensitized to donor human leukocyte antigens class I and/or II antigens with a detectable DSA. A positive cross-match was seen in 14 (77.8%) cases and twelve of 18 patients (66.7%) produced newly-formed DSA following ITx. Histological characteristics of acute ABMR include endothelial C4d deposits, interstitial hemorrhage, and severe congestion with focal fibrin thrombin in the lamina propria capillaries. Multivariate analysis identified a liver-free graft and high level of panel reactive antibody as a significant independent risk factor. Despite initial improvement after therapy, eleven recipients (61.1%) lost transplant secondary to rejection. Of those, 9 (50%) underwent graft removal and 4 (22.2%) received second transplantation following acute ABMR. At an average follow-up of 32.3 mo (range, 13.3-76.4), 8 (44.4%) recipients died. CONCLUSION Our results indicate that acute ABMR is an important cause of intestine graft dysfunction, particularly in a liver-exclusive graft and survivors are at an increased risk of developing refractory acute rejection and chronic rejection. More effective strategies to prevent and manage acute ABMR are needed to improve outcomes. PMID:28058223

  3. Diagnostic criteria of antibody-mediated rejection in kidney transplants.

    PubMed

    Mosquera Reboredo, J M; Vázquez Martul, E

    2011-01-01

    The diagnosis and treatment of anti-donor antibody-mediated rejection or humoral rejection (ABMR) is one of the main discussions at the moment in kidney transplantation. The search for histopathological markers that help us to diagnose ABMR has been more problematic, in contrast to the histological expression of cellular or tubulointerstitial rejection. Although the relationship between post-transplant anti-donor antibodies and the allograft's prognosis has been a topic of discussion for a long time, led in the main by P.Terasaki, it was not until the beginning of 1990s when P. Halloran studied the humoral mechanisms of rejection in greater depth. Feutch described the importance of C4d deposits as a marker that shows a humoral mechanism of allograft rejection in 1993. As a result of many studies carried out, the Banff consensus group established some diagnostic histopathological criteria of acute (ABMR) in 2003. These have been modified slightly in later meetings of the group. Furthermore, in 2005 this same working group looked at the physiopathological mechanisms causing chronic allograft failure in more detail and established the criteria defining chronic humoral rejection. In this review, we are trying to update any useful histopathological criteria for diagnosing acute and chronic ABMR.

  4. Antibody-mediated delivery of therapeutics for cancer therapy.

    PubMed

    Parakh, Sagun; Parslow, Adam C; Gan, Hui K; Scott, Andrew M

    2016-01-01

    Antibody-conjugated therapies (ACTs) combine the specificity of monoclonal antibodies to target cancer cells directly with highly potent payloads, often resulting in superior efficacy and/or reduced toxicity. This represents a new approach to the treatment of cancer. There have been highly promising clinical trial results using this approach with improvements in linker and payload technology. The breadth of current trials examining ACTs in haematological malignancies and solid tumours indicate the potential for clinical impact. This review will provide an overview of ACTs currently in clinical development as well as the principles of antibody delivery and types of payloads used, including cytotoxic drugs, radiolabelled isotopes, nanoparticle-based siRNA particles and immunotoxins. The focus of much of the clinical activity in ACTs has, understandably, been on their use as a monotherapy or in combination with standard of care drugs. This will continue, as will the search for better targets, linkers and payloads. Increasingly, as these drugs enter routine clinical care, important questions will arise regarding how to optimise ACT treatment approaches, including investigation of resistance mechanisms, biomarker and patient selection strategies, understanding of the unique toxicities of these drugs, and combinatorial approaches with standard therapies as well as emerging therapeutic agents like immunotherapy.

  5. Design considerations for liposomal vaccines: Influence of formulation parameters on antibody and cell-mediated immune responses to liposome associated antigens

    PubMed Central

    Watson, Douglas S.; Endsley, Aaron N.; Huang, Leaf

    2012-01-01

    Liposomes (phospholipid bilayer vesicles) are versatile and robust delivery systems for induction of antibody and T lymphocyte responses to associated subunit antigens. In the last 15 years, liposome vaccine technology has matured and now several vaccines containing liposome-based adjuvants have been approved for human use or have reached late stages of clinical evaluation. Given the intensifying interest in liposome-based vaccines, it is important to understand precisely how liposomes interact with the immune system and stimulate immunity. It has become clear that the physicochemical properties of liposomal vaccines – method of antigen attachment, lipid composition, bilayer fluidity, particle charge, and other properties – exert dramatic effects on the resulting immune response. Here, we present a comprehensive review of the physicochemical properties of liposomal vaccines and how they influence immune responses. A discussion of novel and emerging immunomodulators that are suitable for inclusion in liposomal vaccines is also presented. Through a comprehensive analysis of the body of liposomal vaccine literature, we enumerate a series of principles that can guide the rational design of liposomal vaccines to elicit immune responses of a desired magnitude and quality. We also identify major unanswered questions in the field, pointing the direction for future study. PMID:22306376

  6. Antibody-mediated enzyme replacement therapy targeting both lysosomal and cytoplasmic glycogen in Pompe disease.

    PubMed

    Yi, Haiqing; Sun, Tao; Armstrong, Dustin; Borneman, Scott; Yang, Chunyu; Austin, Stephanie; Kishnani, Priya S; Sun, Baodong

    2017-02-02

    Pompe disease is characterized by accumulation of both lysosomal and cytoplasmic glycogen primarily in skeletal and cardiac muscles. Mannose-6-phosphate receptor-mediated enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) targets the enzyme to lysosomes and thus is unable to digest cytoplasmic glycogen. Studies have shown that anti-DNA antibody 3E10 penetrates living cells and delivers "cargo" proteins to the cytosol or nucleus via equilibrative nucleoside transporter ENT2. We speculate that 3E10-mediated ERT with GAA will target both lysosomal and cytoplasmic glycogen in Pompe disease. A fusion protein (FabGAA) containing a humanized Fab fragment derived from the murine 3E10 antibody and the 110 kDa human GAA precursor was constructed and produced in CHO cells. Immunostaining with an anti-Fab antibody revealed that the Fab signals did not co-localize with the lysosomal marker LAMP2 in cultured L6 myoblasts or Pompe patient fibroblasts after incubation with FabGAA. Western blot with an anti-GAA antibody showed presence of the 150 kDa full-length FabGAA in the cell lysates, in addition to the 95- and 76 kDa processed forms of GAA that were also seen in the rhGAA-treated cells. Blocking of mannose-6-phosphate receptor with mannose-6-phosphate markedly reduced the 95- and the 76 kDa forms but not the 150 kDa form. In GAA-KO mice, FabGAA achieved similar treatment efficacy as rhGAA at an equal molar dose in reducing tissue glycogen contents. Our data suggest that FabGAA retains the ability of rhGAA to treat lysosomal glycogen accumulation and has the beneficial potential over rhGAA to reduce cytoplasmic glycogen storage in Pompe disease.

  7. Protein disulfide isomerases are antibody targets during immune-mediated tumor destruction

    PubMed Central

    Fonseca, Catia; Soiffer, Robert; Ho, Vincent; Vanneman, Matthew; Jinushi, Masahisa; Ritz, Jerome; Neuberg, Donna; Stone, Richard; DeAngelo, Dan

    2009-01-01

    The identification of cancer antigens that contribute to transformation and are linked with immune-mediated tumor destruction is an important goal for immunotherapy. Toward this end, we screened a murine renal cell carcinoma cDNA expression library with sera from mice vaccinated with irradiated tumor cells engineered to secrete granulocyte macrophage colony-stimulating factor (GM-CSF). Multiple nonmutated, overexpressed proteins that function in tumor cell migration, protein/nucleic acid homeostasis, metabolism, and stress responses were detected. Among these, the most frequently recognized clone was protein disulfide isomerase (PDI). High titer antibodies to human PDI were similarly induced in an acute myeloid leukemia patient who achieved a complete response after vac-cination with irradiated, autologous GM-CSF–secreting tumor cells in the setting of nonmyeloablative allogeneic bone marrow transplantation. Moreover, ERp5, a closely related disulfide isomerase involved in major histocompatibility complex (MHC) class I chain-related protein A (MICA) shedding, also evoked potent humoral reactions in diverse solid and hematologic malignancy patients who responded to GM-CSF–secreting tumor cell vaccines or antibody blockade of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4). Together, these findings reveal the unexpected immunogenicity of PDIs and raise the possibility that these gene products might serve as targets for therapeutic monoclonal antibodies. PMID:19008459

  8. Antibodies to human fetal erythroid cells from a nonimmune phage antibody library

    PubMed Central

    Huie, Michael A.; Cheung, Mei-Chi; Muench, Marcus O.; Becerril, Baltazar; Kan, Yuet W.; Marks, James D.

    2001-01-01

    The ability to isolate fetal nucleated red blood cells (NRBCs) from the maternal circulation makes possible prenatal genetic analysis without the need for diagnostic procedures that are invasive for the fetus. Such isolation requires antibodies specific to fetal NRBCs. To generate a panel of antibodies to antigens present on fetal NRBCs, a new type of nonimmune phage antibody library was generated in which multiple copies of antibody fragments are displayed on each phage. Antibody fragments specific for fetal NRBCs were isolated by extensive predepletion of the phage library on adult RBCs and white blood cells (WBCs) followed by positive selection and amplification on fetal liver erythroid cells. After two rounds of selection, 44% of the antibodies analyzed bound fetal NRBCs, with two-thirds of these showing no binding of WBCs. DNA fingerprint analysis revealed the presence of at least 16 unique antibodies. Antibody specificity was confirmed by flow cytometry, immunohistochemistry, and immunofluorescence of total fetal liver and adult RBCs and WBCs. Antibody profiling suggested the generation of antibodies to previously unknown fetal RBC antigens. We conclude that multivalent display of antibodies on phage leads to efficient selection of panels of specific antibodies to cell surface antigens. The antibodies generated to fetal RBC antigens may have clinical utility for isolating fetal NRBCs from maternal circulation for noninvasive prenatal genetic diagnosis. Some of the antibodies may also have possible therapeutic utility for erythroleukemia. PMID:11226299

  9. Leukemia in AKR mice. I. Effects of leukemic cells on antibody-forming potential of syngeneic and allogeneic normal cells

    PubMed Central

    1976-01-01

    Cells from the spleen, thymus, lymph node, and liver of leukemic AKR mice suppress in vitro antibody responses of normal syngeneic and semiallogeneic cells. This suppression can be mediated by irradiated leukemic cells, requires cell contact between leukemic and normal cells, and may occur at any time during the in vitro culture period. Leukemic AKR cells do not suppress antibody responses of allogeneic cells, even when allogeneic cells have H-2 or background genes homologous with AKR. Leukemic cells do, however, suppress cells that are unable to respond allogeneically to leukemic AKR cells, such as cells of the F1s of AKR. Suppression of normal AKR antibody responses by leukemic AKR cells may be overcome by addition of irradiated allogeneic cells. The fact that leukemic AKR cells are able to suppress normal lymphocyte responses may be of significance in pathogenesis of leukemia in these mice. PMID:942993

  10. A refractory case of subclinical antibody-mediated rejection due to anti-HLA-DQ antibody in a kidney transplant patient.

    PubMed

    Fujimoto, Toshinari; Nakada, Yasuyuki; Yamamoto, Izumi; Kobayashi, Akimitsu; Tanno, Yudo; Yamada, Hiroki; Miki, Jun; Ohkido, Ichiro; Tsuboi, Nobuo; Yamamoto, Hiroyasu; Yokoo, Takashi

    2015-07-01

    We herein report a refractory case of subclinical antibody-mediated rejection (AMR) due to anti-HLA-DQ antibody in a kidney transplant patient. A 45-year-old man was admitted for a protocol biopsy; he had a serum creatinine (S-Cr) level of 1.8 mg/dL 3 years following primary kidney transplantation. Histological examination revealed moderate to severe inflammatory cell infiltration in the peritubular capillaries. Thorough laboratory examination showed that the patient had donor-specific antibodies (DSAbs) to DR9 and DQ9. Considering both the histological and laboratory findings, we diagnosed acute antibody-mediated rejection. The patient underwent 3 days of consecutive steroid pulse therapy, intravenous immunoglobulin (IVIG), and plasma exchange. We also administered rituximab (200 mg/body). Six months after the treatment, a second allograft biopsy revealed the progression of interstitial fibrosis and tubular atrophy and persistence of mild peritubular capillaritis. Further analysis showed that the anti-DR9 antibodies had disappeared, but that the mean fluorescence intensity value of the anti-DQ9 antibodies had increased. Therefore, we repeated the plasma exchange and IVIG. Allograft function was stable throughout the course of treatment, and the S-Cr level remained at 1.8 mg/dL. This case report demonstrates the difficulty of treating AMR due to the presence of anti-DQ DSAbs and the necessity for subsequent therapies in refractory cases.

  11. BLyS-mediated modulation of naïve B cell subsets impacts HIV Env-induced antibody responses1

    PubMed Central

    Dosenovic, Pia; Soldemo, Martina; Scholz, Jean L.; O’Dell, Sijy; Grasset, Emilie K.; Pelletier, Nadège; Karlsson, Mikael C. I.; Mascola, John R.; Wyatt, Richard T.; Cancro, Michael P.; Karlsson Hedestam, Gunilla B.

    2012-01-01

    Neutralizing Abs provide the protective effect of the majority of existing human vaccines. For a prophylactic vaccine against HIV-1, broadly neutralizing Abs (bNAbs) targeting conserved epitopes of the viral envelope glycoproteins (Env) are likely required, as the pool of circulating HIV-1 variants is extremely diverse. The failure to efficiently induce bNAbs by vaccination may be due to the use of sub-optimal immunogens or immunization regimens, or it may indicate that B cells specific for broadly neutralizing Env determinants are selected against during peripheral checkpoints, either before or after antigen encounter. To investigate if perturbation of B cell subsets prior to immunization with recombinant Env protein affects the vaccine-induced Ab response in mice, we used B Lymphocyte Stimulator (BLyS), a cytokine that regulates survival and selection of peripheral B cells. We show that the transient BLyS treatment used here substantially affected naïve B cell populations; in particular, it resulted in an increased number of B cells surviving counter-selection at the transitional stages. We also observed an increased number of mature naïve B cells, especially marginal zone B cells, in BLyS-treated mice. Intriguingly, provision of excess BLyS prior to immunization led to a consistent improvement in the frequency and potency of HIV-1 Env vaccine-induced neutralizing Ab responses, without increasing the number of Env-specific Ab-secreting cells or the Ab binding titers measured after boosting. The results presented here suggest that an increased understanding of BLyS-regulated processes may help the design of vaccine regimens aimed at eliciting improved neutralizing Ab responses against HIV-1. PMID:22561155

  12. Engineering human cells for in vivo secretion of antibody and non-antibody therapeutic proteins.

    PubMed

    Sánchez-Martín, David; Sanz, Laura; Álvarez-Vallina, Luis

    2011-12-01

    Purified proteins such as antibodies are widely used as therapeutic agents in clinical medicine. However, clinical-grade proteins for therapeutic use require sophisticated technologies and are extremely expensive to produce. In vivo secretion of therapeutic proteins by genetically engineered human cells may advantageously replace injection of highly purified proteins. The use of gene transfer methods circumvents problems related to large-scale production and purification and offers additional benefits by achieving sustained concentrations of therapeutic protein with a syngenic glycosylation pattern that make the protein potentially less immunogenic. The feasibility of the in vivo production of therapeutic proteins by diverse cells/tissues has now been demonstrated using different techniques, such as ex vivo genetically modified cells and in vivo gene transfer mediated by viral vectors.

  13. Polyreactive Antibodies Plus Complement Enhance the Phagocytosis of Cells Made Apoptotic by UV-Light or HIV

    PubMed Central

    Zhou, Zhao-hua; Wild, Teresa; Xiong, Ying; Sylvers, Peter; Zhang, Yahong; Zhang, Luxia; Wahl, Larry; Wahl, Sharon M.; Kozlowski, Steven; Notkins, Abner L.

    2013-01-01

    Polyreactive antibodies are a major component of the natural antibody repertoire and are capable of binding a variety of structurally unrelated antigens. Many of the properties attributed to natural antibodies, in fact, are turning out to be due to polyreactive antibodies. In humans, each day, billions of cells undergo apoptosis. In the present experiments, we show by ImageStream technology that although polyreactive antibodies do not bind to live T cells they bind to both the plasma membrane and cytoplasm of late apoptotic cells, fix complement, generate the anaphylatoxin C5a and increase by as much as 5 fold complement-mediated phagocytosis by macrophages. Of particular importance, T cells undergoing apoptosis following infection with HIV also bind polyreactive antibodies and are phagocytosed. We conclude that the polyreactive antibodies in the natural antibody repertoire contribute in a major way to the clearance of cells made apoptotic by a variety of natural and infectious processes. PMID:23881356

  14. The persistent elimination of B cells responding to blood group A carbohydrates by synthetic group A carbohydrates and B-1 cell differentiation blockade: novel concept in preventing antibody-mediated rejection in ABO-incompatible transplantation.

    PubMed

    Irei, Toshimitsu; Ohdan, Hideki; Zhou, Wendy; Ishiyama, Kohei; Tanaka, Yuka; Ide, Kentaro; Asahara, Toshimasa

    2007-12-15

    We demonstrated a novel strategy for specific and persistent inhibition of antibody (Ab) production against blood group A or B carbohydrate determinants necessary for successful ABO-incompatible transplantation. Similar to human blood group O or B individuals, mice have naturally occurring Abs against human blood group A carbohydrates in their sera. B cells with receptors for A carbohydrates in mice belonging to the CD5(+)CD11b(+)B-1a subset have phenotypic properties similar to those of human B cells. These cells could be temporarily eliminated by injecting synthetic A carbohydrates (GalNAcalpha1-3, Fucalpha1-2Gal) conjugated to bovine serum albumin (A-BSA) and anti-BSA Abs. In mice that received the injection of A-BSA/anti-BSA Abs, the serum levels of anti-A IgM were reduced, but immunization with human A erythrocytes resulted in increased serum levels of anti-A Abs. When combined with cyclosporin A (CsA) treatment, which blocks B-1a cell differentiation, and treatment with A-BSA/anti-BSA Abs, the serum levels of anti-A Abs were persistently undetectable in the mice even after the immunization. B cells with receptors for A carbohydrates were markedly reduced in these mice. These results are consistent with the hypotheses that treatment with A-BSA/anti-BSA Abs temporarily depletes B cells responding to A determinants, and CsA treatment prevents the replenishment of these cells.

  15. Unraveling Effector Functions of B Cells During Infection: The Hidden World Beyond Antibody Production

    PubMed Central

    León, Beatriz; Ballesteros-Tato, André; Misra, Ravi S.; Wojciechowski, Wojciech; Lund, Frances E.

    2015-01-01

    Antibodies made by B cells are critically important for immune protection to a variety of infectious agents. However, it is becoming increasingly clear that B cells do more than make antibodies and that B cells can both enhance and suppress immune responses. Furthermore, there is growing evidence that B cells modulate cellular immune responses by antibody dependent and independent mechanisms. Although we have a good understanding of the roles played by antibody-secreting effector B cells during immune responses, we know very little about the Ab independent “effector” functions of B cells in either health or disease. Given the recent data suggesting that B cells may contribute to autoimmune disease pathogenesis via an antibody independent mechanism and the increasing use of B cell depletion therapy in autoimmune patients, investigators are beginning to reassess the multiple roles for B cells during immune responses. In this article, we review data describing how B cells mediate protection to pathogens independently of antibody production. In particular, we will focus on the role that B cells play in facilitating dendritic cell and T cell interactions in lymph nodes, the importance of antigen-presenting B cells in sustaining effector T cell and T follicular helper responses to pathogens and the relevance of cytokine-producing effector and regulatory B cells in immune responses. PMID:22394173

  16. Decitabine enhances anti-CD33 monoclonal antibody BI 836858-mediated natural killer ADCC against AML blasts.

    PubMed

    Vasu, Sumithira; He, Shun; Cheney, Carolyn; Gopalakrishnan, Bhavani; Mani, Rajeswaran; Lozanski, Gerard; Mo, Xiaokui; Groh, Veronica; Whitman, Susan P; Konopitzky, Renate; Kössl, Christian; Bucci, Donna; Lucas, David M; Yu, Jianhua; Caligiuri, Michael A; Blum, William; Adam, Paul J; Borges, Eric; Rueter, Bjoern; Heider, Karl-Heinz; Marcucci, Guido; Muthusamy, Natarajan

    2016-06-09

    Acute myeloid leukemia (AML) is the most common type of acute leukemia, affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence, novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (gemtuzumab ozogamicin) have been shown to improve overall survival, validating CD33 as a target for antibody-based therapy of AML. Here, we report the in vitro efficacy of BI 836858, a fully human, Fc-engineered, anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858-opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine, 2 hypomethylating agents that show efficacy in older patients, did not compromise BI 836858-induced NK-cell-mediated ADCC. Evaluation of BI 836858-mediated ADCC in serial marrow AML aspirates in patients who received a 10-day course of DAC (pre-DAC, days 4, 11, and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared with pre-DAC treatment. Analysis of ligands to activating receptors (NKG2D) showed significantly increased NKG2D ligand [NKG2DL] expression in day 28 post-DAC samples compared with pre-DAC samples; when NKG2DL receptor was blocked using antibodies, BI 836858-mediated ADCC was significantly decreased, suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML.

  17. Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonadherence.

    PubMed

    Sellarés, J; de Freitas, D G; Mengel, M; Reeve, J; Einecke, G; Sis, B; Hidalgo, L G; Famulski, K; Matas, A; Halloran, P F

    2012-02-01

    We prospectively studied kidney transplants that progressed to failure after a biopsy for clinical indications, aiming to assign a cause to every failure. We followed 315 allograft recipients who underwent indication biopsies at 6 days to 32 years posttransplant. Sixty kidneys progressed to failure in the follow-up period (median 31.4 months). Failure was rare after T-cell-mediated rejection and acute kidney injury and common after antibody-mediated rejection or glomerulonephritis. We developed rules for using biopsy diagnoses, HLA antibody and clinical data to explain each failure. Excluding four with missing information, 56 failures were attributed to four causes: rejection 36 (64%), glomerulonephritis 10 (18%), polyoma virus nephropathy 4 (7%) and intercurrent events 6 (11%). Every rejection loss had evidence of antibody-mediated rejection by the time of failure. Among rejection losses, 17 of 36 (47%) had been independently identified as nonadherent by attending clinicians. Nonadherence was more frequent in patients who progressed to failure (32%) versus those who survived (3%). Pure T-cell-mediated rejection, acute kidney injury, drug toxicity and unexplained progressive fibrosis were not causes of loss. This prospective cohort indicates that many actual failures after indication biopsies manifest phenotypic features of antibody-mediated or mixed rejection and also underscores the major role of nonadherence.

  18. Social Support Mediates Loneliness and Human Herpesvirus Type 6 (HHV-6) Antibody Titers.

    PubMed

    Dixon, Denise; Cruess, Stacy; Kilbourn, Kristin; Klimas, Nancy; Fletcher, Mary Ann; Ironson, Gail; Baum, Andrew; Schneiderman, Neil; Antoni, Michael H

    2006-07-31

    The current study investigated the impact of a severe environmental stressor and the role that declining social integration played in mediating its effect on loneliness and immune status. Increased loneliness and decreased social support in the months following the stressor (storm) were significantly associated with increased HHV-6 antibody titers, reflecting poorer control over the virus. Poorer social integration mediated the relationship between loneliness and HHV-6, even after controlling for nonspecific polyclonal B-cell activation, disease status (CD3+CD4+ cell counts), living arrangements, acute social losses (bereavement), and potential disruptions in social-support resources. These findings suggest that specific elements of social support may explain the oft-noted negative effects of loneliness on the immune system, and generalized to a medically vulnerable population.

  19. Social Support Mediates Loneliness and Human Herpesvirus Type 6 (HHV-6) Antibody Titers

    PubMed Central

    Dixon, Denise; Cruess, Stacy; Kilbourn, Kristin; Klimas, Nancy; Fletcher, Mary Ann; Ironson, Gail; Baum, Andrew; Schneiderman, Neil; Antoni, Michael H.

    2009-01-01

    The current study investigated the impact of a severe environmental stressor and the role that declining social integration played in mediating its effect on loneliness and immune status. Increased loneliness and decreased social support in the months following the stressor (storm) were significantly associated with increased HHV-6 antibody titers, reflecting poorer control over the virus. Poorer social integration mediated the relationship between loneliness and HHV-6, even after controlling for nonspecific polyclonal B-cell activation, disease status (CD3+CD4+ cell counts), living arrangements, acute social losses (bereavement), and potential disruptions in social-support resources. These findings suggest that specific elements of social support may explain the oft-noted negative effects of loneliness on the immune system, and generalized to a medically vulnerable population. PMID:20407593

  20. Electron microscopic demonstration of lesions in target cell membranes associated with antibody-dependent cellular cytotoxicity.

    PubMed Central

    Dourmashkin, R R; Deteix, P; Simone, C B; Henkart, P

    1980-01-01

    To test the hypothesis that complement-mediated cell lysis and cell-mediated cytotoxicity operate by analogous mechanisms, cell membranes from two antibody-dependent cytotoxicity systems were examined by electron microscopy after negative staining. Ring-shaped membrane lesions generally similar to, but larger than, those previously described for complement lysis were observed. These findings are in agreement with recent measurements of larger functional pores for ADCC than complement. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7214742

  1. CD47 limits antibody dependent phagocytosis against non-malignant B cells.

    PubMed

    Gallagher, Sandra; Turman, Sean; Lekstrom, Kristen; Wilson, Susan; Herbst, Ronald; Wang, Yue

    2017-05-01

    Recent studies have demonstrated the importance of CD47 in protecting malignant B cells from antibody dependent cellular phagocytosis (ADCP). Combined treatment of anti-CD47 and -CD20 antibodies synergistically augment elimination of tumor B cells in xenograft mouse models. This has led to the development of novel reagents that can potentially enhance killing of malignant B cells in patients. B cell depleting therapy is also a promising treatment for autoimmune patients. In the current study, we aimed to investigate whether or not CD47 protects non-malignant B cells from ADCP. We show that CD47 is expressed on all B cells in mice, with the highest level on plasma cells in bone marrow and spleen. Although its expression is dispensable for B cell development in mice, CD47 on B cells limits antibody mediated phagocytosis. B cell depletion following in vivo anti-CD19 treatment is more efficient in CD47-/- mice than in wild type mice. In vitro, both naïve and activated B cells from CD47-/- mice are more sensitive to ADCP than wild type B cells. Lastly, we show in an ADCP assay that blocking CD47 can enhance anti-CD19 antibody mediated phagocytosis of wild type B cells. These results suggest that in addition to its already demonstrated benefit in cancer, targeting CD47 may be used as an adjunct in combination with B cell depletion antibodies for treatment of autoimmune diseases.

  2. Differential Detection of Tumor Cells Using a Combination of Cell Rolling, Multivalent Binding, and Multiple Antibodies

    PubMed Central

    2015-01-01

    Effective quantification and in situ identification of circulating tumor cells (CTCs) in blood are still elusive because of the extreme rarity and heterogeneity of the cells. In our previous studies, we developed a novel platform that captures tumor cells at significantly improved efficiency in vitro using a unique biomimetic combination of two physiological processes: E-selectin-induced cell rolling and poly(amidoamine) (PAMAM) dendrimer-mediated strong multivalent binding. Herein, we have engineered a novel multifunctional surface, on the basis of the biomimetic cell capture, through optimized incorporation of multiple antibodies directed to cancer cell-specific surface markers, such as epithelial cell adhesion molecule (EpCAM), human epidermal growth factor receptor-2 (HER-2), and prostate specific antigen (PSA). The surfaces were tested using a series of tumor cells, MDA-PCa-2b, MCF-7, and MDA-MB-361, both in mixture in vitro and after being spiked into human blood. Our multifunctional surface demonstrated highly efficient capture of tumor cells in human blood, achieving up to 82% capture efficiency (∼10-fold enhancement than a surface with the antibodies alone) and up to 90% purity. Furthermore, the multipatterned antibodies allowed differential capturing of the tumor cells. These results support that our multifunctional surface has great potential as an effective platform that accommodates virtually any antibodies, which will likely lead to clinically significant, differential detection of CTCs that are rare and highly heterogeneous. PMID:24892731

  3. Annexin A2 is involved in antiphospholipid antibody-mediated pathogenic effects in vitro and in vivo.

    PubMed

    Romay-Penabad, Zurina; Montiel-Manzano, Maria Guadalupe; Shilagard, Tuya; Papalardo, Elizabeth; Vargas, Gracie; Deora, Arun B; Wang, Michael; Jacovina, Andrew T; Garcia-Latorre, Ethel; Reyes-Maldonado, Elba; Hajjar, Katherine A; Pierangeli, Silvia S

    2009-10-01

    Antiphospholipid (aPL) antibodies recognize receptor-bound beta(2) glycoprotein I (beta(2)GPI) on target cells, and induce an intracellular signaling and a procoagulant/proinflammatory phenotype that leads to thrombosis. Evidence indicates that annexin A2 (A2), a receptor for tissue plasminogen activator and plasminogen, binds beta(2)GPI on target cells. However, whether A2 mediates pathogenic effects of aPL antibodies in vivo is unknown. In this work, we studied the effects of human aPL antibodies in A2-deficient (A2(-/-)) mice. A2(-/-) and A2(+/+) mice were injected with immunoglobulin G (IgG) isolated from either a patient with antiphospholipid syndrome (IgG-APS), a healthy control subject (IgG-normal human serum), a monoclonal anti-beta(2)GPI antibody (4C5), an anti-A2 monoclonal antibody, or monoclonal antibody of irrelevant specificity as control. We found that, after IgG-APS or 4C5 injections and vascular injury, mean thrombus size was significantly smaller and tissue factor activity was significantly less in A2(-/-) mice compared with A2(+/+) mice. The expression of vascular cell adhesion molecule-1 induced by IgG-APS or 4C5 in explanted A2(-/-) aorta was also significantly reduced compared with A2(+/+) mice. Interestingly, anti-A2 monoclonal antibody significantly decreased aPL-induced expression of intercellular cell adhesion molecule-1, E-selectin, and tissue factor activity on cultured endothelial cells. Together, these data indicate for the first time that A2 mediates the pathogenic effects of aPL antibodies in vivo and in vitro APS.

  4. Establishment of Stable, Cell-Mediated Immunity that Makes "Susceptible" Mice Resistant to Leishmania major

    NASA Astrophysics Data System (ADS)

    Bretscher, Peter A.; Wei, Guojian; Menon, Juthika N.; Bielefeldt-Ohmann, Helle

    1992-07-01

    Cell-mediated, but not antibody-mediated, immune responses protect humans against certain pathogens that produce chronic diseases such as leishmaniasis. Effective vaccination against such pathogens must therefore produce an immunological "imprint" so that stable, cell-mediated immunity is induced in all individuals after natural infection. BALB/c mice "innately susceptible" to Leishmania major produce antibodies after substantial infection. In the present study, "susceptible" mice injected with a small number of parasites mounted a cell-mediated response and acquired resistance to a larger, normally pathogenic, challenge. This vaccination strategy may be applicable in diseases in which protection is dependent on cell-mediated immunity.

  5. Antibody-protein A conjugated quantum dots for multiplexed imaging of surface receptors in living cells.

    PubMed

    Jin, Takashi; Tiwari, Dhermendra K; Tanaka, Shin-Ichi; Inouye, Yasushi; Yoshizawa, Keiko; Watanabe, Tomonobu M

    2010-11-01

    To use quantum dots (QDs) as fluorescent probes for receptor imaging, QD surface should be modified with biomolecules such as antibodies, peptides, carbohydrates, and small-molecule ligands for receptors. Among these QDs, antibody conjugated QDs are the most promising fluorescent probes. There are many kinds of coupling reactions that can be used for preparing antibody conjugated QDs. Most of the antibody coupling reactions, however, are non-selective and time-consuming. In this paper, we report a facile method for preparing antibody conjugated QDs for surface receptor imaging. We used ProteinA as an adaptor protein for binding of antibody to QDs. By using ProteinA conjugated QDs, various types of antibodies are easily attached to the surface of the QDs via non-covalent binding between the F(c) (fragment crystallization) region of antibody and ProteinA. To show the utility of ProteinA conjugated QDs, HER2 (anti-human epidermal growth factor receptor 2) in KPL-4 human breast cancer cells were stained by using anti-HER2 antibody conjugated ProteinA-QDs. In addition, multiplexed imaging of HER2 and CXCR4 (chemokine receptor) in the KPL-4 cells was performed. The result showed that CXCR4 receptors coexist with HER2 receptors in the membrane surface of KPL-4 cells. ProteinA mediated antibody conjugation to QDs is very useful to prepare fluorescent probes for multiplexed imaging of surface receptors in living cells.

  6. Myelin-reactive antibodies mediate the pathology of MBP-PLP fusion protein MP4-induced EAE.

    PubMed

    Kuerten, Stefanie; Pauly, Robert; Rottlaender, Andrea; Rodi, Michael; Gruppe, Traugott L; Addicks, Klaus; Tary-Lehmann, Magdalena; Lehmann, Paul V

    2011-07-01

    Experimental autoimmune encephalomyelitis (EAE) is frequently used for studies of multiple sclerosis (MS). Because in most EAE models T cells mediate the pathology in the absence of B cells/autoantibodies, the notion has evolved that also MS may be a primarily T cell-mediated disease. We have previously introduced MBP-PLP fusion protein (MP4)-induced EAE in C57BL/6 mice. Here we show that the disease in this model is antibody-dependent. Immunization of B cell-deficient mice did not induce EAE. When such B cell-deficient mice were, however, injected with MBP/PLP-specific antibodies in addition to the immunization with MP4, they developed disease of a severity and course that was similar to the wild-type mice. The deposition of antibodies in demyelinated lesions provided further evidence for the contribution of MBP/PLP-specific antibodies to CNS lesion formation. Based upon these data we suggest a two-stage model for the involvement of MBP/PLP-specific antibodies in autoimmune CNS pathology.

  7. Both Notch1 and its ligands in B cells promote antibody production.

    PubMed

    Zhu, Gaizhi; Wang, Xiaoqian; Xiao, He; Liu, Xiaoling; Fang, Ying; Zhai, Bing; Xu, Ruonan; Han, Gencheng; Chen, Guojiang; Hou, Chunmei; Shen, Beifen; Li, Yan; Ma, Ning; Wu, Haitao; Liu, Guangchao; Wang, Renxi

    2017-08-29

    Notch1 signaling regulates B and T lymphocyte development and also in vitro promotes antibody secretion upon B cell activation. However, it is still unclear about the role of Notch1 in antibody production upon in vitro and in vivo mixture lymphocytes activation. We first showed that Notch1 expressed in LPS-activated CD19(hi) B cells and CD19(cre) mediated Notch1 knock-down in LPS-activated B cells. Furthermore, we demonstrated that Notch1 knock-down in B cells reduced antibody production in LPS-stimulated B cells but did not affect antibody production in LPS-stimulated splenocytes and in experimental allergic encephalomyelitis (EAE) mice. Importantly, Notch1 ligands Dll1 and Jag1 expressed in B cells and pre-coated Notch1 protein promotes Notch1-knocked down B cells to produce antibody in LPS-stimulated B cells suggesting that Notch1 in other cells may promote antibody production by binding its ligands Dll1 and Jag1 in B cells. Together, our results suggest that both Notch1 and its ligands in B cells play an important role in antibody production. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Natural Killer (NK) Cell Education Differentially Influences HIV Antibody-Dependent NK Cell Activation and Antibody-Dependent Cellular Cytotoxicity.

    PubMed

    Bernard, Nicole F; Kiani, Zahra; Tremblay-McLean, Alexandra; Kant, Sanket A; Leeks, Christopher E; Dupuy, Franck P

    2017-01-01

    Immunotherapy using broadly neutralizing antibodies (bNAbs) endowed with Fc-mediated effector functions has been shown to be critical for protecting or controlling viral replication in animal models. In human, the RV144 Thai trial was the first trial to demonstrate a significant protection against HIV infection following vaccination. Analysis of the correlates of immune protection in this trial identified an association between the presence of antibody-dependent cellular cytotoxicity (ADCC) mediated by immunoglobulin G (IgG) antibodies (Abs) to HIV envelope (Env) V1/V2 loop structures and protection from infection, provided IgA Abs with competing specificity were not present. Systems serology analyses implicated a broader range of Ab-dependent functions in protection from HIV infection, including but not limited to ADCC and Ab-dependent NK cell activation (ADNKA) for secretion of IFN-γ and CCL4 and expression of the degranulation marker CD107a. The existence of such correlations in the absence of bNAbs in the RV144 trial suggest that NK cells could be instrumental in protecting against HIV infection by limiting viral spread through Fc-mediated functions such as ADCC and the production of antiviral cytokines/chemokines. Beside the engagement of FcγRIIIa or CD16 by the Fc portion of anti-Env IgG1 and IgG3 Abs, natural killer (NK) cells are also able to directly kill infected cells and produce cytokines/chemokines in an Ab-independent manner. Responsiveness of NK cells depends on the integration of activating and inhibitory signals through NK receptors, which is determined by a process during their development known as education. NK cell education requires the engagement of inhibitory NK receptors by their human leukocyte antigen ligands to establish tolerance to self while allowing NK cells to respond to self cells altered by virus infection, transformation, stress, and to allogeneic cells. Here, we review recent findings regarding the impact of inter

  9. Persistent expression of biologically active anti-HER2 antibody by AAVrh.10-mediated gene transfer.

    PubMed

    Wang, G; Qiu, J; Wang, R; Krause, A; Boyer, J L; Hackett, N R; Crystal, R G

    2010-08-01

    Trastuzumab (Herceptin) is a recombinant humanized monoclonal antibody (mAb) directed against an extracellular region of the human epidermal growth-factor receptor type 2 (HER2) protein. We hypothesized that a single adeno-associated virus (AAV)-mediated genetic delivery of an anti-HER2 antibody should be effective in mediating long-term production of anti-HER2 and in suppressing the growth of human tumors in a xenograft model in nude mice. The adeno-associated virus gene transfer vector AAVrh.10alphaHER2 was constructed based on a non-human primate AAV serotype rh.10 to express the complementary DNAs for the heavy and light chains of mAb 4D5, the murine precursor to trastuzumab. The data show that genetically transferred anti-HER2 selectively bound human HER2 protein and suppressed the proliferation of HER2(+) tumor cell lines. A single administration of AAVrh.10alphaHER2 provided long-term therapeutic levels of anti-HER2 antibody expression without inducing an anti-idiotype response, suppressed the growth of HER2(+) tumors and increased the survival of tumor bearing mice. In the context that trastuzumab therapy requires frequent and repeated administration, this strategy might be developed as an alternate platform for delivery of anti-HER2 therapy.

  10. VZV T cell-mediated immunity.

    PubMed

    Weinberg, Adriana; Levin, Myron J

    2010-01-01

    Primary varicella-zoster virus (VZV) infection (varicella) induces VZV-specific antibody and VZV-specific T cell-mediated immunity. T cell-mediated immunity, which is detected within 1-2 weeks after appearance of rash, and consists of both CD4 and CD8 effector and memory T cells, is essential for recovery from varicella. Administration of a varicella vaccine also generates VZV-specific humoral and cellular immune responses. The memory cell responses that develop during varicella or after vaccination contribute to protection following re-exposure to VZV. These responses are subsequently boosted either by endogenous re-exposure (silent reactivation of latent virus) or exogenous re-exposure (environmental). VZV-specific T cell-mediated immunity is also necessary to maintain latent VZV in a subclinical state in sensory ganglia. When these responses decline, as occurs with aging or iatrogenic immune suppression, reactivation of VZV leads to herpes zoster. Similarly, the magnitude of these responses early after the onset of herpes zoster correlates with the extent of zoster-associated pain. These essential immune responses are boosted by the VZV vaccine developed to prevent herpes zoster.

  11. Quantifying renal allograft loss following early antibody-mediated rejection.

    PubMed

    Orandi, B J; Chow, E H K; Hsu, A; Gupta, N; Van Arendonk, K J; Garonzik-Wang, J M; Montgomery, J R; Wickliffe, C; Lonze, B E; Bagnasco, S M; Alachkar, N; Kraus, E S; Jackson, A M; Montgomery, R A; Segev, D L

    2015-02-01

    Unlike antibody-mediated rejection (AMR) with clinical features, it remains unclear whether subclinical AMR should be treated, as its effect on allograft loss is unknown. It is also uncertain if AMR's effect is homogeneous across donor (deceased/live) and (HLA/ABO) antibody types. We compared 219 patients with AMR (77 subclinical, 142 clinical) to controls matched on HLA/ABO-compatibility, donor type, prior transplant, panel reactive antibody (PRA), age and year. One and 5-year graft survival in subclinical AMR was 95.9% and 75.7%, compared to 96.8% and 88.4% in matched controls (p = 0.0097). Subclinical AMR was independently associated with a 2.15-fold increased risk of graft loss (95% CI: 1.19-3.91; p = 0.012) compared to matched controls, but not different from clinical AMR (p = 0.13). Fifty three point two percent of subclinical AMR patients were treated with plasmapheresis within 3 days of their AMR-defining biopsy. Treated subclinical AMR patients had no difference in graft loss compared to matched controls (HR 1.73; 95% CI: 0.73-4.05; p = 0.21), but untreated subclinical AMR patients did (HR 3.34; 95% CI: 1.37-8.11; p = 0.008). AMR's effect on graft loss was heterogeneous when stratified by compatible deceased donor (HR = 4.73; 95% CI: 1.57-14.26; p = 0.006), HLA-incompatible deceased donor (HR = 2.39; 95% CI: 1.10-5.19; p = 0.028), compatible live donor (no AMR patients experienced graft loss), ABO-incompatible live donor (HR = 6.13; 95% CI: 0.55-67.70; p = 0.14) and HLA-incompatible live donor (HR = 6.29; 95% CI: 3.81-10.39; p < 0.001) transplant. Subclinical AMR substantially increases graft loss, and treatment seems warranted.

  12. Role of Fc in Antibody-Mediated Protection from Ricin Toxin

    PubMed Central

    Pincus, Seth. H.; Das, Anushka; Song, Kejing; Maresh, Grace A.; Corti, Miriam; Berry, Jody

    2014-01-01

    We have studied the role of the antibody (Ab) Fc region in mediating protection from ricin toxicity. We compared the in vitro and in vivo effects of intact Ig and of Fab fragments derived from two different neutralizing Ab preparations, one monoclonal, the other polyclonal. Consistent results were obtained from each, showing little difference between Ig and Fab in terms of antigen binding and in vitro neutralization, but with relatively large differences in protection of animals. We also studied whether importing Ab into the cell by Fc receptors enhanced the intracellular neutralization of ricin toxin. We found that the imported Ab was found in the ER and Golgi, a compartment traversed by ricin, as it traffics through the cell, but intracellular Ab did not contribute to the neutralization of ricin. These results indicate that the Fc region of antibody is important for in vivo protection, although the mechanism of enhanced protection by intact Ig does not appear to operate at the single cell level. When using xenogeneic antibodies, the diminished immunogenicity of Fab/F(ab’)2 preparations should be balanced against possible loss of protective efficacy. PMID:24811206

  13. Role of Fc in antibody-mediated protection from ricin toxin.

    PubMed

    Pincus, Seth H; Das, Anushka; Song, Kejing; Maresh, Grace A; Corti, Miriam; Berry, Jody

    2014-05-07

    We have studied the role of the antibody (Ab) Fc region in mediating protection from ricin toxicity. We compared the in vitro and in vivo effects of intact Ig and of Fab fragments derived from two different neutralizing Ab preparations, one monoclonal, the other polyclonal. Consistent results were obtained from each, showing little difference between Ig and Fab in terms of antigen binding and in vitro neutralization, but with relatively large differences in protection of animals. We also studied whether importing Ab into the cell by Fc receptors enhanced the intracellular neutralization of ricin toxin. We found that the imported Ab was found in the ER and Golgi, a compartment traversed by ricin, as it traffics through the cell, but intracellular Ab did not contribute to the neutralization of ricin. These results indicate that the Fc region of antibody is important for in vivo protection, although the mechanism of enhanced protection by intact Ig does not appear to operate at the single cell level. When using xenogeneic antibodies, the diminished immunogenicity of Fab/F(ab')2 preparations should be balanced against possible loss of protective efficacy.

  14. Current and future challenges in therapy for antibody-mediated rejection.

    PubMed

    Nair, Nandini; Ball, Timothy; Uber, Patricia A; Mehra, Mandeep R

    2011-06-01

    Antibody-mediated rejection (AMR) continues to present a challenge for the survival of the cardiac allograft. AMR appears to be on the rise, likely secondary to changing trends in clinical practice, including selection of patients for transplantation on mechanical circulatory support and development of more effective combinations of immunosuppressive drugs against acute cellular rejection. Most current strategies are aimed at treating acute AMR, but the treatment of chronic AMR is still not well defined. Clinically, AMR can often be more severe than cellular rejection and more difficult to treat, often not responding to typical protocols of increased immunosuppression. Complex steps involved in the antibody response allows for several potential targets for therapeutic intervention, including suppression of T and B cells, elimination of circulating antibodies, and inhibition of residual antibodies. Existing evidence suggests a multiregimen approach is the best option. Sustenance of accommodation and induction of tolerance could be viewed as viable options if adequate immune surveillance can be achieved in this setting. This review discusses the challenges in treating AMR and provides a critical analysis of current and possible future therapies.

  15. An analysis of gastric parietal cell antibodies and thyroid cell antibodies in patients with pernicious anaemia and thyroid disorders

    PubMed Central

    Cruchaud, A.; Juditz, E.

    1968-01-01

    An analysis of immunoglobulins responsible for gastric parietal cell antibodies and for antibodies to thyroid cell has been performed in twenty-four patients with pernicious anaemia and thirteen patients with thyroid disorders. Immunofluorescent technique with conjugated antisera specific to each of the three main immunoglobulins and to β1c-globulin was used. IgG-globulin was responsible for gastric parietal cell antibodies in all patients investigated; IgA was found in gastric parietal cell antibodies of four thyroid patients but in none of the pernicious anaemia patients; IgM antibodies were found in two pernicious anaemia patients and in six thyroid patients. With regard to thyroid cell antibodies, IgG and IgA were both found responsible for antibody activity in thirteen cases and IgM in ten cases. In six patients, antibodies to thyroid cell were not β1c-fixing, whereas all sera with gastric parietal cell antibodies fixed β1c-globulin. An obvious relationship could not be found either between the titres of the different types of antibodies within the same category of antibody, or between the titres of gastric parietal cell antibodies and thyroid cell antibodies in the same individual. An occasional relationship was found between the titres of IgG-parietal cell or thyroid cell antibodies and the capacity to bind β1c-globulin. These results demonstrate that each of the three main immunoglobulins may contribute to gastric parietal cell antibodies and to antibodies to thyroid cell. PMID:4180858

  16. ABO-compatible Liver Allograft Antibody-mediated Rejection: an update

    PubMed Central

    Demetris, Anthony J.; Zeevi, Adriana; O’Leary, Jacqueline G.

    2015-01-01

    Purpose Liver allograft antibody-mediated rejection (AMR) studies have lagged behind parallel efforts in kidney and heart because of a comparative inherent hepatic resistance to AMR. Three developments, however, have increased interest: 1) solid phase antibody testing enabled more precise antibody characterization; 2) increased expectations for long-term, morbidity-free survival; and 3) immunosuppression minimization trials. Recent Findings Two overlapping liver allograft AMR phenotypic expressions are beginning to emerge: acute and chronic AMR. Acute AMR usually occurs within the several weeks after transplantation and characterized clinically by DSA persistence, allograft dysfunction, thrombocytopenia, and hypocomplementemia. Acute AMR appears histopathologically similar to acute AMR in other organs: diffuse microvascular endothelial cell hypertrophy, C4d deposits, neutrophilic, eosinophilic, and macrophage-mediated microvasculitis/capillaritis, along with liver-specific ductular reaction, centrilobular hepatocyte swelling and hepatocanalicular cholestasis often combined with T cell-mediated rejection (TCMR). Chronic AMR is less well-defined, but strongly linked to serum class II DSA and associated with late-onset acute TCMR, fibrosis, chronic rejection and decreased survival. Unlike acute AMR, chronic AMR is a slowly evolving insult with a number of potential manifestations, but most commonly appears as low-grade lymphoplasmacytic portal and perivenular inflammation accompanied by unusual fibrosis patterns and variable microvascular C4d deposition; capillaritis is more difficult to identify than in acute AMR. Summary More precise DSA characterization, increasing expectations for long-term survival, and immunosuppression weaning precipitated a re-emergence of liver allograft AMR interest. Pathophysiological similarities exist between heart, kidney, and liver allografts, but liver-specific considerations may prove critical to our ultimate understanding of all

  17. Fluorometric assay for red blood cell antibodies

    SciTech Connect

    Schreiber, A.B.; Lambermont, M.; Strosberg, A.D.; Wybran, J.

    1981-03-01

    A fluorometric assay is described for the detection of red blood cell antibodies. The assay reveals as little as 600 molecules of bound, fluoroesceinated rabbit anti-human IgG antibodies per erythrocyte. Eleven patients with possible autoimmune erythrocyte disorder and negative direct antiglobulin test were studied by the fluorometric assay. The outcome of the fluorometric assay was compared with that of the human allogeneic rosette test. Results obtained by the two methods were in complete agreement. Five of the patients were shown to possess unexpectedly high levels of erythrocyte-bound IgG in spite of a negative, direct antiglobulin test. These findings and the validity of the fluorometric assay are discussed.

  18. Probable C4d-negative accelerated acute antibody-mediated rejection due to non-HLA antibodies.

    PubMed

    Niikura, Takahito; Yamamoto, Izumi; Nakada, Yasuyuki; Kamejima, Sahoko; Katsumata, Haruki; Yamakawa, Takafumi; Furuya, Maiko; Mafune, Aki; Kobayashi, Akimitsu; Tanno, Yudo; Miki, Jun; Yamada, Hiroki; Ohkido, Ichiro; Tsuboi, Nobuo; Yamamoto, Hiroyasu; Yokoo, Takashi

    2015-07-01

    We report a case of probable C4d-negative accelerated acute antibody-mediated rejection due to non-HLA antibodies. A 44 year-old male was admitted to our hospital for a kidney transplant. The donor, his wife, was an ABO minor mismatch (blood type O to A) and had Gitelman syndrome. Graft function was delayed; his serum creatinine level was 10.1 mg/dL at 3 days after transplantation. Open biopsy was performed immediately; no venous thrombosis was observed during surgery. Histology revealed moderate peritubular capillaritis and mild glomerulitis without C4d immunoreactivity. Flow cytometric crossmatching was positive, but no panel-reactive antibodies against HLA or donor-specific antibodies (DSAbs) to major histocompatibility complex class I-related chain A (MICA) were detected. Taken together, we diagnosed him with probable C4d-negative accelerated antibody-mediated rejection due to non-HLA, non-MICA antibodies, the patient was treated with steroid pulse therapy (methylprednisolone 500 mg/day for 3 days), plasma exchange, intravenous immunoglobulin (40 g/body), and rituximab (200 mg/body) were performed. Biopsy at 58 days after transplantation, at which time S-Cr levels were 1.56 mg/dL, found no evidence of rejection. This case, presented with a review of relevant literature, demonstrates that probable C4d-negative accelerated acute AMR can result from non-HLA antibodies.

  19. JC polyomavirus mutants escape antibody-mediated neutralization.

    PubMed

    Ray, Upasana; Cinque, Paola; Gerevini, Simonetta; Longo, Valeria; Lazzarin, Adriano; Schippling, Sven; Martin, Roland; Buck, Christopher B; Pastrana, Diana V

    2015-09-23

    JC polyomavirus (JCV) persistently infects the urinary tract of most adults. Under conditions of immune impairment, JCV causes an opportunistic brain disease, progressive multifocal leukoencephalopathy (PML). JCV strains found in the cerebrospinal fluid of PML patients contain distinctive mutations in surface loops of the major capsid protein, VP1. We hypothesized that VP1 mutations might allow the virus to evade antibody-mediated neutralization. Consistent with this hypothesis, neutralization serology revealed that plasma samples from PML patients neutralized wild-type JCV strains but failed to neutralize patient-cognate PML-mutant JCV strains. This contrasted with serological results for healthy individuals, most of whom robustly cross-neutralized all tested JCV variants. Mice administered a JCV virus-like particle (VLP) vaccine initially showed neutralizing "blind spots" (akin to those observed in PML patients) that closed after booster immunization. A PML patient administered an experimental JCV VLP vaccine likewise showed markedly increased neutralizing titer against her cognate PML-mutant JCV. The results indicate that deficient humoral immunity is a common aspect of PML pathogenesis and that vaccination may overcome this humoral deficiency. Thus, vaccination with JCV VLPs might prevent the development of PML.

  20. Successful rescue of refractory, severe antibody mediated rejection with splenectomy.

    PubMed

    Kaplan, Bruce; Gangemi, Antonio; Thielke, James; Oberholzer, José; Sankary, Howard; Benedetti, Enrico

    2007-01-15

    Antibody-mediated rejection (AMR) commonly occurs after transplantation of ABO-incompatible and sensitized renal transplant. Treatment regimens commonly include a combination of plasmapheresis (PL) and intravenous immunoglobulin (IVIG). However, some cases of AMR remain refractory to treatment. We report a case series of four patients with AMR refractory to standard therapy (ST) who resolved after splenectomy. Four living donor kidney transplant recipients were diagnosed with AMR. Two patients were ABO incompatible, one was cross-match positive and one had no obvious predisposing factors. After failure of therapy with corticosteroids, PL, IVIG, Thymoglobulin, and Rituximab (three patients) or Campath (one patient), AMR was treated with laparoscopic splenectomy. After an average of 11 days of ST, laparoscopic splenectomy was performed for rescue. The urinary output improved immediately in all patients, serum creatinine levels decreased within 48 hr, and ABO titers fell in the ABO-incompatible patient and the cross-match became negative in the two sensitized patients. Splenectomy may play a role in the treatment of AMR refractory to ST.

  1. Decitabine enhances anti-CD33 monoclonal antibody BI 836858–mediated natural killer ADCC against AML blasts

    PubMed Central

    Vasu, Sumithira; He, Shun; Cheney, Carolyn; Gopalakrishnan, Bhavani; Mani, Rajeswaran; Lozanski, Gerard; Mo, Xiaokui; Groh, Veronica; Whitman, Susan P.; Konopitzky, Renate; Kössl, Christian; Bucci, Donna; Lucas, David M.; Yu, Jianhua; Caligiuri, Michael A.; Blum, William; Adam, Paul J.; Borges, Eric; Rueter, Bjoern; Heider, Karl-Heinz; Marcucci, Guido

    2016-01-01

    Acute myeloid leukemia (AML) is the most common type of acute leukemia, affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence, novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (gemtuzumab ozogamicin) have been shown to improve overall survival, validating CD33 as a target for antibody-based therapy of AML. Here, we report the in vitro efficacy of BI 836858, a fully human, Fc-engineered, anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858–opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK-cell–mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine, 2 hypomethylating agents that show efficacy in older patients, did not compromise BI 836858–induced NK-cell–mediated ADCC. Evaluation of BI 836858–mediated ADCC in serial marrow AML aspirates in patients who received a 10-day course of DAC (pre-DAC, days 4, 11, and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared with pre-DAC treatment. Analysis of ligands to activating receptors (NKG2D) showed significantly increased NKG2D ligand [NKG2DL] expression in day 28 post-DAC samples compared with pre-DAC samples; when NKG2DL receptor was blocked using antibodies, BI 836858–mediated ADCC was significantly decreased, suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML. PMID:27013443

  2. B Cell-Based Seamless Engineering of Antibody Fc Domains

    PubMed Central

    Murayama, Akiho; Ohta, Kunihiro

    2016-01-01

    Engineering of monoclonal antibodies (mAbs) enables us to obtain mAbs with additional functions. In particular, modifications in antibody’s Fc (fragment, crystallizable) region can provide multiple benefits such as added toxicity by drug conjugation, higher affinity to Fc receptors on immunocytes, or the addition of functional modules. However, the generation of recombinant antibodies requires multiple laborious bioengineering steps. We previously developed a technology that enables rapid in vitro screening and isolation of specific mAb-expressing cells from the libraries constructed with chicken B-cell line DT40 (referred to as the ‘ADLib system’). To upgrade this ADLib system with the ability to generate customized mAbs, we developed a novel and rapid engineering technology that enables seamless exchanges of mAbs’ Fc domains after initial selections of mAb-producing clones by the ADLib system, using a gene-replacement unit for recombinase-mediated cassette exchange (RMCE). In this system, Cre-recombinase recognition sites were inserted into the Fc region of the active DT40 IgM allele, allowing the replacement of the Fc domain by the sequences of interest upon co-transfection of a Cre recombinase and a donor DNA, enabling the rapid exchange of Fc regions. Combining this method with the ADLib system, we demonstrate rapid Fc engineering to generate fluorescent antibodies and to enhance affinity to Fc receptors. PMID:27907066

  3. Cell adhesion molecules: detection with univalent second antibody

    PubMed Central

    1980-01-01

    Identification of cell surface molecules that play a role in cell-cell adhesion (here called cell adhesion molecules) has been achieved by demonstrating the inhibitory effect of univalent antibodies that bind these molecules in an in vitro assay of cell-cell adhesion. A more convenient reagent, intact (divalent) antibody, has been avoided because it might agglutinate the cells rather than blocking cell-cell adhesion. In this report, we show that intact rabbit immunoglobulin directed against certain cell surface molecules of Dictyostelium discoideum blocks cell-cell adhesion when the in vitro assay is performed in the presence of univalent goat anti-rabbit antibody. Under appropriate experimental conditions, the univalent second antibody blocks agglutination induced by the rabbit antibody without significantly interfering with its effect on cell-cell adhesion. This method promises to be useful for screening monoclonal antibodies raised against potential cell adhesion molecules because: (a) it allows for the screening of large numbers of antibody samples without preparation of univalent fragments; and (b) it requires much less antibody because of the greater affinity of divalent antibodies for antigens. PMID:6970200

  4. Antiendothelial Cell Antibodies in Patients With COPD.

    PubMed

    Karayama, Masato; Inui, Naoki; Suda, Takafumi; Nakamura, Yutaro; Nakamura, Hirotoshi; Chida, Kingo

    2010-12-01

    Circulating antiendothelial cell antibodies (AECA) bind to endothelial antigens and induce endothelial cell damage. These antibodies have been detected in patients with collagen vascular diseases and systemic vasculitis. Recently, autoimmune mechanisms and vascular involvement have attracted attention in COPD. This study aimed to investigate the expression of AECA in patients with COPD. A total of 116 patients with COPD, whose condition was established based on the Global Initiative for Chronic Obstructive Lung Disease criteria, were evaluated. Serum samples were examined for AECA by a cellular enzyme-linked immunosorbent assay using human umbilical vein endothelial cells. In addition, 157 subjects without any clinical or radiologic evidence of COPD or pulmonary disease served as a reference population. The patients with COPD exhibited significantly higher serum AECA concentrations than subjects in the reference population. The expression of AECA was significantly elevated in the patients with COPD, even when compared with that in smokers among the reference population who had similar smoking habits to the patients with COPD but normal spirometry. These findings suggest that an autoimmune component associated with endothelial cell damage is possibly involved in COPD.

  5. Late Failing Heart Allografts: Pathology of Cardiac Allograft Vasculopathy and Association With Antibody-Mediated Rejection.

    PubMed

    Loupy, A; Toquet, C; Rouvier, P; Beuscart, T; Bories, M C; Varnous, S; Guillemain, R; Pattier, S; Suberbielle, C; Leprince, P; Lefaucheur, C; Jouven, X; Bruneval, P; Duong Van Huyen, J P

    2016-01-01

    In heart transplantation, there is a lack of robust evidence of the specific causes of late allograft failure. We hypothesized that a substantial fraction of failing heart allografts may be associated with antibody-mediated injury and immune-mediated coronary arteriosclerosis. We included all patients undergoing a retransplantation for late terminal heart allograft failure in three referral centers. We performed an integrative strategy of heart allograft phenotyping by assessing the heart vascular tree including histopathology and immunohistochemistry together with circulating donor-specific antibodies. The main analysis included 40 explanted heart allografts patients and 402 endomyocardial biopsies performed before allograft loss. Overall, antibody-mediated rejection was observed in 19 (47.5%) failing heart allografts including 16 patients (40%) in whom unrecognized previous episodes of subclinical antibody-mediated rejection occurred 4.5 ± 3.5 years before allograft loss. Explanted allografts with evidence of antibody-mediated rejection demonstrated higher endothelitis and microvascular inflammation scores (0.89 ± 0.26 and 2.25 ± 0.28, respectively) compared with explanted allografts without antibody-mediated rejection (0.42 ± 0.11 and 0.36 ± 0.09, p = 0.046 and p < 0.0001, respectively). Antibody-mediated injury was observed in 62.1% of failing allografts with pure coronary arteriosclerosis and mixed (arteriosclerosis and atherosclerosis) pattern, while it was not observed in patients with pure coronary atherosclerosis (p = 0.0076). We demonstrate that antibody-mediated rejection is operating in a substantial fraction of failing heart allografts and is associated with severe coronary arteriosclerosis. Unrecognized subclinical antibody-mediated rejection episodes may be observed years before allograft failure.

  6. Mechanism of human antibody-mediated neutralization of Marburg virus.

    PubMed

    Flyak, Andrew I; Ilinykh, Philipp A; Murin, Charles D; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L; Hashiguchi, Takao; Bornholdt, Zachary A; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Ward, Andrew B; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E

    2015-02-26

    The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition.

  7. Mechanism of Human Antibody-Mediated Neutralization of Marburg Virus

    PubMed Central

    Flyak, Andrew I.; Ilinykh, Philipp A.; Murin, Charles D.; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L.; Hashiguchi, Takao; Bornholdt, Zachary A.; Slaughter, James C.; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G.; Ward, Andrew B.; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E.

    2015-01-01

    Summary The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of Antibody-GP complexes reveals that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP but not to MARV GP. The data suggest that MARV neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition. PMID:25723164

  8. Integrins in epithelial cell polarity: using antibodies to analyze adhesive function and morphogenesis.

    PubMed

    Matlin, Karl S; Haus, Brian; Zuk, Anna

    2003-07-01

    Epithelial cells polarize in response to cell-substratum and cell-cell adhesive interactions. Contacts between cells and proteins of the extracellular matrix are mediated by integrin receptors. Of the 24 recognized integrin heterodimers, epithelial cells typically express four or more distinct integrins, with the exact complement dependent on the tissue of origin. Investigation of the roles of integrins in epithelial cell polarization has depended on the use of function-blocking antibodies both to determine ligand specificity of individual integrins and to disrupt and redirect normal morphogenesis. In this article we describe techniques for employing function-blocking anti-integrin antibodies in adhesion assays of the polarized Madin-Darby canine kidney (MDCK) cell line and to demonstrate the involvement of beta1 integrins in collagen-induced tubulocyst formation. These techniques can be easily expanded to other antibodies and epithelial cell lines to characterize specific functions of individual integrins in epithelial morphogenesis.

  9. TU-100 (Daikenchuto) and ginger ameliorate anti-CD3 antibody induced T cell-mediated murine enteritis: microbe-independent effects involving Akt and NF-κB suppression.

    PubMed

    Ueno, Nobuhiro; Hasebe, Takumu; Kaneko, Atsushi; Yamamoto, Masahiro; Fujiya, Mikihiro; Kohgo, Yutaka; Kono, Toru; Wang, Chong-Zhi; Yuan, Chun-Su; Bissonnette, Marc; Chang, Eugene B; Musch, Mark W

    2014-01-01

    The Japanese traditional medicine daikenchuto (TU-100) has anti-inflammatory activities, but the mechanisms remain incompletely understood. TU-100 includes ginger, ginseng, and Japanese pepper, each component possessing bioactive properties. The effects of TU-100 and individual components were investigated in a model of intestinal T lymphocyte activation using anti-CD3 antibody. To determine contribution of intestinal bacteria, specific pathogen free (SPF) and germ free (GF) mice were used. TU-100 or its components were delivered by diet or by gavage. Anti-CD3 antibody increased jejunal accumulation of fluid, increased TNFα, and induced intestinal epithelial apoptosis in both SPF and GF mice, which was blocked by either TU-100 or ginger, but not by ginseng or Japanese pepper. TU-100 and ginger also blocked anti-CD3-stimulated Akt and NF-κB activation. A co-culture system of colonic Caco2BBE and Jurkat-1 cells was used to examine T-lymphocyte/epithelial cells interactions. Jurkat-1 cells were stimulated with anti-CD3 to produce TNFα that activates epithelial cell NF-κB. TU-100 and ginger blocked anti-CD3 antibody activation of Akt in Jurkat cells, decreasing their TNFα production. Additionally, TU-100 and ginger alone blocked direct TNFα stimulation of Caco2BBE cells and decreased activation of caspase-3 and polyADP ribose. The present studies demonstrate a new anti-inflammatory action of TU-100 that is microbe-independent and due to its ginger component.

  10. Increasing FcγRIIa affinity of an FcγRIII-optimized anti-EGFR antibody restores neutrophil-mediated cytotoxicity

    PubMed Central

    Derer, Stefanie; Glorius, Pia; Schlaeth, Martin; Lohse, Stefan; Klausz, Katja; Muchhal, Umesh; Desjarlais, John R; Humpe, Andreas; Valerius, Thomas; Peipp, Matthias

    2014-01-01

    Antibody-dependent cell-mediated cytotoxicity (ADCC) has been suggested as an essential mechanism for the in vivo activity of cetuximab, an epidermal growth factor receptor (EGFR)-targeting therapeutic antibody. Thus, enhancing the affinity of human IgG1 antibodies to natural killer (NK) cell-expressed FcγRIIIa by glyco- or protein-engineering of their Fc portion has been demonstrated to improve NK cell-mediated ADCC and to represent a promising strategy to improve antibody therapy. However, human polymorphonuclear (PMN) effector cells express the highly homologous FcγRIIIb isoform, which is described to be ineffective in triggering ADCC. Here, non-fucosylated or protein-engineered anti-EGFR antibodies with optimized FcγRIIIa affinities demonstrated the expected benefit in NK cell-mediated ADCC, but did not mediate ADCC by PMN, which could be restored by FcγRIIIb blockade. Furthermore, eosinophils and PMN from paroxysmal nocturnal hemoglobinuria patients that expressed no or low levels of FcγRIIIb mediated effective ADCC with FcγRIII-optimized anti-EGFR antibody. Additional experiments with double FcγRIIa/FcγRIII-optimized constructs demonstrated enhanced PMN-mediated ADCC compared with single FcγRIII-optimized antibody. In conclusion, our data demonstrate that FcγRIIIb engagement impairs PMN-mediated ADCC activity of FcγRIII-optimized anti-EGFR antibodies, while further optimization of FcγRIIa binding significantly restores PMN recruitment. PMID:24492248

  11. Efficacy of anti-CD47 antibody-mediated phagocytosis with macrophages against primary effusion lymphoma.

    PubMed

    Goto, Hiroki; Kojima, Yuki; Matsuda, Kouki; Kariya, Ryusho; Taura, Manabu; Kuwahara, Kazuhiko; Nagai, Hirokazu; Katano, Harutaka; Okada, Seiji

    2014-07-01

    Recently, the critical role of CD47 on the surface of resistant cancer cells has been proposed in their evasion of immunosurveillance. Primary effusion lymphoma (PEL) is a subtype of aggressive non-Hodgkin lymphoma that shows serous lymphomatous effusion in body cavities, especially in advanced acquired immunodeficiency syndrome (AIDS). PEL is resistant to conventional chemotherapy and has a poor prognosis. In this study, we evaluated the effect of anti-CD47 antibody (Ab) on PEL in vitro and in vivo. Surface CD47 of PEL cell lines was examined by flow cytometry. Efficacy of knocking down CD47 or anti-CD47 Ab-mediated phagocytosis against PEL was evaluated using mouse peritoneal macrophages and human macrophages in vitro. Primary PEL cells were injected intraperitoneally into NOD/Rag-2/Jak3 double-deficient (NRJ) mice to establish a direct xenograft mouse model. Surface CD47 of PEL cell lines was highly expressed. Knocking down CD47 and anti-CD47 Ab promoted phagocytic activities of macrophages in a CD47 expression-dependent manner in vitro. Treatment with anti-CD47 Ab inhibited ascite formation and organ invasion completely in vivo compared with control IgG-treated mice. CD47 plays the pivotal role in the immune evasion of PEL cells in body cavities. Therapeutic antibody targeting of CD47 could be an effective therapy for PEL. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Notch-Mediated Cell Adhesion

    PubMed Central

    Murata, Akihiko; Hayashi, Shin-Ichi

    2016-01-01

    Notch family members are generally recognized as signaling molecules that control various cellular responses in metazoan organisms. Early fly studies and our mammalian studies demonstrated that Notch family members are also cell adhesion molecules; however, information on the physiological roles of this function and its origin is limited. In this review, we discuss the potential present and ancestral roles of Notch-mediated cell adhesion in order to explore its origin and the initial roles of Notch family members dating back to metazoan evolution. We hypothesize that Notch family members may have initially emerged as cell adhesion molecules in order to mediate multicellularity in the last common ancestor of metazoan organisms. PMID:26784245

  13. A reappraisal of humoral immunity based on mechanisms of antibody-mediated protection against intracellular pathogens.

    PubMed

    Casadevall, Arturo; Pirofski, Liise-anne

    2006-01-01

    Sometime in the mid to late twentieth century the study of antibody-mediated immunity (AMI) entered the doldrums, as many immunologists believed that the function of AMI was well understood, and was no longer deserving of intensive investigation. However, beginning in the 1990s studies using monoclonal antibodies (mAbs) revealed new functions for antibodies, including direct antimicrobial effects and their ability to modify host inflammatory and cellular responses. Furthermore, the demonstration that mAbs to several intracellular bacterial and fungal pathogens were protective issued a serious challenge to the paradigm that host defense against such microbes was strictly governed by cell-mediated immunity (CMI). Hence, a new view of AMI is emerging. This view is based on the concept that a major function of antibody (Ab) is to amplify or subdue the inflammatory response to a microbe. In this regard, the "damage-response framework" of microbial pathogenesis provides a new conceptual viewpoint for understanding mechanisms of AMI. According to this view, the ability of an Ab to affect the outcome of a host-microbe interaction is a function of its capacity to modify the damage ensuing from such an interaction. In fact, it is increasingly apparent that the efficacy of an Ab cannot be defined either by immunoglobulin or epitope characteristics alone, but rather by a complex function of Ab variables, such as specificity, isotype, and amount, host variables, such as genetic background and immune status, and microbial variables, such as inoculum, mechanisms of avoiding host immune surveillance and pathogenic strategy. Consequently, far from being understood, recent findings in AMI imply a system with unfathomable complexity and the field is poised for a long overdue renaissance.

  14. Tumour cell-antibody interactions. II. In vitro studies.

    PubMed Central

    Froese, G; Berczi, I; Israels, L G

    1982-01-01

    The interaction of L5178Y thymic lymphoma cells syngeneic to DBA/2 mice and of normal thymocytes with goat IgG antibodies was studied in vitro. Viable tumour and normal cells exerted a rapid, continuous and temperature-dependent destruction of antibody activity. Fractionation studies of culture supernatants from antibody-coated cells revealed that a significant portion of the antibody was completely degraded to amino acids. Tumour cells digested antibody more effectively than did normal lymphocytes. This observed degradation of antibody was most extensive at 37 degrees, significantly less at room temperature (23 degrees) and not detectable at 0 degrees. Undegraded antibody released from antibody-coated cells had also lost its antibody activity to a considerable extent. This was due to the formation of soluble antigen-antibody complexes, which was observed even at 0 degrees. Cells fixed with 10% formalin bound maximum amounts of antibody were incapable of digesting antibody even at 37 degrees and did not release immune complexes. These findings are of relevance to cancer immunodiagnosis and immunotherapy. PMID:6977481

  15. Cellular Uptake of α-Synuclein Oligomer-Selective Antibodies is Enhanced by the Extracellular Presence of α-Synuclein and Mediated via Fcγ Receptors.

    PubMed

    Gustafsson, Gabriel; Eriksson, Fredrik; Möller, Christer; da Fonseca, Tomás Lopes; Outeiro, Tiago F; Lannfelt, Lars; Bergström, Joakim; Ingelsson, Martin

    2017-01-01

    Immunotherapy targeting aggregated α-synuclein has emerged as a potential treatment strategy against Parkinson's disease and other α-synucleinopathies. We have developed α-synuclein oligomer/protofibril selective antibodies that reduce toxic α-synuclein in a human cell line and, upon intraperitoneal administration, in spinal cord of transgenic mice. Here, we investigated under which conditions and by which mechanisms such antibodies can be internalized by cells. For this purpose, human neuroglioma H4 cells were treated with either monoclonal oligomer/protofibril selective α-synuclein antibodies, linear epitope monoclonal α-synuclein antibodies, or with a control antibody. The oligomer/protofibril selective antibody mAb47 displayed the highest cellular uptake and was therefore chosen for additional analyses. Next, α-synuclein overexpressing cells were incubated with mAb47, which resulted in increased antibody internalization as compared to non-transfected cells. Similarly, regular cells exposed to mAb47 together with media containing α-synuclein displayed a higher uptake as compared to cells incubated with regular media. Finally, different Fcγ receptors were targeted and we then found that blockage of FcγRI and FcγRIIB/C resulted in reduced antibody internalization. Our data thus indicate that the robust uptake of the oligomer/protofibril selective antibody mAb47 by human CNS-derived cells is enhanced by extracellular α-synuclein and mediated via Fcγ receptors. Altogether, our finding lend further support to the belief that α-synuclein pathology can be modified by monoclonal antibodies and that these can target toxic α-synuclein species in the extracellular milieu. In the context of immunotherapy, antibody binding of α-synuclein would then not only block further aggregation but also mediate internalization and subsequent degradation of antigen-antibody complexes.

  16. A Highly Conserved Residue of the HIV-1 gp120 Inner Domain Is Important for Antibody-Dependent Cellular Cytotoxicity Responses Mediated by Anti-cluster A Antibodies

    PubMed Central

    Ding, Shilei; Veillette, Maxime; Coutu, Mathieu; Prévost, Jérémie; Scharf, Louise; Bjorkman, Pamela J.; Ferrari, Guido; Robinson, James E.; Stürzel, Christina; Hahn, Beatrice H.; Sauter, Daniel; Kirchhoff, Frank; Lewis, George K.; Pazgier, Marzena

    2015-01-01

    Previous studies have shown that sera from HIV-1-infected individuals contain antibodies able to mediate antibody-dependent cellular cytotoxicity (ADCC). These antibodies preferentially recognize envelope glycoprotein (Env) epitopes induced upon CD4 binding. Here, we show that a highly conserved tryptophan at position 69 of the gp120 inner domain is important for ADCC mediated by anti-cluster A antibodies and sera from HIV-1-infected individuals. PMID:26637462

  17. [Antibody-mediated rejection of renal allograft and the update Banff classification 2013].

    PubMed

    Honsová, Eva

    2015-01-01

    The view on the role of donor-specific antibodies in organ transplantation has been changed during the last several decades. Today, it is considered that the majority of cases of the late renal allograft dysfunction and loss are caused by the presence of donor-specific antibodies to HLA antigens. The real breakthrough in the diagnosis of antibody-mediated rejection was represented by the discovery of C4d, which enabled the determination of the diagnostic criteria of acute and later chronic antibody-mediated rejection. Although detection of C4d has been the cornerstone in the diagnosis of antibody-mediated rejection for over 10 years, it has become clear that some cases with similar morphological and clinical features do not have detectable C4d. Outcomes of key studies concerning presence of donor specific antibodies and morphological features in the graft biopsy samples resulted in the modification of Banff classification of 2013, which includes integrating C4d negative antibody-mediated rejection and also that acute vascular rejection (v1, v2) can be a part of the antibody-mediated rejection.

  18. Natural killer cell mediated cytotoxic responses in the Tasmanian devil.

    PubMed

    Brown, Gabriella K; Kreiss, Alexandre; Lyons, A Bruce; Woods, Gregory M

    2011-01-01

    The Tasmanian devil (Sarcophilus harrisii), the world's largest marsupial carnivore, is under threat of extinction following the emergence of an infectious cancer. Devil facial tumour disease (DFTD) is spread between Tasmanian devils during biting. The disease is consistently fatal and devils succumb without developing a protective immune response. The aim of this study was to determine if Tasmanian devils were capable of forming cytotoxic antitumour responses and develop antibodies against DFTD cells and foreign tumour cells. The two Tasmanian devils immunised with irradiated DFTD cells did not form cytotoxic or humoral responses against DFTD cells, even after multiple immunisations. However, following immunisation with xenogenic K562 cells, devils did produce cytotoxic responses and antibodies against this foreign tumour cell line. The cytotoxicity appeared to occur through the activity of natural killer (NK) cells in an antibody dependent manner. Classical NK cell responses, such as innate killing of DFTD and foreign cancer cells, were not observed. Cells with an NK-like phenotype comprised approximately 4 percent of peripheral blood mononuclear cells. The results of this study suggest that Tasmanian devils have NK cells with functional cytotoxic pathways. Although devil NK cells do not directly recognise DFTD cancer cells, the development of antibody dependent cell-mediated cytotoxicity presents a potential pathway to induce cytotoxic responses against the disease. These findings have positive implications for future DFTD vaccine research.

  19. Natural Killer Cell Mediated Cytotoxic Responses in the Tasmanian Devil

    PubMed Central

    Brown, Gabriella K.; Kreiss, Alexandre; Lyons, A. Bruce; Woods, Gregory M.

    2011-01-01

    The Tasmanian devil (Sarcophilus harrisii), the world's largest marsupial carnivore, is under threat of extinction following the emergence of an infectious cancer. Devil facial tumour disease (DFTD) is spread between Tasmanian devils during biting. The disease is consistently fatal and devils succumb without developing a protective immune response. The aim of this study was to determine if Tasmanian devils were capable of forming cytotoxic antitumour responses and develop antibodies against DFTD cells and foreign tumour cells. The two Tasmanian devils immunised with irradiated DFTD cells did not form cytotoxic or humoral responses against DFTD cells, even after multiple immunisations. However, following immunisation with xenogenic K562 cells, devils did produce cytotoxic responses and antibodies against this foreign tumour cell line. The cytotoxicity appeared to occur through the activity of natural killer (NK) cells in an antibody dependent manner. Classical NK cell responses, such as innate killing of DFTD and foreign cancer cells, were not observed. Cells with an NK-like phenotype comprised approximately 4 percent of peripheral blood mononuclear cells. The results of this study suggest that Tasmanian devils have NK cells with functional cytotoxic pathways. Although devil NK cells do not directly recognise DFTD cancer cells, the development of antibody dependent cell-mediated cytotoxicity presents a potential pathway to induce cytotoxic responses against the disease. These findings have positive implications for future DFTD vaccine research. PMID:21957452

  20. Relationship between natural and heme-mediated antibody polyreactivity

    SciTech Connect

    Hadzhieva, Maya; Vassilev, Tchavdar; Bayry, Jagadeesh; Kaveri, Srinivas; Lacroix-Desmazes, Sébastien; Dimitrov, Jordan D.

    2016-03-25

    Polyreactive antibodies represent a considerable fraction of the immune repertoires. Some antibodies acquire polyreactivity post-translationally after interaction with various redox-active substances, including heme. Recently we have demonstrated that heme binding to a naturally polyreactive antibody (SPE7) results in a considerable broadening of the repertoire of recognized antigens. A question remains whether the presence of certain level of natural polyreactivity of antibodies is a prerequisite for heme-induced further extension of antigen binding potential. Here we used a second monoclonal antibody (Hg32) with unknown specificity and absence of intrinsic polyreactivity as a model to study the potential of heme to induce polyreactivity of antibodies. We demonstrated that exposure to heme greatly extends the antigen binding potential of Hg32, suggesting that the intrinsic binding promiscuity is not a prerequisite for the induction of polyreactivity by heme. In addition we compared the kinetics and thermodynamics of the interaction of heme-exposed antibodies with a panel of unrelated antigens. These analyses revealed that the two heme-sensitive antibodies adopt different mechanisms of binding to the same set of antigens. This study contributes to understanding the phenomenon of induced antibody polyreactivity. The data may also be of importance for understanding of physiological and pathological roles of polyreactive antibodies. - Highlights: • Exposure of certain monoclonal IgE antibodies to heme results in gain of antigen binding polyreactivity. • Natural polyreactivity of antibodies is dispensable for acquisition of polyreactivity through interaction with heme. • Heme-induced monoclonal IgE antibodies differ in their thermodynamic mechanisms of antigen recognition.

  1. Prevention trumps treatment of antibody-mediated transplant rejection.

    PubMed

    Knechtle, Stuart J; Kwun, Jean; Iwakoshi, Neal

    2010-04-01

    Belying the spectacular success of solid organ transplantation and improvements in immunosuppressive therapy is the reality that long-term graft survival rates remain relatively unchanged, in large part due to chronic and insidious alloantibody-mediated graft injury. Half of heart transplant recipients develop chronic rejection within 10 years - a daunting statistic, particularly for young patients expecting to achieve longevity by enduring the rigors of a transplant. The current immunosuppressive pharmacopeia is relatively ineffective in preventing late alloantibody-associated chronic rejection. In this issue of the JCI, Kelishadi et al. report that preemptive deletion of B cells prior to heart transplantation in cynomolgus monkeys, in addition to conventional posttransplant immunosuppressive therapy with cyclosporine, markedly attenuated not only acute graft rejection but also alloantibody elaboration and chronic graft rejection. The success of this preemptive strike implies a central role for B cells in graft rejection, and this approach may help to delay or prevent chronic rejection after solid organ transplantation.

  2. Siglecs as targets for therapy in immune cell mediated disease

    PubMed Central

    O’Reilly, Mary K.; Paulson, James C.

    2010-01-01

    The sialic acid-binding immunoglobulin-like lectins (siglecs) comprise a family of receptors that are differentially expressed on leukocytes and other immune cells. The restricted expression of several siglecs to one or a few cell types makes them attractive targets for cell-directed therapies. The anti-CD33 (Siglec-3) antibody Gemtuzumab (Mylotarg™) is approved for treatment of acute myeloid leukemia (AML), and antibodies targeting CD22 (Siglec-2) are currently in clinical trials for treatment of B cell non-Hodgkins lymphomas and autoimmune diseases. Because siglecs are endocytic receptors, they are well suited for a ‘Trojan horse’ strategy, whereby therapeutic agents conjugated to an antibody, or multimeric glycan ligand, bind to the siglec and are efficiently carried into the cell. Although the rapid internalization of unmodified siglec antibodies reduces their utility for induction of antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC), antibody binding of Siglec-8, Siglec-9, and CD22 have been demonstrated to induce apoptosis of eosinophils, neutrophils, and depletion of B cells, respectively. Here we review the properties of siglecs that make them attractive for cell-targeted therapies. PMID:19359050

  3. Antibody secreting cell assay for influenza A virus in swine

    USDA-ARS?s Scientific Manuscript database

    An ELISPOT assay to enumerate B-cells producing antibodies specific to a given antigen, also known as an antibody secreting cell (ASC) assay, was adapted to detect B-cells specific for influenza A virus (IAV). The assay is performed ex vivo and enumerates ASC at a single cell level. A simple ASC det...

  4. Anti-Inositolglycan Antibodies Selectively Block Some of the Actions of Insulin in Intact BC_3H1 Cells

    NASA Astrophysics Data System (ADS)

    Romero, Guillermo; Gamez, Graciela; Huang, Laura C.; Lilley, Kevin; Luttrell, Louis

    1990-02-01

    We have studied the mechanism of generation of insulin mediators by using specific antibodies raised against the oligosaccharide anchor of membrane proteins. These antibodies (i) block the in vitro effects of purified insulin mediators and (ii) block the insulin-induced stimulation of pyruvate dehydrogenase in intact BC_3H1 myocytes but not insulin-stimulated glucose uptake, generation of diacylglycerol, or generation of insulin mediators. When added to intact cells in the presence of insulin, these antibodies induce the accumulation of insulin mediator activity in the extracellular medium. We therefore conclude that these anti-inositolglycan antibodies block some of the effects of insulin by inhibiting the uptake of specific insulin mediators generated outside the cell.

  5. Detection of anti-liver cell membrane antibody using a human hepatocellular carcinoma cell line

    SciTech Connect

    Lobo-Yeo, A.; McSorley, C.; McFarlane, B.M.; Mieli-Vergani, G.; Mowat, A.P.; Vergani, D.

    1989-02-01

    A radioimmunometric technique for the detection of autoantibodies to liver membrane antigens has been developed using Alexander cells, a human hepatocellular carcinoma cell line. After incubation of Alexander cells with serum, antimembrane antibodies were detected by addition of /sup 125/I-labeled Protein A. Binding ratios in 15 children with uncontrolled autoimmune chronic active hepatitis and in seven children with primary sclerosing cholangitis were significantly higher than in 18 age-matched normal controls. Nine patients with inactive autoimmune chronic active hepatitis, 13 with alpha 1-antitrypsin deficiency and five with fulminant hepatic failure had ratios similar to controls. In nine patients with Wilson's disease, there was a modest but significant increase in binding ratio. In four children with autoimmune chronic active hepatitis, binding ratios fell during effective immunosuppressive therapy. Sera from patients with systemic lupus erythematosus or rheumatoid arthritis gave normal results, excluding that binding derives from Fc-mediated immune complex capture. A positive correlation was found between Alexander cell binding values and anti-liver-specific protein antibody titers, suggesting that the two assays detect antibodies against shared antigenic determinants. The Alexander cell assay is a simple, rapid and sensitive technique to detect antibody to liver cell membrane antigens.

  6. Circulating CD4(+)CXCR5(+) T Cells Exacerbate B Cell Antibody Production in Crohn's Disease Through IL-21 Secretion.

    PubMed

    Wang, Zhenlong; Ding, Lan; Wang, Zhiming; Wang, Jinjing; Zhu, Nan; Diao, Yanqing; Demmy, Todd A; Haitsma, Jack M; Lech-Maranda, Ewa E; Bangia, Naveen N; Czuczman, Myron A; Qian, Xiaoli; Kovacs, Kalman L

    2015-01-01

    Interleukin-21 (IL-21) upregulation was observed in Crohn's disease (CD) patients and was shown to contribute to ongoing mucosal inflammation in CD patients through stabilizing Th1 cell differentiation and IFN-γ production. Given the role of IL-21 in mediating adaptive B cell antibody responses in healthy individuals, we examined the effect of IL-21 upregulation in B cell responses in patients with active CD, including ileum, ileocolonic and colon subtypes, defined by the primary site of CD involvement. We first observed an upregulation of blood plasma IL-21 concentration and IL-21 production from CD4(+) T cells in CD patients compared to healthy individuals. The IL-21-expressing T cells were more concentrated in the CD4(+)CXCR5(+) compartment, both in unstimulated medium and after stimulation with SEB. ICOS and PD-1 expressions were also concentrated in the CD4(+)CXCR5(+) subset in CD patients. Since peripheral blood CD4(+)CXCR5(+) T cell-mediated antibody secretion is IL-21-dependent, we examined the plasma antibody concentration in CD patients and healthy controls. We found that CD patients had significantly higher plasma Ig level than healthy patients, with no significant differences between different CD subtypes. Higher plasma IL-21 concentration and increased IL-21 production from CD4(+) T cells were directly correlated with higher plasma antibody levels. Moreover, we found that IL-21 and CD4(+)CXCR5(+) T cells can directly enhance B cell antibody response in CD patients. Depletion of secreted IL-21 by sIL-21R addition compromised the CD4(+)CXCR5(+) T cell-mediated increase in antibody production. Together, our results demonstrated a novel role of IL-21 in mediating B cell inflammation in CD development.

  7. Antibody-mediated rejection despite inhibition of terminal complement.

    PubMed

    Bentall, Andrew; Tyan, Dolly B; Sequeira, Flavia; Everly, Matthew J; Gandhi, Manish J; Cornell, Lynn D; Li, Han; Henderson, Nicole A; Raghavaiah, Suresh; Winters, Jeffrey L; Dean, Patrick G; Stegall, Mark D

    2014-12-01

    Terminal complement blockade has been shown to decrease the incidence of early acute antibody-mediated rejection (eAMR) in the first month after positive cross-match kidney transplant recipients, yet some patients still develop eAMR. The current study investigated possible mechanisms of eAMR despite eculizumab treatment. Of the 26 patients treated with eculizumab, two developed clinical eAMR and another patient developed histologic signs of eAMR without graft dysfunction ('subclinical eAMR'). Twenty-three did not have histologic injury on early surveillance biopsies. All 26 patients had therapeutic levels of eculizumab and showed complete blockade of complement in hemolytic assays. High levels of donor-specific alloantibody (DSA) including total IgG, IgG3, and C1q+ DSA were present in patients with and without eAMR, and none correlated well with eAMR. In contrast, IgM DSA was present in only four patients after transplantation: the two patients with clinical eAMR, one patient with subclinical AMR, and one patient without eAMR (P = 0.006 correlation with eAMR). Both clinical eAMR episodes were easily treated with plasma exchange which removed IgM more completely and rapidly than IgG, resulting in normalization of function and histology. These data suggest a possible role of antidonor IgM DSA in the pathogenesis of eAMR in patients treated with terminal complement blockade (ClinicalTrials.gov Identifier: NCT00670774). © 2014 Steunstichting ESOT.

  8. Bortezomib-based treatment of acute antibody-mediated rejection: a case report.

    PubMed

    Wang, Q; Li, X L; Xu, X G; Shi, B Y; Zhang, Z M; Li, Z L; Han, Y; Zhou, W Q; Chen, C Q; Cai, M; Zhang, X

    2015-12-22

    Antibody-mediated rejection (AMR) is an important factor affecting survival after renal transplantation. A highly selective proteasome inhibitor, bortezomib, clears activated plasma cells from the body and has important therapeutic effect on AMR. We investigated the effects of bortezomib on AMR in a patient after a second renal transplant. Biopsy confirmed the diagnosis of mixed cellular rejection and AMR. Bortezomib was administered on day 1 (1.3 mg/m(2)), day 4 (1.0 mg/m(2)), and day 8 (1.0 mg/m(2)). On the same days, 250 mg methylprednisolone was administered once, and cyclosporine dose (5 mg·kg(-1)·day(-1)) was reduced by 50%. Oral mycophenolate mofetil and steroid were withdrawn on day 1 of bortezomib treatment. Intermittent double-filtration plasmapheresis was also performed. We monitored parameters, including T lymphocyte subsets, CD139 and CD19 expression, panel reactive antibody (PRA), and serum creatinine concentration. At follow-up 6 months after bortezomib treatment, we observed: 1) serum creatinine stabilized at 130 μM from a peak level of 337 μM; 2) PRA decreased from a maximum of 66.7 to 0%; 3) blood plasma cell percentage rebounded after significantly decreasing following the first dose of bortezomib; 4) in renal allograft biopsy, immunohistochemical staining for C4d shifted from strongly positive to negative, and cellular rejection shifted from type IIA to borderline; and 5) adverse effects such as platelet suppression, hypotension, and grade 3 peripheral neuropathy emerged. Bortezomib effectively treated antibody-mediated renal transplantation rejection in this case study, but clinical trials with large sample sizes are still needed to explore clinical safety and tolerability.

  9. Regulation of anti-HLA antibody-dependent natural killer cell activation by immunosuppressive agents.

    PubMed

    Shin, Bong-Ha; Ge, Shili; Mirocha, James; Karasyov, Artur; Vo, Ashley; Jordan, Stanley C; Toyoda, Mieko

    2014-02-15

    It was demonstrated that human natural killer (NK) cells, via antibody-dependent cellular cytotoxicity (ADCC)-like mechanism, increase IFNγ production after exposure to alloantigens. This finding was associated with an increased risk for antibody-mediated rejection (ABMR). Although the effects of various immunosuppressive drugs on T cells and B cells have been extensively studied, their effects on NK cells are less clear. This study reports the effect of immunosuppressive agents on antibody-mediated NK cell activation in vitro. Whole blood from normal individuals was incubated with irradiated peripheral blood mononuclear cells (PBMCs) pretreated with anti-HLA antibody+ sera (in vitro ADCC), with or without immunosuppressive agents. The %IFNγ+ and CD107a+ (degranulation marker) in CD56+ NK cells were enumerated by flow cytometry. Cyclosporine A and tacrolimus significantly reduced IFNγ production in a dose-dependent manner (53%-83%), but showed minimal effect on degranulation (20%). Prednisone significantly reduced both IFNγ production and degranulation (50%-66% reduction at maximum therapeutic levels). Calcineurin inhibitors (CNIs) in combination with prednisone additively suppressed IFNγ production and degranulation. The effect of sirolimus or mycophenolate mofetil on NK cells was minimal. These results suggest that potent suppressive effects of CNIs and prednisone on antibody-mediated NK cell activation may contribute to the reduction of ADCC in sensitized patients and possibly reduce the risk for ADCC-mediated ABMR. These further underscore the importance of medication compliance in prevention of ABMR and possibly chronic rejection, and suggest that ADCC-mediated injury may increase in strategies aimed at CNI or steroid minimization or avoidance.

  10. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells.

    PubMed

    Jäger, Volker; Büssow, Konrad; Wagner, Andreas; Weber, Susanne; Hust, Michael; Frenzel, André; Schirrmann, Thomas

    2013-06-26

    The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility. In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average. Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.

  11. Superficial dsg2 expression limits epidermal blister formation mediated by pemphigus foliaceus antibodies and exfoliative toxins.

    PubMed

    Brennan, Donna; Hu, Ying; Medhat, Walid; Dowling, Alicia; Mahoney, My G

    2010-01-01

    Cell-cell adhesion mediated by desmosomes is crucial for maintaining proper epidermal structure and function, as evidenced by several severe and potentially fatal skin disorders involving impairment of desmosomal proteins. Pemphigus foliaceus (PF) and staphylococcal scalded skin syndrome (SSSS) are subcorneal blistering diseases resulting from loss of function of the desmosomal cadherin, desmoglein 1 (Dsg1). To further study the pathomechanism of these diseases and to assess the adhesive properties of Dsg2, we employed a recently established transgenic (Tg) mouse model expressing Dsg2 in the superficial epidermis. Neonatal Tg and wild type (WT) mice were injected with purified ETA or PF Ig. We showed that ectopic expression of Dsg2 reduced the extent of blister formation in response to both ETA and PF Ig. In response to PF Ig, we observed either a dramatic loss or a reorganization of Dsg1-alpha, Dsg1-beta, and, to a lesser extent, Dsg1-gamma, in WT mice. The Inv-Dsg2 Tg mice showed enhanced retention of Dsg1 at the cell-cell border. Collectively, our data support the role for Dsg2 in cell adhesion and suggest that ectopic superficial expression of Dsg2 can increase membrane preservation of Dsg1 and limit epidermal blister formation mediated by PF antibodies and exfoliative toxins.

  12. Superficial Dsg2 Expression Limits Epidermal Blister Formation Mediated by Pemphigus Foliaceus Antibodies and Exfoliative Toxins

    PubMed Central

    Brennan, Donna; Hu, Ying; Medhat, Walid; Dowling, Alicia; Mahoney, Mỹ G.

    2010-01-01

    Cell-cell adhesion mediated by desmosomes is crucial for maintaining proper epidermal structure and function, as evidenced by several severe and potentially fatal skin disorders involving impairment of desmosomal proteins. Pemphigus foliaceus (PF) and staphylococcal scalded skin syndrome (SSSS) are subcorneal blistering diseases resulting from loss of function of the desmosomal cadherin, desmoglein 1 (Dsg1). To further study the pathomechanism of these diseases and to assess the adhesive properties of Dsg2, we employed a recently established transgenic (Tg) mouse model expressing Dsg2 in the superficial epidermis. Neonatal Tg and wild type (WT) mice were injected with purified ETA or PF Ig. We showed that ectopic expression of Dsg2 reduced the extent of blister formation in response to both ETA and PF Ig. In response to PF Ig, we observed either a dramatic loss or a reorganization of Dsg1-α, Dsg1-β, and, to a lesser extent, Dsg1-γ, in WT mice. The Inv-Dsg2 Tg mice showed enhanced retention of Dsg1 at the cell-cell border. Collectively, our data support the role for Dsg2 in cell adhesion and suggest that ectopic superficial expression of Dsg2 can increase membrane preservation of Dsg1 and limit epidermal blister formation mediated by PF antibodies and exfoliative toxins. PMID:20631906

  13. Effector-mediated eradication of precursor B acute lymphoblastic leukemia with a novel Fc engineered monoclonal antibody targeting the BAFF-R

    PubMed Central

    Parameswaran, Reshmi; Lim, Min; Fei, Fei; Abdel-Azim, Hisham; Arutyunyan, Anna; Schiffer, Isabelle; McLaughlin, Margaret E.; Gram, Hermann; Huet, Heather; Groffen, John; Heisterkamp, Nora

    2014-01-01

    B-cell activating factor receptor (BAFF-R) is expressed on precursor B acute lymphoblastic leukemia ALL (pre-B ALL) cells but not on their pre-B normal counterparts. Thus, selective killing of ALL cells is possible by targeting this receptor. Here we have further examined therapeutic targeting of pre-B ALL based on the presence of the BAFF-R. Mouse pre-B ALL cells lacking BAFF-R function had comparable viability and proliferation to wild type cells but were more sensitive to drug treatment. Viability of human pre-B ALL cells was further reduced when antibodies to the BAFF-R were combined with other drugs, even in the presence of stromal protection. This indicates that inhibition of BAFF-R function reduces fitness of stressed pre-B ALL cells. We tested a novel humanized anti-BAFF-R monoclonal antibody optimalized for FcRγIII mediated, antibody-dependent cell killing by effector cells. Antibody binding to human ALL cells was inhibitable, in a dose-dependent manner, by recombinant human BAFF. There was no evidence for internalization of the antibodies. The antibodies significantly stimulated NK cell-mediated killing of different human patient-derived ALL cells. Moreover, incubation of such ALL cells with these antibodies stimulated phagocytosis by macrophages. When this was tested in an immunodeficient transplant model, mice that were treated with the antibody had a significantly decreased leukemia burden in bone marrow and spleen. In view of the restricted expression of the BAFF-R on normal cells and the multiple anti-pre-B ALL activities stimulated by this antibody, a further examination of its use for treatment of pre-B ALL is warranted. PMID:24825858

  14. Naturally occurring anti-band-3 antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes

    SciTech Connect

    Lutz, H.U.; Bussolino, F.; Flepp, R.; Fasler, S.; Stammler, P.; Kazatchkine, M.D.; Arese, P.

    1987-11-01

    Treatment of erythrocytes with the thiol-specific oxidant azodicarboxylic acid bis(dimethylamide) (diamide) enhances their phagocytosis by adherent monocytes. Phagocytosis of diamide-treated erythrocytes required that the cells were opsonized with whole serum, since complement inactivation abolished phagocytosis. Opsonization with whole serum containing 20-100 times the physiological concentration of naturally occurring anti-band-3- antibodies enhanced phagocytosis of diamide-treated erythrocytes. High inputs of anti-band-3 also restored phagocytosis of erythrocytes that had been incubated with complement-inactivated serum. Elevated concentrations of anti-spectrin antibodies were ineffective in whole and complement-inactivated serum. Specific recognition of diamide-treated erythrocytes by anti-band-3 antibodies may be due to generation of anti-band-3 reactive protein oligomers on intact diamide-treated erythrocytes. Generation of such oligomers was dose-dependent with respect to diamide. Bound anti-band-3 alone was not sufficient to mediate phagocytosis. It resulted in deposition of complement component C3b on the cells through activation of the alternative complement pathway in amounts exceeding that of bound antibodies by two orders of magnitude. Thus, anti-band-3 and complement together mediate phagocytosis of oxidatively stressed erythrocytes, which simulate senescent erythrocytes with respect to bound antibody and complement.

  15. Lymphocyte Display: A Novel Antibody Selection Platform Based on T Cell Activation

    PubMed Central

    Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Álvarez-Vallina, Laura Sanz, Luis

    2009-01-01

    Since their onset, display technologies have proven useful for the selection of antibodies against a variety of targets; however, most of the antibodies selected with the currently available platforms need to be further modified for their use in humans, and are restricted to accessible antigens. Furthermore, these platforms are not well suited for in vivo selections. We present here a novel cell based antibody display platform, which takes advantage of the functional capabilities of T lymphocytes. The display of antibodies on the surface of T lymphocytes, as a part of a chimeric-immune receptor (CIR) mediating signaling, may ideally link the antigen-antibody interaction to a demonstrable change in T cell phenotype, due to subsequent expression of the early T cell activation marker CD69. In this proof-of-concept, an in vitro selection was carried out using a human T cell line lentiviral-transduced to express a tumor-specific CIR on the surface, against a human tumor cell line expressing the carcinoembryonic antigen. Based on an effective interaction between the CIR and the tumor antigen, we demonstrated that combining CIR-mediated activation with FACS sorting of CD69+ T cells, it is possible to isolate binders to tumor specific cell surface antigen, with an enrichment factor of at least 103-fold after two rounds, resulting in a homogeneous population of T cells expressing tumor-specific CIRs. PMID:19777065

  16. HIV-1 Vpu Antagonism of Tetherin Inhibits Antibody-Dependent Cellular Cytotoxic Responses by Natural Killer Cells

    PubMed Central

    Alvarez, Raymond A.; Hamlin, Rebecca E.; Monroe, Anthony; Moldt, Brian; Hotta, Mathew T.; Rodriguez Caprio, Gabriela; Fierer, Daniel S.; Simon, Viviana

    2014-01-01

    ABSTRACT The type I interferon-inducible factor tetherin retains virus particles on the surfaces of cells infected with vpu-deficient human immunodeficiency virus type 1 (HIV-1). While this mechanism inhibits cell-free viral spread, the immunological implications of tethered virus have not been investigated. We found that surface tetherin expression increased the antibody opsonization of vpu-deficient HIV-infected cells. The absence of Vpu also stimulated NK cell-activating FcγRIIIa signaling and enhanced NK cell degranulation and NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). The deletion of vpu in HIV-1-infected primary CD4+ T cells enhanced the levels of antibody binding and Fc receptor signaling mediated by HIV-positive-patient-derived antibodies. The magnitudes of antibody binding and Fc signaling were both highly correlated to the levels of tetherin on the surfaces of infected primary CD4 T cells. The affinity of antibody binding to FcγRIIIa was also found to be critical in mediating efficient Fc activation. These studies implicate Vpu antagonism of tetherin as an ADCC evasion mechanism that prevents antibody-mediated clearance of virally infected cells. IMPORTANCE The ability of the HIV-1 accessory factor to antagonize tetherin has been considered to primarily function by limiting the spread of virus by preventing the release of cell-free virus. This study supports the hypothesis that a major function of Vpu is to decrease the recognition of infected cells by anti-HIV antibodies at the cell surface, thereby reducing recognition by antibody-dependent clearance by natural killer cells. PMID:24623433

  17. Anti-DNA antibody mediated catalysis is isotype dependent.

    PubMed

    Xia, Yumin; Eryilmaz, Ertan; Zhang, Qiuting; Cowburn, David; Putterman, Chaim

    2016-01-01

    Anti-DNA antibodies are the serological hallmark of systemic lupus erythematosus, and participate in the pathogenesis of lupus nephritis by cross-reacting with multiple renal antigens. Previously, using a panel of murine anti-DNA IgGs that share identical variable regions but that differ in the constant regions, we demonstrated that the cross-reaction and renal pathogenicity of anti-DNA antibodies are isotype dependent. In this study, we investigated the catalytic potential of this anti-DNA antibody panel, and determined its isotype dependency. The three isotype switch variants (IgG1, IgG2a, IgG2b) and the parent IgG3 PL9-11 anti-DNA antibodies were compared in their catalysis of 500 base pair linear double stranded DNA and a 12-mer peptide (ALWPPNLHAWVP), by gel analysis, MALDI-TOF mass spectrometry, and nuclear magnetic resonance spectroscopy. The binding affinity of anti-DNA antibodies to double stranded DNA and peptide antigens were assessed by ELISA and surface plasmon resonance. We found that the PL9-11 antibody isotypes vary significantly in their potential to catalyze the cleavage of both linear and double stranded DNA and the proteolysis of peptides. The degree of the cleavage and proteolysis increases with the incubation temperature and time. While different PL9-11 isotypes have the same initial attack sites within the ALWPPNLHAWVP peptide, there was no correlation between binding affinity to the peptide and proteolysis rates. In conclusion, the catalytic properties of anti-DNA antibodies are isotype dependent. This finding provides further evidence that antibodies that share the same variable region, but which have different constant regions, are functionally distinct. The catalytic effects modulated by antibody constant regions need to be considered in the design of therapeutic antibodies (abzymes) and peptides designed to block pathogenic autoantibodies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Anti-DNA antibody mediated catalysis is isotype dependent

    PubMed Central

    Xia, Yumin; Eryilmaz, Ertan; Zhang, Qiuting; Cowburn, David; Putterman, Chaim

    2015-01-01

    Anti-DNA antibodies are the serological hallmark of systemic lupus erythematosus, and participate in the pathogenesis of lupus nephritis by cross-reacting with multiple renal antigens. Previously, using a panel of murine anti-DNA IgGs that share identical variable regions but that differ in the constant regions, we demonstrated that the cross-reaction and renal pathogenicity of anti-DNA antibodies are isotype dependent. In this study, we investigated the catalytic potential of this anti-DNA antibody panel, and determined its isotype dependency. The three isotype switch variants (IgG1, IgG2a, IgG2b) and the parent IgG3 PL9-11 anti-DNA antibodies were compared in their catalysis of 500 base pair linear double stranded DNA and a 12-mer peptide (ALWPPNLHAWVP), by gel analysis, MALDI-TOF mass spectrometry, and nuclear magnetic resonance spectroscopy. The binding affinity of anti-DNA antibodies to double stranded DNA and peptide antigens were assessed by ELISA and surface plasmon resonance. We found that the PL9-11 antibody isotypes vary significantly in their potential to catalyze the cleavage of both linear and double stranded DNA and the proteolysis of peptides. The degree of the cleavage and proteolysis increases with the incubation temperature and time. While different PL9-11 isotypes have the same initial attack sites within the ALWPPNLHAWVP peptide, there was no correlation between binding affinity to the peptide and proteolysis rates. In conclusion, the catalytic properties of anti-DNA antibodies are isotype dependent. This finding provides further evidence that antibodies that share the same variable region, but which have different constant regions, are functionally distinct. The catalytic effects modulated by antibody constant regions need to be considered in the design of therapeutic antibodies (abzymes) and peptides designed to block pathogenic autoantibodies. PMID:26655427

  19. What Lies Beneath: Antibody Dependent Natural Killer Cell Activation by Antibodies to Internal Influenza Virus Proteins.

    PubMed

    Vanderven, Hillary A; Ana-Sosa-Batiz, Fernanda; Jegaskanda, Sinthujan; Rockman, Steven; Laurie, Karen; Barr, Ian; Chen, Weisan; Wines, Bruce; Hogarth, P Mark; Lambe, Teresa; Gilbert, Sarah C; Parsons, Matthew S; Kent, Stephen J

    2016-06-01

    The conserved internal influenza proteins nucleoprotein (NP) and matrix 1 (M1) are well characterised for T cell immunity, but whether they also elicit functional antibodies capable of activating natural killer (NK) cells has not been explored. We studied NP and M1-specific ADCC activity using biochemical, NK cell activation and killing assays with plasma from healthy and influenza-infected subjects. Healthy adults had antibodies to M1 and NP capable of binding dimeric FcγRIIIa and activating NK cells. Natural symptomatic and experimental influenza infections resulted in a rise in antibody dependent NK cell activation post-infection to the hemagglutinin of the infecting strain, but changes in NK cell activation to M1 and NP were variable. Although antibody dependent killing of target cells infected with vaccinia viruses expressing internal influenza proteins was not detected, opsonising antibodies to NP and M1 likely contribute to an antiviral microenvironment by stimulating innate immune cells to secrete cytokines early in infection. We conclude that effector cell activating antibodies to conserved internal influenza proteins are common in healthy and influenza-infected adults. Given the significance of such antibodies in animal models of heterologous influenza infection, the definition of their importance and mechanism of action in human immunity to influenza is essential.

  20. Microarray diagnosis of antibody-mediated rejection in kidney transplant biopsies: an international prospective study (INTERCOM).

    PubMed

    Halloran, P F; Pereira, A B; Chang, J; Matas, A; Picton, M; De Freitas, D; Bromberg, J; Serón, D; Sellarés, J; Einecke, G; Reeve, J

    2013-11-01

    In a reference set of 403 kidney transplant biopsies, we recently developed a microarray-based test that diagnoses antibody-mediated rejection (ABMR) by assigning an ABMR score. To validate the ABMR score and assess its potential impact on practice, we performed the present prospective INTERCOM study (clinicaltrials.gov NCT01299168) in 300 new biopsies (264 patients) from six centers: Baltimore, Barcelona, Edmonton, Hannover, Manchester and Minneapolis. We assigned ABMR scores using the classifier created in the reference set and compared it to conventional assessment as documented in the pathology reports. INTERCOM documented uncertainty in conventional assessment: In 41% of biopsies where ABMR features were noted, the recorded diagnoses did not mention ABMR. The ABMR score correlated with ABMR histologic lesions and donor-specific antibodies, but not with T cell-mediated rejection lesions. The agreement between ABMR scores and conventional assessment was identical to that in the reference set (accuracy 85%). The ABMR score was more strongly associated with failure than conventional assessment, and when the ABMR score and conventional assessment disagreed, only the ABMR score was associated with early progression to failure. INTERCOM confirms the need to reduce uncertainty in the diagnosis of ABMR, and demonstrates the potential of the ABMR score to impact practice.

  1. A game of numbers: the stoichiometry of antibody-mediated neutralization of flavivirus infection

    PubMed Central

    Pierson, Theodore C.; Diamond, Michael S.

    2016-01-01

    The humoral response contributes to the protection against viral pathogens. Although antibodies have the potential to inhibit viral infections via several mechanisms, an ability to neutralize viruses directly may be particularly important. Neutralizing antibody titers are commonly used as predictors of protection from infection, especially in the context of vaccine responses and immunity. Despite the simplicity of the concept, how antibody binding results in virus inactivation is incompletely understood despite decades of research. Flaviviruses have been an attractive system in which to seek a structural and quantitative understanding of how antibody interactions with virions modulate infection because of the contribution of antibodies to both protection and pathogenesis. This review will present a stoichiometric model of antibody-mediated neutralization of flaviviruses and discuss how these concepts can inform the development of vaccines and antibody-based therapeutics. PMID:25595803

  2. The molecular landscape of antibody-mediated kidney transplant rejection: evidence for NK involvement through CD16a Fc receptors.

    PubMed

    Venner, J M; Hidalgo, L G; Famulski, K S; Chang, J; Halloran, P F

    2015-05-01

    The recent recognition that antibody-mediated rejection (ABMR) is the major cause of kidney transplant loss creates strong interest in its pathogenesis. We used microarray analysis of kidney transplant biopsies to identify the changes in pure ABMR. We found that the ABMR transcript changes in the initial Discovery Set were strongly conserved in a subsequent Validation Set. In the Combined Set of 703 biopsies, 2603 transcripts were significantly changed (FDR < 0.05) in ABMR versus all other biopsies. In cultured cells, the transcripts strongly associated with ABMR were expressed in endothelial cells, e.g. cadherins CDH5 and CDH13; IFNG-treated endothelial cells, e.g. phospholipase PLA1A and chemokine CXCL11; or NK cells, e.g. cytotoxicity molecules granulysin (GNLY) and FGFBP2. Other ABMR transcripts were expressed in normal kidney but not cell lines, either increased e.g. Duffy chemokine receptor (DARC) or decreased e.g. sclerostin (SOST). Pathway analysis of ABMR transcripts identified angiogenesis, with roles for angiopoietin and vascular endothelial growth factors; leukocyte-endothelial interactions; and NK signaling, including evidence for CD16a Fc receptor signaling elements shared with T cells. These data support a model of ABMR involving injury-repair in the microcirculation induced by cognate recognition involving antibody and CD16a, triggering IFNG release and antibody-dependent NK cell-mediated cytotoxicity.

  3. Monoclonal antibodies against plant cell wall polysaccharides

    SciTech Connect

    Hahn, M.G.; Bucheli, E.; Darvill, A.; Albersheim, P. )

    1989-04-01

    Monoclonal antibodies (McAbs) are useful tools to probe the structure of plant cell wall polysaccharides and to localize these polysaccharides in plant cells and tissues. Murine McAbs were generated against the pectic polysaccharide, rhamnogalacturonan I (RG-I), isolated from suspension-cultured sycamore cells. The McAbs that were obtained were grouped into three classes based upon their reactivities with a variety of plant polysaccharides and membrane glycoproteins. Eleven McAbs (Class I) recognize epitope(s) that appear to be immunodominant and are found in RG-I from sycamore and maize, citrus pectin, polygalacturonic acid, and membrane glycoproteins from suspension-cultured cells of sycamore, maize, tobacco, parsley, and soybean. A second group of five McAbs (Class II) recognize epitope(s) present in sycamore RG-I, but do not bind to any of the other polysaccharides or glycoproteins recognized by Class I. Lastly, one McAb (Class III) reacts with sycamore RG-I, sycamore and tamarind xyloglucan, and sycamore and rice glucuronoarabinoxylan, but does not bind to maize RG-I, polygalacturonic acid or the plant membrane glycoproteins recognized by Class I. McAbs in Classes II and III are likely to be useful in studies of the structure, biosynthesis and localization of plant cell wall polysaccharides.

  4. A Recombinant Bispecific CD20×CD95 Antibody With Superior Activity Against Normal and Malignant B-cells

    PubMed Central

    Nalivaiko, Kristina; Hofmann, Martin; Kober, Karina; Teichweyde, Nadine; Krammer, Peter H; Rammensee, Hans-Georg; Grosse-Hovest, Ludger; Jung, Gundram

    2016-01-01

    Monoclonal antibodies directed to the B-cell-specific CD20-antigen are successfully used for the treatment of lymphomas and autoimmune diseases. Here, we compare the anti-B-cell activity of three different antibodies directed to CD20: (i) a chimeric, monospecific antibody, (ii) an Fc-optimized variant thereof, and (iii) a bispecific CD20×CD95-antibody in a newly developed recombinant format, termed Fabsc. The bispecific antibody specifically triggers the CD95 death receptor on malignant, as well as activated, normal B-cells. We found that the capability of this antibody to suppress the growth of malignant B-cells in vitro and in vivo and to specifically deplete normal, activated B-cells from peripheral blood mononuclear cell (PBMC) cultures was superior to that of the Fc-optimized monospecific antibody. This antibody in turn was more effective than its nonoptimized variant. Moreover, the bispecific antibody was the only reagent capable of significantly suppressing antibody production in vitro. Our findings imply that the bispecific CD20×CD95-antibody might become a new, prototypical reagent for the treatment of B-cell-mediated autoimmune disease. PMID:26581163

  5. Antibody-mediated p53 protein therapy prevents liver metastasis in vivo.

    PubMed

    Hansen, James E; Fischer, Laurice K; Chan, Grace; Chang, Sophia S; Baldwin, Scott W; Aragon, Robert J; Carter, Jacqueline J; Lilly, Michael; Nishimura, Robert N; Weisbart, Richard H; Reeves, Mark E

    2007-02-15

    To evaluate the clinical efficacy of monoclonal antibody (mAb) 3E10 Fv antibody-mediated p53 protein therapy, an Fv-p53 fusion protein produced in Pichia pastoris was tested on CT26.CL25 colon cancer cells in vitro and in vivo in a mouse model of colon cancer metastasis to the liver. In vitro experiments showed killing of CT26.CL25 cells by Fv-p53 but not Fv or p53 alone, and immunohistochemical staining confirmed that Fv was required for transport of p53 into cells. Prevention of liver metastasis in vivo was tested by splenic injection of 100 nmol/L Fv-p53 10 min and 1 week after injection of CT26.CL25 cancer cells into the portal vein of BALB/c mice. Mice were sacrificed 1 week after the second injection of Fv-p53 and assigned a quantitative metastasis score. Control mice had an average metastasis score of 3.3 +/- 1.3, whereas mice treated with Fv-p53 had an average metastasis score of 0.8 +/- 0.4 (P = 0.004). These results indicate that Fv-p53 treatment had a profound effect on liver metastasis and represent the first demonstration of effective full-length p53 protein therapy in vivo. mAb 3E10 Fv has significant clinical potential as a mediator of intracellular and intranuclear delivery of p53 for prevention and treatment of cancer metastasis.

  6. A new synthesis for antibody-mediated immunity

    PubMed Central

    Casadevall, Arturo; Pirofski, Liise-anne

    2013-01-01

    The view that immunoglobulins function largely by potentiating neutralization, cytotoxicity or phagocytosis is being replaced by a new synthesis whereby antibodies participate in all aspects of the immune response, from protecting the host at the earliest time of encounter with a microbe to later challenges. Perhaps the most transformative concept is that immunoglobulins manifest emergent properties, from their structure and function as individual molecules to their interactions with microbial targets and the host immune system. Given that emergent properties are neither reducible to first principles nor predictable, there is a need for new conceptual approaches for understanding antibody function and mechanisms of antibody immunity. PMID:22179281

  7. Antibody-mediated rejection across solid organ transplants: manifestations, mechanisms, and therapies.

    PubMed

    Valenzuela, Nicole M; Reed, Elaine F

    2017-06-30

    Solid organ transplantation is a curative therapy for hundreds of thousands of patients with end-stage organ failure. However, long-term outcomes have not improved, and nearly half of transplant recipients will lose their allografts by 10 years after transplant. One of the major challenges facing clinical transplantation is antibody-mediated rejection (AMR) caused by anti-donor HLA antibodies. AMR is highly associated with graft loss, but unfortunately there are few efficacious therapies to prevent and reverse AMR. This Review describes the clinical and histological manifestations of AMR, and discusses the immunopathological mechanisms contributing to antibody-mediated allograft injury as well as current and emerging therapies.

  8. Flexibility in surface-exposed loops in a virus capsid mediates escape from antibody neutralization.

    PubMed

    Kolawole, Abimbola O; Li, Ming; Xia, Chunsheng; Fischer, Audrey E; Giacobbi, Nicholas S; Rippinger, Christine M; Proescher, Jody B G; Wu, Susan K; Bessling, Seneca L; Gamez, Monica; Yu, Chenchen; Zhang, Rebecca; Mehoke, Thomas S; Pipas, James M; Wolfe, Joshua T; Lin, Jeffrey S; Feldman, Andrew B; Smith, Thomas J; Wobus, Christiane E

    2014-04-01

    New human norovirus strains emerge every 2 to 3 years, partly due to mutations in the viral capsid that allow escape from antibody neutralization and herd immunity. To understand how noroviruses evolve antibody resistance, we investigated the structural basis for the escape of murine norovirus (MNV) from antibody neutralization. To identify specific residues in the MNV-1 protruding (P) domain of the capsid that play a role in escape from the neutralizing monoclonal antibody (MAb) A6.2, 22 recombinant MNVs were generated with amino acid substitutions in the A'B' and E'F' loops. Six mutations in the E'F' loop (V378F, A382K, A382P, A382R, D385G, and L386F) mediated escape from MAb A6.2 neutralization. To elucidate underlying structural mechanisms for these results, the atomic structure of the A6.2 Fab was determined and fitted into the previously generated pseudoatomic model of the A6.2 Fab/MNV-1 virion complex. Previously, two distinct conformations, A and B, of the atomic structures of the MNV-1 P domain were identified due to flexibility in the two P domain loops. A superior stereochemical fit of the A6.2 Fab to the A conformation of the MNV P domain was observed. Structural analysis of our observed escape mutants indicates changes toward the less-preferred B conformation of the P domain. The shift in the structural equilibrium of the P domain toward the conformation with poor structural complementarity to the antibody strongly supports a unique mechanism for antibody escape that occurs via antigen flexibility instead of direct antibody-antigen binding. Human noroviruses cause the majority of all nonbacterial gastroenteritis worldwide. New epidemic strains arise in part by mutations in the viral capsid leading to escape from antibody neutralization. Herein, we identify a series of point mutations in a norovirus capsid that mediate escape from antibody neutralization and determine the structure of a neutralizing antibody. Fitting of the antibody structure into the

  9. Treatment of simultaneous acute antibody-mediated rejection and acute cellular rejection with alemtuzumab in kidney transplantation: a case report.

    PubMed

    Jirasiritham, S; Khunprakant, R; Techawathanawanna, N; Jirasiritham, Si; Mavichak, V

    2010-04-01

    This is a case report of a living related donor kidney transplantation using basiliximab induction and maintenance immunosuppression with cyclosporine, mycophenolate sodium, and steroid. On the second posttransplant day, the patient developed acute antibody-mediated rejection, which was treated with plasmapheresis and intravenous immunoglobulin (IVIG). Five days later, the graft had still not responded to the treatment. Another biopsy revealed additional acute cellular rejection (Banff IIA). As alemtuzumab can rapidly deplete T and B lymphocytes, monocytes, and natural killer cells, the patient was treated with alemtuzumab (30 mg subcutaneously) together with methylprednisolone (500 mg) and two more plasmaphereses. The kidney graft responded within 48 hours, producing more than 4 L of urine per day. The total lymphocyte decreased from 530/microL to 50/microL remaining in the 50 to 220/microL range. The patient received valgancyclovir and cotrimoxazole as infection prophylaxis. The kidney graft responded well to the rescue treatment and the patient was discharged with a serum creatinine of 1.1 mg/mL and has been uneventfully followed in the outpatient clinic for 8 months. Today, with the potent, effective, and selective immunosuppressive regimens, the rate and severity of acute cellular rejection in kidney transplantation has decreased in most centers. However, the rate of acute antibody-mediated rejection has increased to levels greater than those of acute cellular rejection in many centers. Acute antibody-mediated rejection is more difficult and expensive to treat successfully. The treatment of acute antibody-mediated rejection included plasmapheresis and IVIG. Herein we have reported a case of kidney transplantation simultaneously developing acute antibody-mediated and acute cellular rejection; the patient was successfully treated with alemtuzumab.

  10. Monitoring the Systemic Human Memory B Cell Compartment of Melanoma Patients for Anti-Tumor IgG Antibodies

    PubMed Central

    Gilbert, Amy E.; Karagiannis, Panagiotis; Dodev, Tihomir; Koers, Alexander; Lacy, Katie; Josephs, Debra H.; Takhar, Pooja; Geh, Jenny L. C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Rudman, Sarah M.; Beavil, Rebecca L.; Blower, Philip J.; Beavil, Andrew J.; Gould, Hannah J.; Spicer, James; Nestle, Frank O.; Karagiannis, Sophia N.

    2011-01-01

    Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001). Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001). Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. PMID:21559411

  11. Monitoring the systemic human memory B cell compartment of melanoma patients for anti-tumor IgG antibodies.

    PubMed

    Gilbert, Amy E; Karagiannis, Panagiotis; Dodev, Tihomir; Koers, Alexander; Lacy, Katie; Josephs, Debra H; Takhar, Pooja; Geh, Jenny L C; Healy, Ciaran; Harries, Mark; Acland, Katharine M; Rudman, Sarah M; Beavil, Rebecca L; Blower, Philip J; Beavil, Andrew J; Gould, Hannah J; Spicer, James; Nestle, Frank O; Karagiannis, Sophia N

    2011-04-29

    Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001). Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001). Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer.

  12. Antibody-mediated reduction of {alpha}-ketoamides

    DOEpatents

    Schultz, P.G.; Gallop, M.A.

    1998-06-09

    Monoclonal antibodies r