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Sample records for aggregated human igg

  1. Acid-induced aggregation of human monoclonal IgG1 and IgG2: molecular mechanism and the effect of solution composition.

    PubMed

    Hari, Sanjay B; Lau, Hollis; Razinkov, Vladimir I; Chen, Shuang; Latypov, Ramil F

    2010-11-01

    The prevention of aggregation in therapeutic antibodies is of great importance to the biopharmaceutical industry. In our investigation, acid-induced aggregation of monoclonal IgG1 and IgG2 antibodies was studied at pH 3.5 as a function of salt concentration and buffer type. The extent of aggregation was estimated using a native cation-exchange chromatography (CEX) method based on the loss of soluble monomer. This approach allowed quantitative analysis of antibody aggregation kinetics for individual and mixed protein solutions. Information regarding the aggregation mechanism was gained by assessing stabilities of intact antibodies relative to their Fc and Fab fragments. The role of protein thermodynamic stability in aggregation was deduced from differential scanning calorimetry (DSC). The rate of aggregation under conditions mimicking the viral inactivation step during monoclonal antibody (mAb) processing was found to be strongly dependent on the antibody subclass (IgG1 vs IgG2). At 25 °C, IgG1s were resistant to low pH aggregation, but IgG2s aggregated readily in the presence of salt. The observed distinction between IgG1 and IgG2 aggregation resulted from differential stability of the corresponding C(H)2 domains. This was further confirmed by experimenting with an IgG1 molecule containing an aglycosylated C(H)2 domain. Interestingly, comparative analysis of two buffer systems (based on acetic acid vs citric acid) revealed differences in mAb aggregation under identical pH conditions. Evidence is provided for the importance of the total acid concentration for antibody aggregation at low pH. The effects of C(H)2 instability and solution composition on aggregation are significant and deserve careful consideration during the development of mAb- or Fc-based therapeutics. PMID:20843079

  2. Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain.

    PubMed

    Sakurai, Kazumasa; Nakahata, Ryosuke; Lee, Young-Ho; Kardos, József; Ikegami, Takahisa; Goto, Yuji

    2015-10-01

    Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. An IgG1 molecule, which is now mainly used for antibody preparation, consists of a total of 12 immunoglobulin domains. Each domain has one disulfide bond. The CH3 domain is the C-terminal domain of the heavy chain of IgG1. The disulfide bonds of some of the CH3 domains are known to be reduced in recombinant human monoclonal antibodies. The lack of intramolecular disulfide bonds may decrease the stability and increase the aggregation propensity of an antibody molecule. To investigate the effects of a reduced disulfide bond in the CH3 domain on conformational stability and aggregation propensity, we performed several physicochemical measurements including circular dichroism, differential scanning calorimetry (DSC), and 2D NMR. DSC measurements showed that both the stability and reversibility of the reduced form were lower than those of the oxidized form. In addition, detailed analyses of the thermal denaturation data revealed that, although a dominant fraction of the reduced form retained a stable dimeric structure, some fractions assumed a less-specifically associated oligomeric state between monomers. The results of the present study revealed the characteristic aggregation properties of antibody molecules. PMID:25748879

  3. IgG Conformer's Binding to Amyloidogenic Aggregates

    PubMed Central

    Phay, Monichan; Welzel, Alfred T.; Williams, Angela D.; McWilliams-Koeppen, Helen P.; Blinder, Veronika; O'Malley, Tiernan T.; Solomon, Alan; Walsh, Dominic M.; O'Nuallain, Brian

    2015-01-01

    Amyloid-reactive IgGs isolated from pooled blood of normal individuals (pAbs) have demonstrated clinical utility for amyloid diseases by in vivo targeting and clearing amyloidogenic proteins and peptides. We now report the following three novel findings on pAb conformer's binding to amyloidogenic aggregates: 1) pAb aggregates have greater activity than monomers (HMW species > dimers > monomers), 2) pAbs interactions with amyloidogenic aggregates at least partially involves unconventional (non-CDR) interactions of F(ab) regions, and 3) pAb's activity can be easily modulated by trace aggregates generated during sample processing. Specifically, we show that HMW aggregates and dimeric pAbs present in commercial preparations of pAbs, intravenous immunoglobulin (IVIg), had up to ~200- and ~7-fold stronger binding to aggregates of Aβ and transthyretin (TTR) than the monomeric antibody. Notably, HMW aggregates were primarily responsible for the enhanced anti-amyloid activities of Aβ- and Cibacron blue-isolated IVIg IgGs. Human pAb conformer's binding to amyloidogenic aggregates was retained in normal human sera, and mimicked by murine pAbs isolated from normal pooled plasmas. An unconventional (non-CDR) component to pAb's activity was indicated from control human mAbs, generated against non-amyloid targets, binding to aggregated Aβ and TTR. Similar to pAbs, HMW and dimeric mAb conformers bound stronger than their monomeric forms to amyloidogenic aggregates. However, mAbs had lower maximum binding signals, indicating that pAbs were required to saturate a diverse collection of binding sites. Taken together, our findings strongly support further investigations on the physiological function and clinical utility of the inherent anti-amyloid activities of monomeric but not aggregated IgGs. PMID:26367058

  4. The effect of cytochalasin B and vinca alkaloids on EA- and EAC-rosette formation and on the binding of heat-aggregated human IgG by human lymphocytes

    PubMed Central

    Gutierrez, Carmen; Papamichail, M.; Faulk, W. Page

    1976-01-01

    We report the effect of cytochalasin B and vinca alkaloids on EA- and EAC-rosette formation and on the binding of heat-aggregated human IgG by human lymphocytes. Cytochalasin B inhibits EA- but not EAC-rosette formation, and neither colchicine nor vinblastine have a measurable effect on either reaction. Also, cytochalasin B inhibits the binding of heat-aggregated IgG by human lymphocytes. The inhibition caused by cytochalasin B is reversible, and the cells recover to normal values within 30 min.

  5. Phase transitions in human IgG solutions

    PubMed Central

    Wang, Ying; Lomakin, Aleksey; Latypov, Ramil F.; Laubach, Jacob P.; Hideshima, Teru; Richardson, Paul G.; Munshi, Nikhil C.; Anderson, Kenneth C.; Benedek, George B.

    2013-01-01

    Protein condensations, such as crystallization, liquid-liquid phase separation, aggregation, and gelation, have been observed in concentrated antibody solutions under various solution conditions. While most IgG antibodies are quite soluble, a few outliers can undergo condensation under physiological conditions. Condensation of IgGs can cause serious consequences in some human diseases and in biopharmaceutical formulations. The phase transitions underlying protein condensations in concentrated IgG solutions is also of fundamental interest for the understanding of the phase behavior of non-spherical protein molecules. Due to the high solubility of generic IgGs, the phase behavior of IgG solutions has not yet been well studied. In this work, we present an experimental approach to study IgG solutions in which the phase transitions are hidden below the freezing point of the solution. Using this method, we have investigated liquid-liquid phase separation of six human myeloma IgGs and two recombinant pharmaceutical human IgGs. We have also studied the relation between crystallization and liquid-liquid phase separation of two human cryoglobulin IgGs. Our experimental results reveal several important features of the generic phase behavior of IgG solutions: (1) the shape of the coexistence curve is similar for all IgGs but quite different from that of quasi-spherical proteins; (2) all IgGs have critical points located at roughly the same protein concentration at ∼100 mg/ml while their critical temperatures vary significantly; and (3) the liquid-liquid phase separation in IgG solutions is metastable with respect to crystallization. These features of phase behavior of IgG solutions reflect the fact that all IgGs have nearly identical molecular geometry but quite diverse net inter-protein interaction energies. This work provides a foundation for further experimental and theoretical studies of the phase behavior of generic IgGs as well as outliers with large propensity to

  6. Phase transitions in human IgG solutions

    NASA Astrophysics Data System (ADS)

    Wang, Ying; Lomakin, Aleksey; Latypov, Ramil F.; Laubach, Jacob P.; Hideshima, Teru; Richardson, Paul G.; Munshi, Nikhil C.; Anderson, Kenneth C.; Benedek, George B.

    2013-09-01

    Protein condensations, such as crystallization, liquid-liquid phase separation, aggregation, and gelation, have been observed in concentrated antibody solutions under various solution conditions. While most IgG antibodies are quite soluble, a few outliers can undergo condensation under physiological conditions. Condensation of IgGs can cause serious consequences in some human diseases and in biopharmaceutical formulations. The phase transitions underlying protein condensations in concentrated IgG solutions is also of fundamental interest for the understanding of the phase behavior of non-spherical protein molecules. Due to the high solubility of generic IgGs, the phase behavior of IgG solutions has not yet been well studied. In this work, we present an experimental approach to study IgG solutions in which the phase transitions are hidden below the freezing point of the solution. Using this method, we have investigated liquid-liquid phase separation of six human myeloma IgGs and two recombinant pharmaceutical human IgGs. We have also studied the relation between crystallization and liquid-liquid phase separation of two human cryoglobulin IgGs. Our experimental results reveal several important features of the generic phase behavior of IgG solutions: (1) the shape of the coexistence curve is similar for all IgGs but quite different from that of quasi-spherical proteins; (2) all IgGs have critical points located at roughly the same protein concentration at ˜100 mg/ml while their critical temperatures vary significantly; and (3) the liquid-liquid phase separation in IgG solutions is metastable with respect to crystallization. These features of phase behavior of IgG solutions reflect the fact that all IgGs have nearly identical molecular geometry but quite diverse net inter-protein interaction energies. This work provides a foundation for further experimental and theoretical studies of the phase behavior of generic IgGs as well as outliers with large propensity to

  7. Detergent induces the formation of IgG aggregates: a multi-methodological approach.

    PubMed

    Amani, Samreen; Nasim, Faisal; Khan, Taqi Ahmed; Fazili, Naveed Ahmad; Furkan, Mohammad; Bhat, Imtiyaz Ahmad; Khan, Javed Masood; Khan, Rizwan Hasan; Naeem, Aabgeena

    2014-01-01

    Role of micellar environment created by Triton X-100 (TX-100) and CHAPSO on protein conformation using IgG as a model system has been studied in this paper. A substantial amount of secondary structure with the reduction in constant tertiary contacts was obtained in both bovine and human IgG in the presence of 0.12 mM TX-100 where as 6 and 8 mM CHAPSO concentration was required for this type of secondary structure. Further addition of either of the detergents result in the induction of α-helix in both the IgGs as evident by helix specific peaks in the amide I region of FTIR and circular dichroism spectra. Tryptophan and 8-anilino-1-naphthalene-sulphonic acid (ANS) fluorescence confirmed changes in protein conformation upon addition of detergents. Maximum ANS binding at 0.12 mM TX-100 in both while 6 and 8 mM CHAPSO in bovine and human IgG respectively, indicate a compact ''molten-globule''-like conformation. An increase addition of these detergents results in the burial of hydrophobic patches of both IgG owing to aggregation. Presence of aggregates at 0.2 and 0.16 mM TX-100 and 8 and 9 mM CHAPSO, for bovine and human IgG respectively, was further confirmed by reduction in ANS fluorescence, dynamic light scattering study, thioflavin T fluorescence and congo red absorbance. PMID:24184618

  8. Subclass specificity of the Fc receptor for human IgG on K562.

    PubMed

    Chiofalo, M S; Teti, G; Goust, J M; Trifiletti, R; La Via, M F

    1988-07-01

    The erythroleukemic cell line K562 bears a 40-kDa Fc receptor (Fc gamma RII) serologically related to and with a similar molecular weight as the Fc gamma R present on a broad range of leukocytes. The human IgG subclass specificity of the Fc gamma R on K562 was investigated using IgG aggregates of defined size, obtained from purified human myeloma proteins. The monoclonal antibody IV.3, which reacts with the Fc gamma RII present on various cell types, totally prevented binding of 125I-IgG2 trimers to K562. Experiments with radiolabeled IgG2 trimers showed that K562 cells bound a mean of 156,764 +/- 9895 molecules per cell with an association constant (Ka) of 1.8 +/- 0.7 X 10(8) M-1. Similar results were obtained with IgG3 oligomers. IgG3 and IgG2 trimers were about two- to threefold more effective in inhibiting binding of 125I-IgG2 trimers to K562 than IgG1 and IgG4 trimers. These results were confirmed by inhibition experiments using IgG monomers. The subclass specificity of the Fc gamma RII on K562 (i.e., IgG2 = IgG3 greater than IgG1 = IgG4) is quite distinct from the one reported for the Fc gamma RI and III of human cells (i.e., IgG1 = IgG3 greater than IgG4 and IgG2). PMID:2968843

  9. Human IgG4: a structural perspective

    PubMed Central

    Davies, Anna M; Sutton, Brian J

    2015-01-01

    IgG4, the least represented human IgG subclass in serum, is an intriguing antibody with unique biological properties, such as the ability to undergo Fab-arm exchange and limit immune complex formation. The lack of effector functions, such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, is desirable for therapeutic purposes. IgG4 plays a protective role in allergy by acting as a blocking antibody, and inhibiting mast cell degranulation, but a deleterious role in malignant melanoma, by impeding IgG1-mediated anti-tumor immunity. These findings highlight the importance of understanding the interaction between IgG4 and Fcγ receptors. Despite a wealth of structural information for the IgG1 subclass, including complexes with Fcγ receptors, and structures for intact antibodies, high-resolution crystal structures were not reported for IgG4-Fc until recently. Here, we highlight some of the biological properties of human IgG4, and review the recent crystal structures of IgG4-Fc. We discuss the unexpected conformations adopted by functionally important Cγ2 domain loops, and speculate about potential implications for the interaction between IgG4 and FcγRs. PMID:26497518

  10. Assessment of naturally occurring covalent and total dimer levels in human IgG1 and IgG2.

    PubMed

    Yang, Jane; Goetze, Andrew M; Flynn, Gregory C

    2014-03-01

    Antibody dimers, two self-associated monomers, have been detected on both recombinantly expressed and endogenous human IgG proteins. Nearly 10 years ago, Yoo et al. (2003) described low levels of IgG2 covalent dimer, in human serum, but did not quantify the levels. Here we quantify the total and covalent dimer levels of IgG2 and IgG1 in human blood, and study the origin of covalent dimer formation. Low levels (<1%) of total IgG1 and IgG2 dimers were measured in freshly prepared human plasma. Both IgG1 and IgG2 covalent dimers were also found in plasma. Whereas IgG1 covalent dimer levels were significantly reduced by steps intended to eliminate artifacts during sample preparation, IgG2 covalent dimer levels remain stable in such conditions. About 0.4% of IgG2 in plasma was in a covalent dimer form, yet very little (<0.03%) of IgG1 covalent dimer could be considered naturally occurring. IgG2 dimer also formed in vitro under conditions designed to mimic those in blood, suggesting that formation occurs in vivo during circulation. Thus, small amounts of covalent IgG2 dimer do appear to form naturally. PMID:24321397

  11. Proteins aggregation and human diseases

    NASA Astrophysics Data System (ADS)

    Hu, Chin-Kun

    2015-04-01

    Many human diseases and the death of most supercentenarians are related to protein aggregation. Neurodegenerative diseases include Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), frontotemporallobar degeneration, etc. Such diseases are due to progressive loss of structure or function of neurons caused by protein aggregation. For example, AD is considered to be related to aggregation of Aβ40 (peptide with 40 amino acids) and Aβ42 (peptide with 42 amino acids) and HD is considered to be related to aggregation of polyQ (polyglutamine) peptides. In this paper, we briefly review our recent discovery of key factors for protein aggregation. We used a lattice model to study the aggregation rates of proteins and found that the probability for a protein sequence to appear in the conformation of the aggregated state can be used to determine the temperature at which proteins can aggregate most quickly. We used molecular dynamics and simple models of polymer chains to study relaxation and aggregation of proteins under various conditions and found that when the bending-angle dependent and torsion-angle dependent interactions are zero or very small, then protein chains tend to aggregate at lower temperatures. All atom models were used to identify a key peptide chain for the aggregation of insulin chains and to find that two polyQ chains prefer anti-parallel conformation. It is pointed out that in many cases, protein aggregation does not result from protein mis-folding. A potential drug from Chinese medicine was found for Alzheimer's disease.

  12. Human plasma contains cross-reactive Abeta conformer-specific IgG antibodies.

    PubMed

    O'Nuallain, Brian; Acero, Luis; Williams, Angela D; Koeppen, Helen P McWilliams; Weber, Alfred; Schwarz, Hans P; Wall, Jonathan S; Weiss, Deborah T; Solomon, Alan

    2008-11-25

    Two conformers of aggregated Abeta, i.e., fibrils and oligomers, have been deemed important in the pathogenesis of Alzheimer's disease. We now report that intravenous immune globulin (IVIG) derived from pools of human plasma contains IgGs that recognize conformational epitopes present on fibrils and oligomers, but not their soluble monomeric precursor. We have used affinity chromatography to isolate these antibodies and have shown that they cross-reacted with comparable nanomolar avidity with both types of Abeta aggregates; notably, binding was not inhibited by soluble Abeta monomers. Our studies provide further support for investigating the therapeutic use of IVIG in Alzheimer's disease. PMID:18956886

  13. Inhibition by synthetic peptides from human IgG Fc of OKT4 binding and Fc receptor binding

    SciTech Connect

    Gaarde, W.A.; McClurg, M.R.; Hahn, G.S.; Plummer, J.M.

    1986-03-05

    Synthetic peptides from the Fc region of human IgG were tested for the ability to inhibit IgG rosette formation, /sup 125/I-IgG binding to Fc receptors on lymphocytes, and expression of the T4 cell determinant. The Fc/sub ..gamma../ peptides inhibited IgG rosette formation and competitively inhibited both /sup 125/I-IgG binding to Fc receptors and OKT4 binding to the T4 cell determinant. Peptides containing D-amino acids did not inhibit IgG rosettes, OKT4 binding or /sup 125/I-IgG binding. OKT4 binding to T4 on MNC was inhibited by heat aggregated IgG but not by monomeric IgG. OKT3, OKT8 and OKT11 binding to their determinants was not altered by Fc/sub ..gamma../ peptides, aggregated IgG or monomeric IgG. These results suggest that T4 antigen, Fc receptor for IgG and the Fc/sub ..gamma../ peptide binding site are in close proximity on the cell surface and when occupied by their respective ligands may sterically hinder binding to other sites. Alternatively, Fc/sub ..gamma../ peptides may indirectly regulate cell surface expression of T4 and/or Fc receptors for IgG. These Fc/sub ..gamma../ peptides inhibit the MLR, inhibit antigen induced T cell proliferation and reverse animal models of autoimmune disease. The immunoregulatory activities of these peptides may be related to their selective action on T4 helper/inducer lymphocytes expressing Fc receptors.

  14. Prevention of thermally induced aggregation of IgG antibodies by noncovalent interaction with poly(acrylate) derivatives.

    PubMed

    Martin, Nicolas; Ma, Dewang; Herbet, Amaury; Boquet, Didier; Winnik, Françoise M; Tribet, Christophe

    2014-08-11

    Prevention of thermal aggregation of antibodies in aqueous solutions was achieved by noncovalent association with hydrophobically modified poly(acrylate) copolymers. Using a polyclonal immunoglobin G (IgG) as a model system for antibodies, we have studied the mechanisms by which this multidomain protein interacts with polyanions when incubated at physiological pH and at temperatures below and above the protein unfolding/denaturation temperature, in salt-free solutions and in 0.1 M NaCl solutions. The polyanions selected were sodium poly(acrylates), random copolymers of sodium acrylate and N-n-octadecylacrylamide (3 mol %), and a random copolymer of sodium acrylate, N-n-octylacrylamide (25 mol %), and N-isopropylacrylamide (40 mol %). They were derived from two poly(acrylic acid) parent chains of Mw 5000 and 150000 g·mol(-1). The IgG/polyanion interactions were monitored by static and dynamic light scattering, fluorescence correlation spectroscopy, capillary zone electrophoresis, and high sensitivity differential scanning calorimetry. In salt-free solutions, the hydrophilic PAA chains form complexes with IgG upon thermal unfolding of the protein (1:1 w/w IgG/PAA), but they do not interact with native IgG. The complexes exhibit a remarkable protective effect against IgG aggregation and maintain low aggregation numbers (average degree of oligomerization <12 at a temperature up to 85 °C). These interactions are screened in 0.1 M NaCl and, consequently, PAAs lose their protective effect. Amphiphilic PAA derivatives (1:1 w/w IgG/polymer) are able to prevent thermal aggregation (preserving IgG monomers) or retard aggregation of IgG (formation of oligomers and slow growth), revealing the importance of both hydrophobic interactions and modulation of the Coulomb interactions with or without NaCl present. This study leads the way toward the design of new formulations of therapeutic proteins using noncovalent 1:1 polymer/protein association that are transient and require a

  15. Elimination of soluble sup 123 I-labeled aggregates of IgG in patients with systemic lupus erythematosus. Effect of serum IgG and numbers of erythrocyte complement receptor type 1

    SciTech Connect

    Halma, C.; Breedveld, F.C.; Daha, M.R.; Blok, D.; Evers-Schouten, J.H.; Hermans, J.; Pauwels, E.K.; van Es, L.A. )

    1991-04-01

    Using soluble {sup 123}I-labeled aggregates of human IgG ({sup 123}I-AHIgG) as a probe, we examined the function of the mononuclear phagocyte system in 22 patients with systemic lupus erythematosus (SLE) and 12 healthy controls. In SLE patients, a decreased number of erythrocyte complement receptor type 1 was associated with less binding of {sup 123}I-AHIgG to erythrocytes and a faster initial rate of elimination of {sup 123}I-AHIgG (mean +/- SEM half-maximal clearance time 5.23 +/- 0.2 minutes, versus 6.58 +/- 0.2 minutes in the controls), with possible spillover of the material outside the mononuclear phagocyte system of the liver and spleen. However, multiple regression analysis showed that serum concentrations of IgG were the most important factor predicting the rate of {sup 123}I-AHIgG elimination. IgG concentration may thus reflect immune complex clearance, which in turn, would influence the inflammatory reaction, in SLE.

  16. Quantitative characterization by asymmetrical flow field-flow fractionation of IgG thermal aggregation with and without polymer protective agents.

    PubMed

    Ma, Dewang; Martin, Nicolas; Tribet, Christophe; Winnik, Françoise M

    2014-11-01

    Complexes formed between poly(acrylates) and polyclonal immunoglobulin G (IgG) in its native conformation and after heat stress were characterized using asymmetric flow field-flow fractionation (AF4) coupled with on-line UV-Vis spectroscopy and multi-angle light-scattering detection (MALS). Mixtures of IgG and poly(acrylates) of increasing structural complexity, sodium poly(acrylate) (PAA), a sodium poly(acrylate) bearing at random 3 mol % n-octadecyl groups, and a random copolymer of sodium acrylate (35 mol%), N-n-octylacrylamide (25 mol%) and N-isopropylacrylamide (40 mol%), were fractionated in a sodium phosphate buffer (0.02 M, pH 6.8) in the presence, or not, of 0.1 M NaCl. The AF4 protocol developed allowed the fractionation of solutions containing free poly(acrylates), native IgG monomer and dimer, poly(acrylates)/IgG complexes made up of one IgG molecule and a few polymer chains, and/or larger poly(acrylates)/IgG aggregates. The molar mass and recovery of the soluble analytes were obtained for mixed solutions of poly(acrylates) and native IgG and for the same solutions incubated at 65 °C for 10 min. From the combined AF4 results, we concluded that in solutions of low ionic strength, the presence of PAA increased the recovery ratio of IgG after thermal stress because of the formation of electrostatically-driven PAA/IgG complexes, but PAA had no protective effect in the presence of 0.1 M NaCl. Poly(acrylates) bearing hydrophobic groups significantly increased IgG recovery after stress, independently of NaCl concentration, because of the synergistic effect of hydrophobic and electrostatic interactions. The AF4 results corroborate conclusions drawn from a previous study combining four analytical techniques. This study demonstrates that AF4 is an efficient tool for the analysis of protein formulations subjected to stress, an important achievement given the anticipated important role of proteins in near-future human therapies. PMID:25323742

  17. IgG subclass responses to proinflammatory fraction of Brugia malayi in human filariasis

    PubMed Central

    Joseph, S.K.; Verma, S.K.; Sahoo, M.K.; Sharma, A.; Srivastava, M.; Reddy, M.V.R.; Murthy, P.K.

    2012-01-01

    Background & objectives: Earlier we demonstrated that immunization with F6, a proinflammatory molecular fraction isolated from the human filarial parasite Brugia malayi, protected the host and eliminated the infection in Mastomys coucha by a Th1/Th2 response including IgG2a antibody response. Whether F6 molecules become accessible to human host during natural course of infection and elicit similar response is not known. The present study was undertaken to determine the profile of IgG subclasses specifically reactive to F6 in different categories of bancroftian filariasis cases to infer any relationship between the levels of a particular F6-specific IgG subclass and the infection or disease status. Methods: Serum samples of normal individuals from filariasis non-endemic regions of India like Jammu & Kashmir, Uttarakhand, and Chandigarh [(NEN-W; n=10), healthy subjects from USA (NEN-U; n=10) and three categories of bancroftian filariasis cases from endemic areas: endemic normals (EN; n=10) with no symptoms and no microfilariae, asymptomatic microfilaremics (ASM; n=10) and chronic symptomatic amicrofilaremics (CL; n=10) were assayed for F6-specific IgG1, IgG2, IgG3 and IgG4 by ELISA using SDS-PAGE-isolated F6 fraction of B. malayi adult worms. Results: Significantly high levels of F6-specific IgG1, IgG2 and IgG3 were found in CL (P<0.001) and EN (P<0.01-0.001) bancroftian filariasis cases compared to NEN-U. Significant levels of F6-specific IgG1 (P<0.01) and IgG2 (P<0.01) but not IgG3 were found in ASM cases compared to NEN-U. The most abundant was IgG2 which when compared to NEN-U, was significantly high in CL (P<0.001) and EN cases (P<0.001), followed by ASM (P<0.01). F6-specific IgG4 response in EN, ASM and CL subjects was not significantly different from the levels of NEN-U. Among the non-endemic normals, the NEN-W subjects showed significant reactivity with IgG2 (P<0.001) but not with IgG1, IgG3 and IgG4 as compared to NEN-U subjects. IgG subclass levels were

  18. Uptake and degradation of soluble aggregates of IgG by monocytes of patients with rheumatoid arthritis: relation to disease activity.

    PubMed Central

    Heurkens, A H; Westedt, M L; Breedveld, F C; Jonges, E; Cats, A; Stijnen, T; Daha, M R

    1991-01-01

    Monocytes from patients with rheumatoid arthritis (RA) and rheumatoid vasculitis have a diminished ability to degrade soluble complexes of aggregated IgG in the absence (mediated by Fc receptors) as well as in the presence of complement (C) (mediated by (Fc + C) receptors). To investigate whether a relation exists between the receptor mediated degradation of aggregated IgG by adherent monocytes and disease activity a longitudinal study was performed in 79 patients with RA and rheumatoid vasculitis over a period of 16 months. Adherent monocytes were incubated in vitro with 125I labelled IgG aggregates of restricted size in the absence or presence of fresh serum and the percentage of catabolised IgG aggregates was measured. Cross sectionally the degradation of aggregated IgG by monocytes, mediated by Fc and (Fc + C) receptors, correlated significantly with disease activity as scored by the Ritchie articular index, the presence of extra-articular features, and circulating immune complexes. A high number of Fc receptors on monocytes correlated with diminished degradation, whereas high numbers of complement receptors 1 and 3 correlated with enhanced degradation of aggregated IgG mediated by both Fc and (Fc + C) receptors. The degradation of aggregated IgG by monocytes did not correlate with disease activity in individual patients followed up longitudinally. When patient groups were formed according to the results of longitudinal studies, however, degradation of aggregated IgG mediated by Fc and (Fc + C) receptors was significantly decreased in patients with rheumatoid vasculitis and in patients with active RA in comparison with patients with inactive RA and healthy controls. Patients with active RA and rheumatoid vasculitis also expressed significantly more Fc receptors and less complement receptors on the monocytes than patients with inactive RA. Drug treatment did not correlate with receptor expression or the degradation of aggregated IgG by monocytes either in cross

  19. Structural Characterization of IgG1 mAb Aggregates and Particles Generated under Various Stress Conditions

    PubMed Central

    Telikepalli, Srivalli N.; Kumru, Ozan S.; Kalonia, Cavan; Esfandiary, Reza; Joshi, Sangeeta B.; Middaugh, C. Russell; Volkin, David B.

    2014-01-01

    IgG1 mAb solutions were prepared with and without sodium chloride and subjected to different environmental stresses. Formation of aggregates and particles of varying size was monitored by a combination of size exclusion chromatography (SEC), Nanosight Tracking Analysis (NTA), Micro-flow Imaging (MFI), turbidity, and visual assessments. Stirring and heating induced the highest concentration of particles. In general, the presence of NaCl enhanced this effect. The morphology of the particles formed from mAb samples exposed to different stresses was analyzed from TEM and MFI images. Shaking samples without NaCl generated the most fibrillar particles, while stirring created largely spherical particles. The composition of the particles was evaluated for covalent cross-linking by SDS-PAGE, overall secondary structure by FTIR microscopy, and surface apolarity by extrinsic fluorescence spectroscopy. Freeze-thaw and shaking led to particles containing protein with native-like secondary structure. Heating and stirring produced IgG1 containing aggregates and particles with some non-native disulfide crosslinks, varying levels of intermolecular beta sheet content, and increased surface hydrophobicity. These results highlight the importance of evaluating protein particle morphology and composition, in addition to particle number and size distributions, to better understand the effect of solution conditions and environmental stresses on the formation of protein particles in mAb solutions. PMID:24452866

  20. [Lysophosphatidic acid and human erythrocyte aggregation].

    PubMed

    Sheremet'ev, Iu A; Popovicheva, A N; Levin, G Ia

    2014-01-01

    The effects of lysophosphatidic acid on the morphology and aggregation of human erythrocytes has been studied. Morphology of erythrocytes and their aggregates were studied by light microscopy. It has been shown that lysophosphatidic acid changes the shape of red blood cells: diskocyte become echinocytes. Aggregation of red blood cells (rouleaux) was significantly reduced in autoplasma. At the same time there is a strong aggregation of echinocytes. This was accompanied by the formation of microvesicles. Adding normal plasma to echinocytes restores shape and aggregation of red blood cells consisting of "rouleaux". A possible mechanism of action of lysophosphatidic acid on erythrocytes is discussed. PMID:25509147

  1. Effects of Protein Conformation, Apparent Solubility, and Protein-Protein Interactions on the Rates and Mechanisms of Aggregation for an IgG1Monoclonal Antibody.

    PubMed

    Kalonia, Cavan; Toprani, Vishal; Toth, Ronald; Wahome, Newton; Gabel, Ian; Middaugh, C Russell; Volkin, David B

    2016-07-28

    Non-native protein aggregation is a key degradation pathway of immunoglobulins. In this work, the aggregation kinetics of an immunoglobulin gamma-1 monoclonal antibody (IgG1 mAb) in different solution environments was monitored over a range of incubation temperatures for up to seven months using size exclusion chromatography. Histidine and citrate buffers with/without sodium chloride were employed to modulate the mAb's conformational stability, solubility (in the presence of polyethylene glycol, PEG), and protein-protein interactions as measured by differential scanning calorimetry, PEG precipitation, and static light scattering, respectively. The effect of these parameters on the mechanism(s) of mAb aggregation during storage at different temperatures was determined using kinetic models, which were used to fit aggregation data to determine rate constants for aggregate nucleation and growth processes. This approach was used to investigate the effects of colloidal protein-protein interactions and solubility values (in PEG solutions) on the mechanisms and rates of IgG1 mAb aggregation as a function of temperature-induced structural perturbations. Aggregate nucleation and growth pathways for this IgG1 mAb were sensitive to temperature and overall conformational stability. Aggregate growth, on the other hand, was also sensitive to conditions affecting the solubility of the mAb, particularly at elevated temperatures. PMID:27380437

  2. Human immunoglobulin class and IgG subclass regulation: dual action of interleukin-4.

    PubMed

    Kotowicz, K; Callard, R E

    1993-09-01

    Epstein-Barr virus (EBV) was used as a polyclonal human B cell mitogen to investigate the regulation of immunoglobulin class and IgG subclass responses by interleukin-4 (IL-4). Activation of tonsillar B cells with EBV resulted in an early peak of polyclonal immunoglobulin secretion between days 13 and 14 consisting of IgM, IgA, and IgG1, IgG2, IgG3 and IgG4, but not IgE. Addition of IL-4 to EBV-activated B cells at concentrations of 100 U/ml or greater induced the production of IgE and enhanced IgG4 secretion, but had no effect, or more often inhibited the other isotypes. In contrast, low concentrations of IL-4 (1-5 U/ml) significantly increased the production of IgM, IgA, IgG1, IgG2 and IgG3, but had no effect on IgG4 or IgE. The increase in immunoglobulin secretion obtained with low concentrations of IL-4 was found to occur only with high-density (resting) B cells, suggesting that IL-4 was not functioning simply as a late-acting differentiation factor. Low concentrations of IL-4 significantly increased IgG1, IgG2, IgG3, and IgA production by surface (s) IgM+ (sIgG-/sIgA-) B cells which is consistent with heavy chain switching. In some experiments, however, IL-4 enhanced IgM secretion by sIgM+ B cells, and IgA, IgG1, IgG2, IgG3 by sIgM- B cells, suggesting that it may have an additional B cell differentiation factor activity which was not isotype specific. The different effect of IL-4 at high and low concentrations were similar to those observed in B cell activation experiments, and may be due to the existence of high- and low-affinity IL-4 receptors. PMID:8396532

  3. Aggregation Kinetics for IgG1-Based Monoclonal Antibody Therapeutics.

    PubMed

    Singla, A; Bansal, R; Joshi, Varsha; Rathore, Anurag S

    2016-05-01

    Monoclonal antibodies (mAbs) as a class of therapeutic molecules are finding an increasing demand in the biotechnology industry for the treatment of diseases like cancer and multiple sclerosis. A key challenge associated to successful commercialization of mAbs is that from the various physical and chemical instabilities that are inherent to these molecules. Out of all probable instabilities, aggregation of mAbs has been a major problem that has been associated with a change in the protein structure and is a hurdle in various upstream and downstream processes. It can stimulate immune response causing protein misfolding having deleterious and harmful effects inside a cell. Also, the extra cost incurred to remove aggregated mAbs from the rest of the batch is huge. Size exclusion chromatography (SEC) is a major technique for characterizing aggregation in mAbs where change in the aggregates' size over time is estimated. The current project is an attempt to understand the rate and mechanism of formation of higher order oligomers when subjected to different environmental conditions such as buffer type, temperature, pH, and salt concentration. The results will be useful in avoiding the product exposure to conditions that can induce aggregation during upstream, downstream, and storage process. Extended Lumry-Eyring model (ELE), Lumry-Eyring Native Polymerization model (LENP), and Finke-Watzky model (F-W) have been employed in this work to fit the aggregation experimental data and results are compared to find the best fit model for mAb aggregation to connect the theoretical dots with the reality. PMID:26902302

  4. Changes in Healthy Human IgG Fc-Glycosylation after Birth and during Early Childhood.

    PubMed

    de Haan, Noortje; Reiding, Karli R; Driessen, Gertjan; van der Burg, Mirjam; Wuhrer, Manfred

    2016-06-01

    Glycosylation on the fragment crystallizable (Fc) region of immunoglobulin G (IgG) has a large influence on the interaction of the antibody with Fc gamma receptors (FcγRs). IgG consists of four subclasses that all have distinct affinities for the different FcγRs. Knowledge about the Fc-glycosylation in healthy human is valuable as reference for new biomarkers and in the design of biopharmaceuticals that rely on IgG Fc-glycosylation. Previously, subclass-specific characterization of IgG Fc-glycosylation was performed for healthy adults, pregnant women, and newborns. For young healthy children, however, the subclass-specific description of IgG Fc-glycosylation is still lacking. Therefore, we performed the IgG subclass-specific analysis of the Fc-glycosylation of 130 healthy humans between birth and 40 years of age, including 22 samples derived from the umbilical cords of newborns. The analysis was performed by a previously published matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) workflow, including a derivatization step for the linkage-specific stabilization of sialic acids. The characterization revealed that when children start to produce their own IgG they have a decreased galactosylation, sialylation, and bisection and an increased fucosylation compared with newborns. During childhood, the fucosylation and sialylation decrease, whereas bisection increases and galactosylation stays constant. PMID:27161864

  5. Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation.

    PubMed

    Gong, Qian; Hazen, Meredith; Marshall, Brett; Crowell, Susan R; Ou, Qinglin; Wong, Athena W; Phung, Wilson; Vernes, Jean-Michel; Meng, Y Gloria; Tejada, Max; Andersen, Dana; Kelley, Robert F

    2016-01-01

    For some antibodies intended for use as human therapeutics, reduced effector function is desired to avoid toxicities that might be associated with depletion of target cells. Since effector function(s), including antibody-dependent cell-mediated cytotoxicity (ADCC), require the Fc portion to be glycosylated, reduced ADCC activity antibodies can be obtained through aglycosylation of the human IgG1 isotype. An alternative is to switch to an IgG4 isotype in which the glycosylated antibody is known to have reduced effector function relative to glycosylated IgG1 antibody. ADCC activity of glycosylated IgG1 antibodies is sensitive to the fucosylation status of the Fc glycan, with both in vitro and in vivo ADCC activity increased upon fucose removal ("afucosylation"). The effect of afucosylation on activity of IgG4 antibodies is less well characterized, but it has been shown to increase the in vitro ADCC activity of an anti-CD20 antibody. Here, we show that both in vitro and in vivo activity of anti-CD20 IgG4 isotype antibodies is increased via afucosylation. Using blends of material made in Chinese hamster ovary (CHO) and Fut8KO-CHO cells, we show that ADCC activity of an IgG4 version of an anti-human CD20 antibody is directly proportional to the fucose content. In mice transgenic for human FcγRIIIa, afucosylation of an IgG4 anti-mouse CD20 antibody increases the B cell depletion activity to a level approaching that of the mIgG2a antibody. PMID:27216702

  6. Human IgG1 antibodies suppress angiogenesis in a target-independent manner

    PubMed Central

    Bogdanovich, Sasha; Kim, Younghee; Mizutani, Takeshi; Yasuma, Reo; Tudisco, Laura; Cicatiello, Valeria; Bastos-Carvalho, Ana; Kerur, Nagaraj; Hirano, Yoshio; Baffi, Judit Z; Tarallo, Valeria; Li, Shengjian; Yasuma, Tetsuhiro; Arpitha, Parthasarathy; Fowler, Benjamin J; Wright, Charles B; Apicella, Ivana; Greco, Adelaide; Brunetti, Arturo; Ruvo, Menotti; Sandomenico, Annamaria; Nozaki, Miho; Ijima, Ryo; Kaneko, Hiroki; Ogura, Yuichiro; Terasaki, Hiroko; Ambati, Balamurali K; Leusen, Jeanette HW; Langdon, Wallace Y; Clark, Michael R; Armour, Kathryn L; Bruhns, Pierre; Verbeek, J Sjef; Gelfand, Bradley D; De Falco, Sandro; Ambati, Jayakrishna

    2016-01-01

    Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world’s population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in mice. Here we show bevacizumab suppressed angiogenesis in three mouse models not via Vegfa blockade but rather Fc-mediated signaling through FcγRI (CD64) and c-Cbl, impairing macrophage migration. Other approved humanized or human IgG1 antibodies without mouse targets (adalimumab, alemtuzumab, ofatumumab, omalizumab, palivizumab and tocilizumab), mouse IgG2a, and overexpression of human IgG1-Fc or mouse IgG2a-Fc, also inhibited angiogenesis in wild-type and FcγR humanized mice. This anti-angiogenic effect was abolished by Fcgr1 ablation or knockdown, Fc cleavage, IgG-Fc inhibition, disruption of Fc-FcγR interaction, or elimination of FcRγ-initated signaling. Furthermore, bevacizumab’s Fc region potentiated its anti-angiogenic activity in humanized VEGFA mice. Finally, mice deficient in FcγRI exhibited increased developmental and pathological angiogenesis. These findings reveal an unexpected anti-angiogenic function for FcγRI and a potentially concerning off-target effect of hIgG1 therapies. PMID:26918197

  7. In men at risk of HIV infection, IgM, IgG1, IgG3, and IgA reach the human foreskin epidermis.

    PubMed

    Lemos, M P; Karuna, S T; Mize, G J; Fong, Y; Montano, S M; Ganoza, C; Lama, J R; Sanchez, J; McElrath, M J

    2016-05-01

    We profiled the humoral response in the penis, an area that has been minimally explored but may be relevant for protecting insertive men against HIV and other sexually acquired infections. Comparing paired tissue samples from 20 men at risk of HIV infection, foreskin contains less immunoglobulin A (IgA) and more IgG2 than colon. Using foreskin dermal and epidermal explants and paired plasma from 17 men, we examined Ig accumulation by normalizing Ig to human serum albumin (HSA) transudation. Dermal IgM, IgG2, IgA, and IgE ratios were greater than that in plasma, suggesting there is local antibody secretion at the dermis. Local Ig transcription was concentrated at the inner rather than the outer foreskin, and inner foreskin Ig ratios did not correlate with blood, indicating that localized production can contribute to the foreskin response. IgM, IgG1, IgG3, and IgA have preferential access to the foreskin epidermis, whereas IgG2, IgG4, and IgE are restricted to the dermis. Lastly, Ad5-specific IgA was selectively present in the colon, whereas foreskin Ad5 IgG was mainly derived from blood, and reached the inner epidermis at higher ratios than the outer (P<0.002). In summary, the foreskin antibody response combines local and systemic sources, and there is selective isotype accumulation in the epidermis. PMID:26509877

  8. In Men at Risk of HIV Infection, IgM, IgG1, IgG3 and IgA Reach the Human Foreskin Epidermis

    PubMed Central

    Lemos, Maria P.; Karuna, Shelly T.; Mize, Gregory J.; Fong, Youyi; Montano, Silvia M.; Ganoza, Carmela; Lama, Javier R.; Sanchez, Jorge; McElrath, M. Juliana

    2015-01-01

    We profiled the humoral response in the penis, an area that has been minimally explored but may be relevant for protecting insertive men against HIV and other sexually-acquired infections. Comparing paired tissue samples from 20 men at risk of HIV infection, foreskin contains less IgA and more IgG2 than colon. Using foreskin dermal and epidermal explants and paired plasma from 17 men, we examined Ig accumulation by normalizing Ig to human serum albumin (HSA) transudation. Dermal IgM, IgG2, IgA, and IgE ratios were greater than in plasma, suggesting there is local antibody secretion at the dermis. Local Ig transcription was concentrated at the inner rather than the outer foreskin, and inner foreskin Ig ratios did not correlate with blood, indicating that localized production can contribute to the foreskin response. IgM, IgG1, IgG3, and IgA have preferential access to the foreskin epidermis, whereas IgG2, IgG4, and IgE are restricted to the dermis. Lastly, Ad5-specific IgA was selectively in the colon; whereas foreskin Ad5 IgG was mainly derived from blood, and reached the inner epidermis at higher ratios than the outer (p<0.002). In summary, the foreskin antibody response combines local and systemic sources and there is selective isotype accumulation in the epidermis. PMID:26509877

  9. In vitro and in vivo modifications of recombinant and human IgG antibodies

    PubMed Central

    Liu, Hongcheng; Ponniah, Gomathinayagam; Zhang, Hui-Min; Nowak, Christine; Neill, Alyssa; Gonzalez-Lopez, Nidia; Patel, Rekha; Cheng, Guilong; Kita, Adriana Z; Andrien, Bruce

    2014-01-01

    Tremendous knowledge has been gained in the understanding of various modifications of IgG antibodies, driven mainly by the fact that antibodies are one of the most important groups of therapeutic molecules and because of the development of advanced analytical techniques. Recombinant monoclonal antibody (mAb) therapeutics expressed in mammalian cell lines and endogenous IgG molecules secreted by B cells in the human body share some modifications, but each have some unique modifications. Modifications that are common to recombinant mAb and endogenous IgG molecules are considered to pose a lower risk of immunogenicity. On the other hand, modifications that are unique to recombinant mAbs could potentially pose higher risk. The focus of this review is the comparison of frequently observed modifications of recombinant monoclonal antibodies to those of endogenous IgG molecules. PMID:25517300

  10. Enhanced sialylation of a human chimeric IgG1 variant produced in human and rodent cell lines.

    PubMed

    Mimura, Yusuke; Kelly, Ronan M; Unwin, Louise; Albrecht, Simone; Jefferis, Roy; Goodall, Margaret; Mizukami, Yoichi; Mimura-Kimura, Yuka; Matsumoto, Tsuneo; Ueoka, Hiroshi; Rudd, Pauline M

    2016-01-01

    Glycosylation of the IgG-Fc is essential for optimal binding and activation of Fcγ receptors and the C1q component of complement. However, it has been reported that the effector functions are down-regulated when the Fc glycans terminate in sialic acid residues and that sialylated IgG mediates anti-inflammatory effects of intravenous immunoglobulin (IVIG). Although recombinant IgG is hypo-sialylated, Fc sialylation is shown to be markedly increased when a mouse/human chimeric IgG3 Phe243Ala (F243A) variant is expressed in Chinese hamster ovary (CHO)-K1 cells. Here we investigate whether sialylation is increased in IgG1 F243A when expressed in CHO-K1, mouse myeloma J558L and human embryonic kidney (HEK) 293. Although the sialylation level was 2-5% for IgG1 wild type (WT), it was increased to 31%, 10% and 33% for the variant from CHO-K1, J558L and HEK293 cells, respectively. Interestingly, an increased addition of bisecting GlcNAc and α(1-3)-galactose residues to the Fc glycan was observed for HEK293-derived and J558L-derived IgG1 F243A, respectively. Fucosylation of HEK293-derived IgG1 F243A was maintained despite increased bisecting GlcNAc content. Although sialic acid and bisecting GlcNAc residues are reported to have an opposing effect on antibody-dependent cellular cytotoxicity (ADCC), IgG1 F243A showed 7 times lower ADCC activities than IgG1 WT, irrespective of bisecting GlcNAc residue. Thus, highly sialylated, human cell-derived IgG1 F243A with lowered ADCC activity may be of interest for the development of therapeutic antibodies with anti-inflammatory properties as an alternative to IVIG. PMID:26627984

  11. Human IgG detection in serum on polymer based Mach-Zehnder interferometric biosensors.

    PubMed

    Melnik, Eva; Bruck, Roman; Müellner, Paul; Schlederer, Thomas; Hainberger, Rainer; Lämmerhofer, Michael

    2016-03-01

    We report a new method for detecting human IgG (hIgG) in serum on integrated-optical Mach-Zehnder interferometer biosensors realized in a high index contrast polymer material system. In the linear range of the sensor (5-200 nM) we observed excellent signal recoveries (95-110%) in buffer and serum samples, which indicate the absence of matrix effects. Signal enhancement was reached by using secondary anti-human IgG antibodies, which bind to immobilized target IgGs and allow detecting concentrations down to 100 pM. This polymer based optical sensor is fully compatible with cost-efficient mass production technologies, which makes it an attractive alternative to inorganic optical sensors. Graphical abstract of the hIgG measured on polymer based photonic sensors using a direct binding assay and a signal enhancement strategy with secondary antibodies. PMID:26663736

  12. Fully human monoclonal antibody inhibitors of the neonatal fc receptor reduce circulating IgG in non-human primates.

    PubMed

    Nixon, Andrew E; Chen, Jie; Sexton, Daniel J; Muruganandam, Arumugam; Bitonti, Alan J; Dumont, Jennifer; Viswanathan, Malini; Martik, Diana; Wassaf, Dina; Mezo, Adam; Wood, Clive R; Biedenkapp, Joseph C; TenHoor, Chris

    2015-01-01

    The therapeutic management of antibody-mediated autoimmune disease typically involves immunosuppressant and immunomodulatory strategies. However, perturbing the fundamental role of the neonatal Fc receptor (FcRn) in salvaging IgG from lysosomal degradation provides a novel approach - depleting the body of pathogenic immunoglobulin by preventing IgG binding to FcRn and thereby increasing the rate of IgG catabolism. Herein, we describe the discovery and preclinical evaluation of fully human monoclonal IgG antibody inhibitors of FcRn. Using phage display, we identified several potent inhibitors of human-FcRn in which binding to FcRn is pH-independent, with over 1000-fold higher affinity for human-FcRn than human IgG-Fc at pH 7.4. FcRn antagonism in vivo using a human-FcRn knock-in transgenic mouse model caused enhanced catabolism of exogenously administered human IgG. In non-human primates, we observed reductions in endogenous circulating IgG of >60% with no changes in albumin, IgM, or IgA. FcRn antagonism did not disrupt the ability of non-human primates to mount IgM/IgG primary and secondary immune responses. Interestingly, the therapeutic anti-FcRn antibodies had a short serum half-life but caused a prolonged reduction in IgG levels. This may be explained by the high affinity of the antibodies to FcRn at both acidic and neutral pH. These results provide important preclinical proof of concept data in support of FcRn antagonism as a novel approach to the treatment of antibody-mediated autoimmune diseases. PMID:25954273

  13. Crossreacting IgG antibodies against fox mite antigens in human scabies.

    PubMed

    Haas, N; Wagemann, B; Hermes, B; Henz, B M; Heile, C; Schein, E

    2005-01-01

    Scabies continues to be an important parasitic disease of mammals. There remain, however, major gaps in the understanding of the human host immune response, and a simple diagnostic test is lacking. In contrast to human mites, red fox mites (Sarcoptes scabiei var. vulpis) can be collected easily and have been used, due to crossreactivity, for enzyme-linked immunosorbent assay (ELISA) studies in dogs and pigs. We wanted to investigate the possibility that crossreactivity might also exist for the human mite, and determined titers against fox mite antigens by ELISA in 41 patients with scabies. Specific IgG was significantly higher in patients with scabies than in healthy controls (P=0.01). The sensitivity was, however, only 48%, although it increased slightly during treatment (P=0.86). A positive correlation was also noted between disease duration and severity of infestation (r=0.5), with specific IgG titers increasing in parallel with severity of symptoms (P=0.01). Patients with symptomatic scabies for more than 4 weeks had furthermore significantly higher IgG titers than patients with a shorter duration of disease (P=0.007). In conclusion, these findings demonstrate IgG antibodies in human scabies that crossreact with fox mite antigens, thus encouraging the search for improved ELISAs with more specific mite antigens to produce a more sensitive detection system for scabies in humans. PMID:15650895

  14. [IgG antibodies against toxic shock syndrome toxin 1 in human immunoglobulins].

    PubMed

    Dickgiesser, N; Kustermann, B

    1986-07-15

    IgG antibodies against toxic shock syndrome toxin-1 in human immunoglobulins were determined using the ELISA technique. Of the drugs for intramuscular application, hemogamma and beriglobin contained the highest amount of antibodies. The highest concentration of antibodies in drugs for intravenous application was found in Pseudomonas polyglobin and in Venimmun. PMID:3762013

  15. Regulation of in vitro PWM-induced IgG secretion in humans

    SciTech Connect

    O'Gorman, M.R.; Oger, J.J.

    1989-02-01

    PWM-induced differentiation of human PBMC into immunoglobulin (Ig) secreting cells is a widely used model of in vivo IgG secretion. We examined the relationship between the amount of IgG secreted in PBMC cultures obtained from individuals who consistently secrete either very high (HR) or very low amounts (LR) of IgG and various in vitro immune function assays. The PBMC obtained from LR contained a higher ratio of cells expressing the T-suppressor/inducer to T-helper/inducer phenotype. The autologous mixed lymphocyte response was lower and the amount of in vivo radiation sensitive suppression was higher in the LR than in the HR. LR E- cells secreted less IgG than the HR E- cells when both were mixed with heterologous HR E+ cells. Monocyte depletion reduced IgG secretion in both LR and HR. These results suggest that each of the subsets Ts, Th (Thi, Tsi), and B lymphocytes are involved in the regulation of PWM-induced Ig secretion and that the functions of each of these subsets differ in HR and LR individuals.

  16. Transport of the subclasses of human IgG across the yolk-sac of the foetal rabbit

    PubMed Central

    Hemmings, W. A.

    1974-01-01

    The four subclasses of human IgG were labelled either with 125I or 131I and injected into rabbit uteri (24 days pregnant) in pairs. Transmission to the foetal circulation was measured 1 day later. It was found that all four sub-classes are transmitted, though IgG1 enters most readily. Whole labelled human IgG fast and slow electrophoretic fractions prepared by DEAE chromatography were also injected and an isoelectrofocusing analysis carried out on the injected IgG and the foetal serum. There was considerable variation of transmission even between adjacent peaks of the IEF pattern. PMID:4435834

  17. Conformational difference in human IgG2 disulfide isoforms revealed by hydrogen/deuterium exchange mass spectrometry.

    PubMed

    Zhang, Aming; Fang, Jing; Chou, Robert Y-T; Bondarenko, Pavel V; Zhang, Zhongqi

    2015-03-17

    Both recombinant and natural human IgG2 antibodies have several different disulfide bond isoforms, which possess different global structures, thermal stabilities, and biological activities. A detailed mapping of the structural difference among IgG2 disulfide isoforms, however, has not been established. In this work, we employed hydrogen/deuterium exchange mass spectrometry to study the conformation of three major IgG2 disulfide isoforms known as IgG2-B, IgG2-A1, and IgG2-A2 in two recombinant human IgG2 monoclonal antibodies. By comparing the protection factors between amino acid residues in isoforms B and A1 (the classical form), we successfully identified several local regions in which the IgG2-B isoform showed more solvent protection than the IgG2-A1 isoform. On the basis of three-dimensional structural models of IgG2, these identified regions were located on the Fab domains, close to the hinge, centered on the side where the two Fab arms faced each other in spatial proximity. We speculated that in the more solvent-protected B isoform, the two Fab arms were brought into contact by the nonclassical disulfide bonds, resulting in a more compact global structure. Loss of Fab domain flexibility in IgG2-B could limit its ability to access cell-surface epitopes, leading to reduced antigen binding potency. The A2 isoform was previously found to have disulfide linkages similar to those of the classical A1 isoform, but with different biophysical behaviors. Our data indicated that, compared to IgG2-A1, IgG2-A2 had less solvent protection in some heavy-chain Fab regions close the hinge, suggesting that the A2 isoform had more flexible Fab domains. PMID:25730439

  18. Functionalized micro-capillary film for the rapid at-line analysis of IgG aggregates in a cell culture bioreactor.

    PubMed

    Townsend, Matthew J; Gruber, David E; Kuiper, Marcel; Lazar, Radu A; Field, Ray P; Turner, Richard E; Slater, Nigel K H

    2015-01-01

    A micro-capillary film has been developed that offers the potential for an at-line analytical tool for rapid aggregate analysis during biopharmaceutical antibody production. A non-porous walled micro-capillary film (NMCF) with cation exchange functionality was demonstrated to act as a chromatography medium that could be operated with high linear fluid velocities and was highly resistant to blockage by entrained particulates, including cells. The NMCF containing 19 parallel microcapillaries was prepared using a melt extrusion process from poly(ethylene-vinyl alcohol) copolymer (EVOH). The NMCF-EVOH was modified to have cation-exchange functionality (NMCF-EVOH-SP) and shown to differentially bind monomer and aggregated species of IgG antibody directly from a bioreactor. The use of NMCF-EVOH-SP to quantify aggregate concentrations in monoclonal antibody preparations in less than 20 minutes was demonstrated. PMID:26176737

  19. Functionalized micro-capillary film for the rapid at-line analysis of IgG aggregates in a cell culture bioreactor

    PubMed Central

    Townsend, Matthew J; Gruber, David E; Kuiper, Marcel; Lazar, Radu A; Field, Ray P; Turner, Richard E; Slater, Nigel KH

    2015-01-01

    A micro-capillary film has been developed that offers the potential for an at-line analytical tool for rapid aggregate analysis during biopharmaceutical antibody production. A non-porous walled micro-capillary film (NMCF) with cation exchange functionality was demonstrated to act as a chromatography medium that could be operated with high linear fluid velocities and was highly resistant to blockage by entrained particulates, including cells. The NMCF containing 19 parallel microcapillaries was prepared using a melt extrusion process from poly(ethylene-vinyl alcohol) copolymer (EVOH). The NMCF-EVOH was modified to have cation-exchange functionality (NMCF-EVOH-SP) and shown to differentially bind monomer and aggregated species of IgG antibody directly from a bioreactor. The use of NMCF-EVOH-SP to quantify aggregate concentrations in monoclonal antibody preparations in less than 20 minutes was demonstrated. PMID:26176737

  20. Binding of normal human IgG to myelin sheaths, glia and neurons.

    PubMed Central

    Aarli, J A; Aparicio, S R; Lumsden, C E; Tönder, O

    1975-01-01

    The binding of normal human serum, purified IgG and IgG fragments to central nervous tissue was studied by the anti-globulin consumption (AGCT) and immunofluorescence (IF) techniques. In the AGCT, F(ab')2 fragments failed to react, whereas IgG and Fc fragments did so. In IF experiments, the binding was localized to myelin sheaths, glia and neurons; Fab monomers at a protein concentration of 1-3 mg/ml dod not react with the tissue, but purified Fc fragments at 0-0625 mg/ml did. The binding is neither tissue- nor species-specific. Lipid and protein extraction procedures indicated that the factor responsible for binding to myelin was basic protein. It was concluded that the binding of normal IgG to central nervous tissue is medicated by the Fc part of the molecule. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:803915

  1. Inhibition and reversal of endotoxin-, aggregated IgG- and paf-induced hypotension in the rat by SRI 63-072, a paf receptor antagonist.

    PubMed

    Handley, D A; Van Valen, R G; Melden, M K; Flury, S; Lee, M L; Saunders, R N

    1986-08-01

    Platelet activating factor (paf) given intravenously produces systemic hypotension in the rat. Similar effects can be induced using endotoxin or heat-aggregated IgG challenges, which are thought to involve endogenous paf release. Extending this concept, we have examined the ability of the paf antagonist SRI 63-072 to inhibit or reverse systemic hypotension induced with paf, heat-aggregated IgG or endotoxin 0111-B4 in rats. At 100 ng kg-1 paf, there occurred a 38.6 +/- 5.1% decrease in carotid mean arterial pressure (MAP) followed by a 3.2 +/- 0.7 min recovery period (RP) to return to normal pressure values. The ED50 of SRI 63-072 was 0.16 mg kg-1 i.v. (MAP) and 0.25 mg kg-1 (RP) when given 1-5 min before the paf challenge. Endotoxin (15 mg kg-1 i.v.) produced a hypotensive response (54 +/- 8% decrease in MAP) and a corresponding 80% decrease in mesenteric artery blood flow. When given 2-8 min after endotoxin, 1.0 mg kg-1 i.v. SRI 63-072 totally restored blood pressure and artery blood flow. SRI 63-072 similarly reversed heat-aggregated IgG (10 mg kg-1) induced reduction of MAP, with an ED50 of 0.05 mg kg-1 i.v. The observations that SRI 63-072 can inhibit or reverse systemic vascular effects produced from paf and other provocators of endogenous paf release strongly implicates paf as a common final mediator of hypotension and shock. As SRI 63-072 is a competitive receptor antagonist, the hypotensive effects of these provocators appear to be mediated by vascular receptors for paf. PMID:3019921

  2. Conformation of human IgG subclasses in solution. Small-angle X-ray scattering and hydrodynamic studies.

    PubMed

    Kilár, F; Simon, I; Lakatos, S; Vonderviszt, F; Medgyesi, G A; Závodszky, P

    1985-02-15

    The structure of six human myeloma proteins: IgG1(Bal), IgG2(Klu), IgG3(Bak), IgG3(Het), IgG4(Kov) and IgG4(Pol), was studied in solution using small-angle X-ray scattering and hydrodynamic methods. For IgG1(Bal) and IgG3(Het) the experimental data, including radius of gyration (Rg degree), radii of gyration of the cross-section (Rq1, Rq2), intrinsic viscosity [eta], sedimentation coefficient (S degree 20,w) and molecular mass, were interpreted in terms of structural models based on the Fab and Fc conformations, observed in crystal, by varying the relative positions of the Fab and Fc parts, i.e. their relative angles and distances. The values Rg degree = (6.00 +/- 0.05) nm, S degree 20,w = (6.81 +/- 0.10) S and [eta] = 0.0062 +/- 0.0005 cm3/mg obtained for IgG1(Bal) are compatible with a planar model in which the angle between the Fab arms is about 120 degrees. For IgG3(Het) the following data were obtained: Rg degree = (4.90 +/- 0.05) nm, S degree 20,w = (6.32 +/- 0.01) S and [eta] = (0.0065 +/- 0.0005) cm3/mg. The apparent contradiction between the higher molecular mass and lower Rg degree and S degree 20,w values for IgG3(Het) in comparison to IgG1(Bal) can be resolved by proposing a 'non-planar' (tetrahedral) molecular shape, in which the long hinge peptide is in a folded conformation and the two Fab and Fc parts are in a closely packed arrangement. In this model the angle between the two Fab arms is about 90 degrees, in the average position. The X-ray scattering and hydrodynamic behaviour of the IgG2 and IgG4 types of antibodies appeared to be similar to IgG1(Bal). The parameters of the two IgG3 proteins are similar while they are different to the others. PMID:3971974

  3. Normal human IgG prevents endothelial cell activation induced by TNFα and oxidized low-density lipoprotein atherogenic stimuli

    PubMed Central

    RONDA, N; BERNINI, F; GIACOSA, R; GATTI, R; BALDINI, N; BUZIO, C; ORLANDINI, G

    2003-01-01

    Normal human immunoglobulin G (IgG) has anti-inflammatory and immuno-regulatory properties, which are exploited in the therapy of selected diseases. A putative mechanisms of action is the direct regulation of endothelial cell function by natural antiendothelial cell antibodies. Endothelium activation is a critical event in atherosclerosis. We have verified the ability of normal human IgG to modulate endothelial responses to the atherogenic stimuli tumour necrosis factor-α (TNFα) and oxidized low-density lipoproteins (oxLDL) in vitro. Confocal microscopy was used to visualize vascular cell adhesion molecule-1 (CD106) expression on endothelial cells, cytoplasmic free calcium ([Ca++]i) modifications and fluorescein-coupled oxLDL internalization. Cytokine secretion was measured by ELISA on cell supernatants. IgG prevented TNFα induced CD106 membrane expression and an increase in [Ca++]i, and inhibited the secretion of interleukin-6 (IL-6) and macrophage-colony-stimulating factor (M-CSF). IgG also inhibited CD106 expression induced by oxLDL and one pathway of their internalization, but were ineffective on oxLDL induced [Ca++]i rise and apoptosis. F(ab)′2 fragments from IgG, but not monoclonal IgG, reproduce IgG effects. These findings point to a regulatory role for specific antibodies included in circulating normal IgG towards proinflammatory responses of endothelial cells in atherogenesis and suggest possible development of new therapeutic strategies. PMID:12869027

  4. Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

    PubMed Central

    Wu, Haixia; Truncali, Kristopher; Ritchie, Julie; Kroe-Barrett, Rachel; Singh, Sanjaya; Robinson, Anne S; Roberts, Christopher J

    2015-01-01

    The Fc (fragment crystallizable) is a common structural region in immunoglobulin gamma (IgG) proteins, IgG-based multi-specific platforms, and Fc-fusion platform technologies. Changes in conformational stability, protein-protein interactions, and aggregation of NS0-produced human Fc1 were quantified experimentally as a function of pH (4 to 6) and temperature (30 to 77°C), using a combination of differential scanning calorimetry, laser light scattering, size-exclusion chromatography, and capillary electrophoresis. The Fc1 was O-glycosylated at position 3 (threonine), and confirmed to correspond to the intact IgG1 by comparison with Fc1 produced by cleavage of the parent IgG1. Changing the pH caused large effects for thermal unfolding transitions, but it caused surprisingly smaller effects for electrostatic protein-protein interactions. The aggregation behavior was qualitatively similar across different solution conditions, with soluble dimers and larger oligomers formed in most cases. Aggregation rates spanned approximately 5 orders of magnitude and could be divided into 2 regimes: (i) Arrhenius, unfolding-limited aggregation at temperatures near or above the midpoint-unfolding temperature of the CH2 domain; (ii) a non-Arrhenius regime at lower temperatures, presumably as a result of the temperature dependence of the unfolding enthalpy for the CH2 domain. The non-Arrhenius regime was most pronounced for lower temperatures. Together with the weak protein-protein repulsions, these highlight challenges that are expected for maintaining long-term stability of biotechnology products that are based on human Fc constructs. PMID:26267255

  5. Inhibition of erythrocyte invasion and Plasmodium falciparum merozoite surface protein 1 processing by human immunoglobulin G1 (IgG1) and IgG3 antibodies.

    PubMed

    Lazarou, Maria; Guevara Patiño, José A; Jennings, Richard M; McIntosh, Richard S; Shi, Jianguo; Howell, Steven; Cullen, Eilish; Jones, Tarran; Adame-Gallegos, Jaime R; Chappel, Jonathan A; McBride, Jana S; Blackman, Michael J; Holder, Anthony A; Pleass, Richard J

    2009-12-01

    Antigen-specific antibodies (Abs) to the 19-kDa carboxy-terminal region of Plasmodium falciparum merozoite surface protein 1 (MSP1(19)) play an important role in protective immunity to malaria. Mouse monoclonal Abs (MAbs) 12.10 and 12.8 recognizing MSP1(19) can inhibit red cell invasion by interfering with MSP1 processing on the merozoite surface. We show here that this ability is dependent on the intact Ab since Fab and F(ab')(2) fragments derived from MAb 12.10, although capable of binding MSP1 with high affinity and competing with the intact antibody for binding to MSP1, were unable to inhibit erythrocyte invasion or MSP1 processing. The DNA sequences of the variable (V) regions of both MAbs 12.8 and 12.10 were obtained, and partial amino acid sequences of the same regions were confirmed by mass spectrometry. Human chimeric Abs constructed by using these sequences, which combine the original mouse V regions with human gamma1 and gamma3 constant regions, retain the ability to bind to both parasites and recombinant MSP1(19), and both chimeric human immunoglobulin G1s (IgG1s) were at least as good at inhibiting erythrocyte invasion as the parental murine MAbs 12.8 and 12.10. Furthermore, the human chimeric Abs of the IgG1 class (but not the corresponding human IgG3), induced significant NADPH-mediated oxidative bursts and degranulation from human neutrophils. These chimeric human Abs will enable investigators to examine the role of human Fcgamma receptors in immunity to malaria using a transgenic parasite and mouse model and may prove useful in humans for neutralizing parasites as an adjunct to antimalarial drug therapy. PMID:19805526

  6. EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG

    PubMed Central

    Collin, Mattias; Olsén, Arne

    2001-01-01

    Streptococcus pyogenes is an important human pathogen that selectively interacts with proteins involved in the humoral defense system, such as immunoglobulins and complement factors. In this report we show that S.pyogenes has the ability to hydrolyze the chitobiose core of the asparagine-linked glycan on immuno globulin G (IgG) when bacteria are grown in the presence of human plasma. This activity is associated with the secretion of a novel 108 kDa protein denoted EndoS. EndoS has endoglycosidase activity on purified soluble IgG as well as IgG bound to the bacterial surface. EndoS is required for the activity on IgG, as an isogenic EndoS mutant could not hydrolyze the glycan on IgG. In addition, we show that the secreted streptococcal cysteine proteinase SpeB cleaves IgG in the hinge region in a papain-like manner. This is the first example of an endoglycosidase produced by a bacterial pathogen that selectively hydrolyzes human IgG, and reveals a novel mechanism which may contribute to S.pyogenes pathogenesis. PMID:11406581

  7. Glyco-engineering of human IgG1-Fc through combined yeast expression and in vitro chemoenzymatic glycosylation

    PubMed Central

    Wei, Yadong; Li, Cishan; Huang, Wei; Li, Bing; Strome, Scott; Wang, Lai-Xi

    2009-01-01

    The presence and precise structures of the glycans attached at the Fc domain of monoclonal antibodies play an important role in determining antibody's effector functions such as antibody-dependent cell cytotoxicity (ADCC), complement activation, and anti-inflammatory activity. This paper describes a novel approach for glyco-engineering of human IgG1-Fc that combines high-yield expression of human IgG1-Fc in yeast and subsequent in vitro enzymatic glycosylation, using the endoglycosidase-catalyzed transglycosylation as the key reaction. Human IgG1-Fc was first overproduced in Pichia pastoris. Then the heterogeneous yeast glycans were removed by Endo-H treatment to give the GlcNAc-containing IgG1-Fc as a homodimer. Finally, selected homogeneous glycans were attached to the GlcNAc-primer in the IgG1-Fc through an endoglycosidase-catalyzed transglycosylation, using sugar oxazolines as the donor substrates. It was found that the enzymatic transglycosylation was efficient with native GlcNAc-containing IgG1-Fc homodimer without the need to denature the protein, and the reaction could proceed to completion to give homogeneous glycoforms of IgG1-Fc when excess of oligosaccharide oxazolines was used as the donor substrates. The binding of the synthetic IgG1-Fc glycoforms to the FcγIIIa receptor was also investigated. This novel glyco-engineering approach should be useful for providing various homogeneous, natural or synthetic glycoforms of IgG1-Fc for structure-function relationship studies, and for future clinical applications. PMID:18771295

  8. Human placenta: relative content of antibodies of different classes and subclasses (IgG1-IgG4) containing lambda- and kappa-light chains and chimeric lambda-kappa-immunoglobulins.

    PubMed

    Lekchnov, Evgenii A; Sedykh, Sergey E; Dmitrenok, Pavel S; Buneva, Valentina N; Nevinsky, Georgy A

    2015-06-01

    The specific organ placenta is much more than a filter: it is an organ that protects, feeds and regulates the growth of the embryo. Affinity chromatography, ELISA, SDS-PAGE and matrix-assisted laser desorption ionization mass spectrometry were used. Using 10 intact human placentas deprived of blood, a quantitative analysis of average relative content [% of total immunoglobulins (Igs)] was carried out for the first time: (92.7), IgA (2.4), IgM (2.5), kappa-antibodies (51.4), lambda-antibodies (48.6), IgG1 (47.0), IgG2 (39.5), IgG3 (8.8) and IgG4 (4.3). It was shown for the first time that placenta contains sIgA (2.5%). In the classic paradigm, Igs represent products of clonal B-cell populations, each producing antibodies recognizing a single antigen. There is a common belief that IgGs in mammalian biological fluids are monovalent molecules having stable structures and two identical antigen-binding sites. However, similarly to human milk Igs, placenta antibodies undergo extensive half-molecule exchange and the IgG pool consists of 43.5 ± 15.0% kappa-kappa-IgGs and 41.6 ± 17.0% lambda-lambda-IgGs, while 15.0 ± 4.0% of the IgGs contained both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained, respectively (%): IgG1 (47.7 and 34.4), IgG2 (36.3 and 44.5), IgG3 (7.4 and 11.8) and IgG4 (7.5 and 9.1), while chimeric kappa-lambda-IgGs consisted of (%): 43.5 IgG1, 41.0 IgG2, 5.6 IgG3 and 7.9 IgG4. Our data are indicative of the possibility of half-molecule exchange between placenta IgGs of various subclasses, raised against different antigens, which explains a very well-known polyspecificity and cross-reactivity of different human IgGs. PMID:25644595

  9. [AGGREGATION OF METABOLICALLY DEPLETED HUMAN ERYTHROCYTES].

    PubMed

    Sheremet'ev, Yu A; Popovicheva, A N; Rogozin, M M; Levin, G Ya

    2016-01-01

    An aggregation of erythrocytes in autologous plasma after blood storage for 14 days at 4 °C was studied using photometry and light microscopy. The decrease of ATP content, the formation of echinocytes and spheroechinocytes, the decrease of rouleaux form of erythrocyte aggregation were observed during the storage. On the other hand the aggregates of echinocytes were formed in the stored blood. The addition of plasma from the fresh blood didn't restore the normal discocytic shape and aggregation of erythrocytes in the stored blood. The possible mechanisms of erythrocytes and echinocytes aggregation are discussed. PMID:27220249

  10. FYWHCLDE-based affinity chromatography of IgG: effect of ligand density and purifications of human IgG and monoclonal antibody.

    PubMed

    Zhao, Wei-Wei; Shi, Qing-Hong; Sun, Yan

    2014-08-15

    This work reports the development of an octapeptide-based affinity adsorbent for the purification of human IgG (hIgG) and monoclonal antibody (mAb). The octapeptide was FYWHCLDE selected earlier by the biomimetic design of affinity peptide ligands for hIgG. The ligand was coupled to Sepharose gel at four densities from 10.4 to 31.0μmol/mL, and the effect of peptide density on the adsorption of hIgG and bovine serum albumin (BSA) was first investigated. The binding capacity of hIgG increased from 104.2 to 176.4mg/mL within the ligand density range, and the binding affinity (dissociation constant) kept at 2.4-3.7μM. Batch adsorption revealed that the selectivity of FYWHCLDE-Sepharose for IgG was 30-40 times over BSA. The effective pore diffusivity of IgG decreased somewhat with increasing ligand density, but the dynamic binding capacity at 10% breakthrough, measured by using 10-fold diluted human serum as feedstock, doubled with increasing ligand density from 10.4 to 31.0μmol/mL due to the remarkable increase of static binding capacity. By using the affinity column with a ligand density of 23.9μmol/mL, hIgG and humanized mAb purifications from human serum and cell culture supernatant, respectively, were achieved at high purities and recovery yields. Finally, the robustness of the peptide gel was demonstrated by recycled use of the affinity column in 20 breakthrough cycles. PMID:24947889

  11. Different glycosylation pattern of human IgG1 and IgG3 antibodies isolated from transiently as well as permanently transfected cell lines.

    PubMed

    Vestrheim, A C; Moen, A; Egge-Jacobsen, W; Bratlie, D B; Michaelsen, T E

    2013-05-01

    The effector functions of IgG depend on the presence of carbohydrates attached to asparagine 297 in the Fc-portion. In this report, glycosylation profiles of recombinant wild-type and mutant IgG1 and IgG3 antibodies produced from three cell lines were analysed using LC-ESI-Orbitrap. Clear differences were detected between IgG1 and IgG3 glycoforms, where IgG1 generally contained fucosylated glycoforms, whilst IgG3 mainly were non-fucosylated. When using NS-0 and J558L cells for permanent transfection, IgG1 wt glycoforms differed between the two cell lines, whilst IgG3 wt glycoforms did not. Transiently transfected HEK 293E cells were used to produce IgG1 and IgG3 wt and mutants, affecting complement activation. Cell supernatants were harvested at early and late time points and analysed separately. IgGs harvested late showed simpler and less developed glycosylation structure compared to those harvested early. The IgG harvested early was slightly more effective in complement activation than those harvested late, whilst the antibody-dependent cell-mediated cytotoxicity was unaltered. Generally, the glycosylation pattern of the mutants tested, including a hinge truncate mutant of IgG3, did not differ significantly from the wild-type IgGs. The striking difference in glycosylation pattern of IgG1 compared to IgG3 therefore appears not to be due to the long hinge region of IgG3 (62 amino acids) relative to the IgG1 hinge region (15 amino acids). Furthermore, mutation variants at or near the C1q binding site showed similar glycosylation structure and difference in their complement activation activity observed earlier is thus most likely due to differences in protein structure only. PMID:23488770

  12. Human Milk IgGs Contain Various Combinations of Different Antigen-Binding Sites Resulting in Multiple Variants of their Bispecificity

    PubMed Central

    Sedykh, Sergey E.; Buneva, Valentina N.; Nevinsky, Georgy A.

    2012-01-01

    In the classic paradigm, immunoglobulins represent products of clonal B cell populations, each producing antibodies (Abs) recognizing a single antigen. There is a common belief that IgGs in mammalian biological fluids are monovalent molecules having stable structures and two identical antigen-binding sites. However, human milk IgGs to different antigens undergo extensive half-molecule exchange. In the IgGs pool, only 33±5% and 13±5% of Abs contained light chains exclusively of kappa- or lambda-type, respectively, while 54±10% of the IgGs contained both kappa- and lambda- light chains. All Ab preparations contained different amounts of IgGs of all four subclasses. Interestingly, lambda-IgGs contained an increased amount of IgG2 (87%) and only 3–6% of each of IgG1, IgG3, and IgG4, while kappa-IgGs consisted of comparable (17–32%) amounts of all IgG subtypes. Chimeric kappa-lambda-IgGs consisted of ∼74% IgG1, ∼16% IgG2, ∼5% IgG3 and ∼5% IgG4. As the result of the exchange, all IgG fractions eluted from several specific affinity sorbents under the conditions destroying strong immunocomplexes demonstrated high catalytic activities in hydrolysis of ATP, DNA, oligosaccharides, phosphorylation of proteins, lipids, and oligosaccharides. In vitro, an addition of reduced glutathione and milk plasma to two IgG fractions with different affinity for DNA-cellulose led to a transition of 25–60% of Ab of one fraction to the other fraction. Our data are indicative of the possibility of half-molecule exchange between milk IgGs of various subclasses, raised against different antigens (including abzymes), which explains the polyspecificity and cross-reactivity of these IgGs. PMID:22912765

  13. Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus

    PubMed Central

    Gustafsson, Erika; Haas, Pieter-Jan; Walse, Björn; Hijnen, Marcel; Furebring, Christina; Ohlin, Mats; van Strijp, Jos AG; van Kessel, Kok PM

    2009-01-01

    Background The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. Results In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31–121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. Conclusion Conformational epitopes recognized by human antibodies have been mapped on the

  14. A novel in vitro assay to predict neonatal Fc receptor-mediated human IgG half-life.

    PubMed

    Souders, Colby A; Nelson, Stuart C; Wang, Yang; Crowley, Andrew R; Klempner, Mark S; Thomas, William

    2015-01-01

    Immunoglobulin G (IgG) has an unusually long serum half-life in comparison to proteins of a similar size. It is well-known that this phenomenon is due to IgG's ability to bind the neonatal Fc receptor (FcRn) in a pH-dependent manner. FcRn binding properties can vary among IgGs, resulting in altered in vivo half-lives, and therefore it would be beneficial to accurately predict the FcRn binding properties of therapeutic IgG monoclonal antibodies (mAbs). Here we describe the development of an in vitro model capable of predicting the in vivo half-life of human IgG. Using a high-throughput biolayer interferometry (BLI) platform, the human FcRn association rate at acidic pH and subsequent dissociation rate at physiological pH was determined for 5 human IgG1 mAbs. Comparing the combined FcRn association and dissociation rates to the Phase 1 clinical study half-lives of the mAbs resulted in a strong correlation. The correlation was also verified in vivo using mice transgenic for human FcRn. The model was used to characterize various factors that may influence FcRn-mAb binding, including mAb variable region sequence differences and constant region glycosylation patterns. Results indicated that the complementarity-determining regions of the heavy chain significantly influence the mAb's FcRn binding properties, while the absence of glycosylation does not alter mAb-FcRn binding. Development of this high-throughput FcRn binding model could potentially predict the half-life of therapeutic IgGs and aid in selection of lead candidates while also serving as a screening tool for the development of mAbs with desired pharmacokinetic properties. PMID:26018774

  15. A novel in vitro assay to predict neonatal Fc receptor-mediated human IgG half-life

    PubMed Central

    Souders, Colby A; Nelson, Stuart C; Wang, Yang; Crowley, Andrew R; Klempner, Mark S; Thomas, William

    2015-01-01

    Immunoglobulin G (IgG) has an unusually long serum half-life in comparison to proteins of a similar size. It is well-known that this phenomenon is due to IgG's ability to bind the neonatal Fc receptor (FcRn) in a pH-dependent manner. FcRn binding properties can vary among IgGs, resulting in altered in vivo half-lives, and therefore it would be beneficial to accurately predict the FcRn binding properties of therapeutic IgG monoclonal antibodies (mAbs). Here we describe the development of an in vitro model capable of predicting the in vivo half-life of human IgG. Using a high-throughput biolayer interferometry (BLI) platform, the human FcRn association rate at acidic pH and subsequent dissociation rate at physiological pH was determined for 5 human IgG1 mAbs. Comparing the combined FcRn association and dissociation rates to the Phase 1 clinical study half-lives of the mAbs resulted in a strong correlation. The correlation was also verified in vivo using mice transgenic for human FcRn. The model was used to characterize various factors that may influence FcRn-mAb binding, including mAb variable region sequence differences and constant region glycosylation patterns. Results indicated that the complementarity-determining regions of the heavy chain significantly influence the mAb's FcRn binding properties, while the absence of glycosylation does not alter mAb-FcRn binding. Development of this high-throughput FcRn binding model could potentially predict the half-life of therapeutic IgGs and aid in selection of lead candidates while also serving as a screening tool for the development of mAbs with desired pharmacokinetic properties. PMID:26018774

  16. A simultaneous two-colour detection method of human IgG- and IgE-reactive proteins from lactic acid bacteria.

    PubMed

    Markiewicz, Lidia Hanna; Szymkiewicz, Agata; Szyc, Anna; Wróblewska, Barbara

    2016-07-01

    Whole cell extracts of two Lactobacillus strains were tested with primary antibodies from two pooled sera from allergic patients. Fluorescently labelled anti-human IgG and anti-human IgE secondary antibodies applied in Western blotting, together with an appropriate image acquisition protocol facilitated imagining bacterial proteins that reacted with human IgG and IgE. PMID:27184086

  17. Characterization of a Novel Hemolytic Activity of Human IgG Fractions Arising from Diversity in Protein and Oligosaccharide Components

    PubMed Central

    Zhang, Yueling; Ye, Xiangqun; Zhong, Mingqi; Cao, Jinsong; Zou, Haiying; Chen, Jiehui

    2014-01-01

    Human IgG is a well-established multifunctional antigen specific immunoglobulin molecule of the adaptive immune system. However, an antigen nonspecific immunological function of human IgG has never been reported. In this study, human IgG was isolated using ammonium sulfate fractional precipitation and diethylaminoethanol (DEAE) cellulose 52 ion exchange chromatography, from which h-IgG and hs-IgG fractions were purified on the basis of their differential binding to rabbit anti-shrimp hemocyanin antibody (h) and rabbit anti-shrimp hemocyanin's small subunit antibody (hs), respectively. We found that h-IgG had a higher hemolytic activity than hs-IgG against erythrocytes from humans, rabbits, mice and chickens, whereas the control IgG showed negligible activity. h-IgG could interact directly with erythrocyte membranes, and this interaction was suppressed by high molecular weight osmoprotectants, showing that it may follow a colloid-osmotic mechanism. In comparative proteomics and glycomics studies, h-IgG and hs-IgG yielded 20 and 5 significantly altered protein spots, respectively, on a 2-D gel. The mean carbohydrate content of h-IgG and hs-IgG was approximately 3.6- and 2-fold higher than that of IgG, respectively, and the α-d-mannose/α-d-glucose content was in the order of h-IgG>hs-IgG>IgG. In this study, a novel antigen nonspecific immune property of human IgG was investigated, and the diversity in the protein constituents and glycosylation levels may have functional signficance. PMID:24465658

  18. Intrathoracic Rosai Dorfman Disease with Focal Aggregates of IgG4-bearing Plasma Cells. Case Report and Literature Review.

    PubMed

    Apperley, Scott T; Hyjek, Elizabeth M; Musani, Rumina; Thenganatt, John

    2016-05-01

    Rosai Dorfman disease, also known as sinus histiocytosis with massive lymphadenopathy, is a rare histiocytic disorder that typically presents as painless cervical adenopathy. Occasionally, Rosai Dorfman disease may involve extranodal sites and it can be associated with constitutional symptoms. In this report, we present a patient with progressive dyspnea on exertion, obstruction on spirometry, and paratracheal soft tissue thickening extending to the proximal bronchi and hila. After extensive radiologic and interventional measures, including multiple biopsies, the diagnosis was established on pathology showing characteristic features of Rosai Dorfman disease in a paratracheal lymph node and features of extranodal Rosai Dorfman disease with extensive fibrosis in the paratracheal mass. In addition, the involved paratracheal lymph node demonstrated focally increased IgG4-positive plasma cells, a finding that was not present in the tracheal mass. A review of intrathoracic manifestations of Rosai Dorfman disease including the proposed causes and causative triggers is presented. The histological features and the current understanding of the management and prognosis are also discussed. This unusual presentation highlights the need to consider histiocytic disorders in the differential diagnosis of paratracheal disease. PMID:26820713

  19. Selective binding of IgG4 and other negatively charged plasma proteins in normal and diabetic human kidneys.

    PubMed Central

    Melvin, T.; Kim, Y.; Michael, A. F.

    1984-01-01

    Renal tissue from 9 patients with diabetes mellitus (4 with mild and 5 with end-stage disease) and 3 with antiglomerular basement membrane (GBM) nephritis, as well as 5 normal human kidneys, were examined by immunofluorescence microscopy for the presence of plasma proteins of varying isoelectric point (pI). In normal and diabetic kidneys, IgG deposition in basement membranes was restricted to IgG4 (pI 5.5-6.0), the subclass present in lowest concentration in human plasma. IgG1, IgG2, and IgG3 (pI 7.0-9.5) were not detected. In contrast, in anti-GBM nephritis, all four subclasses were present in a linear pattern in GBM. Other plasma proteins of low isoelectric point were detected in basement membranes: albumin (pI 4.9), alpha-1-acid glycoprotein (pI 2.7), amyloid P (pI 3.9-4.8), and alpha-1-antitrypsin (pI 4.5). These studies are consistent with the hypothesis that circulating anionic plasma proteins are electrostatically bound in vivo to positively charged moieties in normal and especially diabetic basement membranes. Images Figure 1 PMID:6375393

  20. The Fab Conformations in the Solution Structure of Human Immunoglobulin G4 (IgG4) Restrict Access to Its Fc Region

    PubMed Central

    Rayner, Lucy E.; Hui, Gar Kay; Gor, Jayesh; Heenan, Richard K.; Dalby, Paul A.; Perkins, Stephen J.

    2014-01-01

    Human IgG4 antibody shows therapeutically useful properties compared with the IgG1, IgG2, and IgG3 subclasses. Thus IgG4 does not activate complement and shows conformational variability. These properties are attributable to its hinge region, which is the shortest of the four IgG subclasses. Using high throughput scattering methods, we studied the solution structure of wild-type IgG4(Ser222) and a hinge mutant IgG4(Pro222) in different buffers and temperatures where the proline substitution suppresses the formation of half-antibody. Analytical ultracentrifugation showed that both IgG4 forms were principally monomeric with sedimentation coefficients s20,w0 of 6.6–6.8 S. A monomer-dimer equilibrium was observed in heavy water buffer at low temperature. Scattering showed that the x-ray radius of gyration Rg was unchanged with concentration in 50–250 mm NaCl buffers, whereas the neutron Rg values showed a concentration-dependent increase as the temperature decreased in heavy water buffers. The distance distribution curves (P(r)) revealed two peaks, M1 and M2, that shifted below 2 mg/ml to indicate concentration-dependent IgG4 structures in addition to IgG4 dimer formation at high concentration in heavy water. Constrained x-ray and neutron scattering modeling revealed asymmetric solution structures for IgG4(Ser222) with extended hinge structures. The IgG4(Pro222) structure was similar. Both IgG4 structures showed that their Fab regions were positioned close enough to the Fc region to restrict C1q binding. Our new molecular models for IgG4 explain its inability to activate complement and clarify aspects of its stability and function for therapeutic applications. PMID:24876381

  1. Acid soluble platelet aggregating material isolated from human umbilical cord

    SciTech Connect

    Schneider, M.D.

    1983-12-27

    An acid soluble, pepsin sensitive platelet aggregating material is isolated from human umbilical cord tissue by extraction with dilute aqueous acid. The method of isolation is disclosed and its use to control bleeding is described. 2 figs.

  2. Increased serum clearance of oligomannose species present on a human IgG1 molecule

    PubMed Central

    Alessandri, Leslie; Ouellette, David; Acquah, Aima; Rieser, Mathew; LeBlond, David; Saltarelli, Mary; Radziejewski, Czeslaw; Fujimori, Taro; Correia, Ivan

    2012-01-01

    The role of Fc glycans on clearance of IgG molecule has been examined by various groups in experiments where specific glycans have been enriched or the entire spectrum of glycans was studied after administration in pre-clinical or clinical pharmacokinetic (PK) studies. The overall conclusions from these studies are inconsistent, which may result from differences in antibody structure or experimental design. In the present study a well-characterized recombinant monoclonal IgG1 molecule (mAb-1) was analyzed from serum samples obtained from a human PK study. mAb-1 was recovered from serum using its ligand cross-linked to Sepharose beads. The overall purity and recovery of all isoforms were carefully evaluated using a variety of methods. Glycans were then enzymatically cleaved, labeled using 2-aminobenzamide and analyzed by normal phase high performance liquid chromatography. The assays for recovering mAb-1 from serum and subsequent glycan analysis were rigorously qualified at a lower limit of quantitation of 15 μg/mL, thus permitting analysis to day 14 of the clinical PK study. Eight glycans were monitored and classified into two groups: (1) the oligomannose type structures (M5, M6 and M7) and (2) fucosylated biantennary oligosaccharides (FBO) structures (NGA2F, NA1F, NA2F, NA1F-GlcNAc and NGA2F-GlcNAc). We observed that the oligomannose species were cleared at a much faster rate (40%) than FBOs and conclude that high mannose species should be carefully monitored and controlled as they may affect PK of the therapeutic; they should thus be considered an important quality attribute. These observations were only possible through the application of rigorous analytical methods that we believe will need to be employed when comparing innovator and biosimilar molecules. PMID:22669558

  3. N-terminal glutamate to pyroglutamate conversion in vivo for human IgG2 antibodies.

    PubMed

    Liu, Y Diana; Goetze, Andrew M; Bass, Randal B; Flynn, Gregory C

    2011-04-01

    Therapeutic proteins contain a large number of post-translational modifications, some of which could potentially impact their safety or efficacy. In one of these changes, pyroglutamate can form on the N terminus of the polypeptide chain. Both glutamine and glutamate at the N termini of recombinant monoclonal antibodies can cyclize spontaneously to pyroglutamate (pE) in vitro. Glutamate conversion to pyroglutamate occurs more slowly than from glutamine but has been observed under near physiological conditions. Here we investigated to what extent human IgG2 N-terminal glutamate converts to pE in vivo. Pyroglutamate levels increased over time after injection into humans, with the rate of formation differing between polypeptide chains. These changes were replicated for the same antibodies in vitro under physiological pH and temperature conditions, indicating that the changes observed in vivo were due to chemical conversion not differential clearance. Differences in the conversion rates between the light chain and heavy chain on an antibody were eliminated by denaturing the protein, revealing that structural elements affect pE formation rates. By enzymatically releasing pE from endogenous antibodies isolated from human serum, we could estimate the naturally occurring levels of this post-translational modification. Together, these techniques and results can be used to predict the exposure of pE for therapeutic antibodies and to guide criticality assessments for this attribute. PMID:21282104

  4. IgG4 but no IgG1 antibody production after intralymphatic immunotherapy with recombinant MAT-Feld1 in human.

    PubMed

    Freiberger, S N; Zehnder, M; Gafvelin, G; Grönlund, H; Kündig, T M; Johansen, P

    2016-09-01

    Allergy immunotherapy (AIT) mediates protection against allergen exposure in part due to allergen-specific antibodies. While immunization typically stimulated IgG1 and IgG2, AIT is often associated with production of IgG4. Here, twenty cat dander-sensitized patients were randomized to receive three injections of intralymphatic immunotherapy (ILIT) with MAT-Feld1 adsorbed to aluminum hydroxide or just aluminum hydroxide (placebo) in a double-blind setting (ClinicalTrials.gov NCT00718679). Whereas the clinical data, showing benefit of Mat-Feld1 ILIT was published in 2012 (Senti et al., J Allergy Clin Immunol, vol 129(5):1290-1296), the current study investigated the cat allergen-specific antibody responses. Blood was drawn prior to ILIT, as well as 1, 3, and 12 months after first ILIT. The sera were analyzed to characterize all IgG subclasses and IgE antibody responses. ILIT with MAT-Feld1 elicited high levels of total IgG that were maintained for at least 12 months. Interestingly, a strong increase in IgG4 and some increase in IgG2 were observed throughout the study, while production of cat-specific IgG1 and IgG3 was not stimulated by MAT-Feld1 ILIT. The IgE levels remained constant. PMID:27253988

  5. Structure of full-length human anti-PD1 therapeutic IgG4 antibody pembrolizumab.

    PubMed

    Scapin, Giovanna; Yang, Xiaoyu; Prosise, Winifred W; McCoy, Mark; Reichert, Paul; Johnston, Jennifer M; Kashi, Ramesh S; Strickland, Corey

    2015-12-01

    Immunoglobulin G4 antibodies exhibit unusual properties with important biological consequences. We report the structure of the human full-length IgG4 S228P anti-PD1 antibody pembrolizumab, solved to 2.3-Å resolution. Pembrolizumab is a compact molecule, consistent with the presence of a short hinge region. The Fc domain is glycosylated at the CH2 domain on both chains, but one CH2 domain is rotated 120° with respect to the conformation observed in all reported structures to date, and its glycan chain faces the solvent. We speculate that this new conformation is driven by the shorter hinge. The structure suggests a role for the S228P mutation in preventing the IgG4 arm exchange. In addition, this unusual Fc conformation suggests possible structural diversity between IgG subclasses and shows that use of isolated antibody fragments could mask potentially important interactions, owing to molecular flexibility. PMID:26595420

  6. Peroxynitrite-induced structural perturbations in human IgG: A physicochemical study.

    PubMed

    Arfat, Mir Yasir; Arif, Zarina; Chaturvedi, Sumit Kumar; Moinuddin; Alam, Khursheed

    2016-08-01

    IgG is an important defence protein. To exhibit optimum function the molecule must maintain its native structure. Peroxynitrite is a potent oxidizing and nitrating agent produced in vivo under pathophysiological conditions. It can oxidize and/or nitrate various amino acids causing changes in the structure and function of proteins. Such proteins may be involved in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis. In the present work, peroxynitrite-induced structural changes in IgG have been studied by UV-visible, fluorescence, CD, FT-IR, DLS spectroscopy and DSC as well as by SDS-PAGE. Peroxynitrite-modified IgG exhibited hyperchromicity at 280 nm, quenching of tryptophan fluorescence, increase in ANS fluorescence, loss of β-sheet, shift in the positions of amide I and amide II bands, appearance of new peak in FT-IR, attachment of nitro residues and increase in melting temperature, compared to native IgG. Furthermore, peroxynitrite-modified IgG exhibited an additional peak at 420 nm, quenching in tyrosine fluorescence and enhancement in dityrosine fluorescence compared to native IgG. Generation of nitrotyrosine, dityrosine and nitrotryptophan was also observed in peroxynitrite-modified IgG. Gross structural changes in IgG caused by peroxynitrite and observed in vitro may favour autoantibodies induction in vivo under similar conditions. PMID:27210739

  7. Low avidity IgG antibodies in diagnosis of recent human schistosomiasis.

    PubMed

    Mostafa, Nahed E; Awad, Adel; Shalaby, Mohsen

    2002-12-01

    One hundred thirty school children from a schistosomiasis endemic area in Sharkia Governorate, were selected on parasitological findings. Seventy persons were negative on the first screen and turned positive after 3 months of the screening (recently infected). Stool examination, ELISA (IgG & IgM), low avid IgG, and circulating antigens were performed for all patients and controls. ELISA detected IgM in all cases. IgG and circulating antigens in 90% of schistosomiasis patients. Low avidity IgG were detected in 85.71% of recent cases. The specificity of ELISA appeared to be >99%. The IgM/IgG ratio was >1 in patients with recent infection. The percentage of fall of O.D. readings of IgG after addition of 6 molar urea was high among cases with recent infection. Low avid lgG appears to be good and valuable in diagnosis of recent schistosomiasis in man. PMID:12512829

  8. Zinc significantly changes the aggregation pathway and the conformation of aggregates of human prion protein.

    PubMed

    Pan, Kai; Yi, Chuan-Wei; Chen, Jie; Liang, Yi

    2015-08-01

    Prion diseases are caused by the conformational change of cellular prion protein PrP(C) into pathological prion protein PrP(Sc). Here we study the effect of zinc on the aggregation and conformational change of human prion protein (PrP). As revealed by thioflavin T binding assays, Sarkosyl-soluble SDS-PAGE, and transmission electron microscopy, aggregation of wild-type PrP in the absence of Zn(2+) undergoes four steps: amorphous aggregates, profibrils, mature fibrils, and fragmented fibrils. When the molar ratio of Zn(2+) to PrP was 9:1, however, aggregation of wild-type PrP undergoes another pathway in which wild-type PrP forms oligomers quickly and then forms short-rod aggregates. Unlike wild-type PrP, the octarepeats deletion mutant PrPΔocta forms typical mature fibrils either with or without zinc. As evidenced by isothermal titration calorimetry, Fourier transform infrared spectroscopy, and proteinase K digestion assays, Zn(2+) strongly binds to wild-type PrP monomers with the first binding constant exceeding 10(7)M(-1) under denaturing conditions, and changes the conformation of wild-type PrP aggregates remarkably, but weakly binds to PrPΔocta with binding affinity around 10(4)M(-1) and has no obvious effects on the conformation of PrPΔocta aggregates. Our data demonstrate that zinc significantly changes the aggregation pathway and the conformation of wild-type PrP aggregates mainly via interaction with its octarepeat region. Our findings could explain how zinc modifies pathological PrP conformation associated with prion diseases. PMID:25922234

  9. The Solution Structures of Two Human IgG1 Antibodies Show Conformational Stability and Accommodate Their C1q and FcγR Ligands*

    PubMed Central

    Rayner, Lucy E.; Hui, Gar Kay; Gor, Jayesh; Heenan, Richard K.; Dalby, Paul A.; Perkins, Stephen J.

    2015-01-01

    The human IgG1 antibody subclass shows distinct properties compared with the IgG2, IgG3, and IgG4 subclasses and is the most exploited subclass in therapeutic antibodies. It is the most abundant subclass, has a half-life as long as that of IgG2 and IgG4, binds the FcγR receptor, and activates complement. There is limited structural information on full-length human IgG1 because of the challenges of crystallization. To rectify this, we have studied the solution structures of two human IgG1 6a and 19a monoclonal antibodies in different buffers at different temperatures. Analytical ultracentrifugation showed that both antibodies were predominantly monomeric, with sedimentation coefficients s20,w0 of 6.3–6.4 S. Only a minor dimer peak was observed, and the amount was not dependent on buffer conditions. Solution scattering showed that the x-ray radius of gyration Rg increased with salt concentration, whereas the neutron Rg values remained unchanged with temperature. The x-ray and neutron distance distribution curves P(r) revealed two peaks, M1 and M2, whose positions were unchanged in different buffers to indicate conformational stability. Constrained atomistic scattering modeling revealed predominantly asymmetric solution structures for both antibodies with extended hinge structures. Both structures were similar to the only known crystal structure of full-length human IgG1. The Fab conformations in both structures were suitably positioned to permit the Fc region to bind readily to its FcγR and C1q ligands without steric clashes, unlike human IgG4. Our molecular models for human IgG1 explain its immune activities, and we discuss its stability and function for therapeutic applications. PMID:25659433

  10. Aggregation of human polymorphonuclear leucocytes during phagocytosis of bacteria.

    PubMed Central

    Henricks, P A; van der Tol, M E; Verhoef, J

    1984-01-01

    The process of aggregation of human polymorphonuclear leucocytes (PMN) during the uptake of bacteria was studied. Radiolabelled S. aureus were opsonized in different sera, washed, resuspended in buffer and added to the PMN. Uptake of the bacteria and aggregation of the PMN were measured simultaneously. Maximal aggregation occurred within 6 min, when 5 X 10(6) PMN had phagocytosed 2.5 X 10(8) S. aureus. Also the effects of serum concentrations and different sera for opsonization of the bacteria on PMN aggregation were studied. Despite normal uptake, aggregation of PMN was low when bacteria were opsonized in complement-deficient sera. Furthermore when PMN were treated with pronase to inactivate complement receptors on the cell surface of the PMN, and bacteria preopsonized in immune serum were added, no change in uptake occurred, although the degree of aggregation halved compared to control PMN. So, interaction between the bacteria and the complement receptor of the PMN cell membrane is needed for triggering the process of aggregation. By using dansylcadaverin and diphenylamine to modulate lysosomal enzyme release, azide or PMN from a chronic granulomatous disease patient to study the effect of the formation of oxygen species, and theophylline, DB-cAMP or 8 Br-cAMP to increase cAMP levels, it was concluded that aggregation of PMN during phagocytosis was not dependent on oxygen metabolism, degranulation or cAMP levels of PMN. PMID:6086503

  11. Antitumor effects of L6, an IgG2a antibody that reacts with most human carcinomas.

    PubMed Central

    Hellström, I; Beaumier, P L; Hellström, K E

    1986-01-01

    Mouse monoclonal antibody L6 (IgG2a subtype) recognizes a ganglioside antigen expressed at the surface of cells from human non-small-cell lung carcinomas, breast carcinomas, and colon carcinomas. We now show that this antibody can lyse L6 antigen-positive human tumor cells in the presence of Leu-11b-positive human lymphocytes (i.e., mediate antibody-dependent cellular cytotoxicity) or human serum (mediate complement-dependent cytotoxicity) and that it can inhibit the outgrowth of an L6 antigen-positive human tumor transplanted onto nude mice. PMID:3462743

  12. Specificity of human anti-carbohydrate IgG antibodies as probed with polyacrylamide-based glycoconjugates.

    PubMed

    Smorodin, E P; Kurtenkov, O A; Sergeyev, B L; Pazynina, G V; Bovin, N V

    2004-01-01

    The TF, Tn, and SiaTn glycotopes are frequently expressed in cancer-associated mucins. Antibodies to these glycotopes were found in human serum. A set of polyacrylamide (PAA)--based glycoconjugates was applied to the direct and competitive enzyme-linked immunosorbent assays (ELISA) to characterize the specificity of serum IgG antibodies. The anti-TF, -Tn and -SiaTn IgG were affinity purified from serum of cancer patients and characterized using PAA-conjugates and free saccharides. The anti-TF and -Tn antibodies were shown to be specific. The anti-TF IgG bound both Galbeta1-3GalNAcalpha- and Galbeta1-3GalNAcbeta-PAA, the latter was three-four times more effective inhibitor of antibody binding. The anti-Tn IgG reacted only with GalNAcalpha-PAA. The anti-SiaTn IgG cross-reacted with Tn-PAA but SiaTn-PAA was five-six times more effective inhibitor in a competitive assay. The IC50 values for PAA-conjugates with the corresponding antibodies typically ranged from 2 to 5 x 10(-8) M. The antibodies display a low specificity to mucin-type glycoconjugates in comparison with PAA-conjugates as was shown for mucins isolated from human malignant tumor tissues, ovine submaxillary mucin (OSM) and asialo-OSM. The unusual IgG-antibody specificity to GalNAcbeta and GalNAcbeta1-3GalNAcbeta ligands was found in human serum. PMID:15001840

  13. Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein.

    PubMed

    Xu, Juan; Zhang, Chunhua

    2016-05-01

    Enterovirus (EV71) can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 (VP1), plays a critical role in the pathogenicity of EV71. High level expression and secretion of VP1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP1 DNA vaccines, including wt-VP1, tPA-VP1, VP1-d, VP1-hFc and VP1-mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different (P<0.05). In this study, we further investigated the protein levels of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc (hFc) to VP1. VP1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VP1-hFc protein for additional studies. PMID:27533931

  14. Human IgG Fc promotes expression, secretion and immunogenicity of enterovirus 71 VP1 protein

    PubMed Central

    Xu, Juan; Zhang, Chunhua

    2016-01-01

    Abstract Enterovirus (EV71) can cause severe neurological diseases, but the underlying pathogenesis remains unclear. The capsid protein, viral protein 1 (VP1), plays a critical role in the pathogenicity of EV71. High level expression and secretion of VP1 protein are necessary for structure, function and immunogenicity in its natural conformation. In our previous studies, 5 codon-optimized VP1 DNA vaccines, including wt-VP1, tPA-VP1, VP1-d, VP1-hFc and VP1-mFc, were constructed and analyzed. They expressed VP1 protein, but the levels of secretion and immunogenicity of these VP1 constructs were significantly different (P<0.05). In this study, we further investigated the protein levels of these constructs and determined that all of these constructs expressed VP1 protein. The secretion level was increased by including a tPA leader sequence, which was further increased by fusing human IgG Fc (hFc) to VP1. VP1-hFc demonstrated the most potent immunogenicity in mice. Furthermore, hFc domain could be used to purify VP1-hFc protein for additional studies.

  15. The N-glycan Glycoprotein Deglycosylation Complex (Gpd) from Capnocytophaga canimorsus Deglycosylates Human IgG

    PubMed Central

    Renzi, Francesco; Manfredi, Pablo; Mally, Manuela; Moes, Suzette; Jenö, Paul; Cornelis, Guy R.

    2011-01-01

    C. canimorsus 5 has the capacity to grow at the expenses of glycan moieties from host cells N-glycoproteins. Here, we show that C. canimorsus 5 also has the capacity to deglycosylate human IgG and we analyze the deglycosylation mechanism. We show that deglycosylation is achieved by a large complex spanning the outer membrane and consisting of the Gpd proteins and sialidase SiaC. GpdD, -G, -E and -F are surface-exposed outer membrane lipoproteins. GpdDEF could contribute to the binding of glycoproteins at the bacterial surface while GpdG is a endo-β-N-acetylglucosaminidase cleaving the N-linked oligosaccharide after the first N-linked GlcNAc residue. GpdC, resembling a TonB-dependent OM transporter is presumed to import the oligosaccharide into the periplasm after its cleavage from the glycoprotein. The terminal sialic acid residue of the oligosaccharide is then removed by SiaC, a periplasm-exposed lipoprotein in direct contact with GpdC. Finally, most likely degradation of the oligosaccharide proceeds sequentially from the desialylated non reducing end by the action of periplasmic exoglycosidases, including β-galactosidases, β-N-Acetylhexosaminidases and α-mannosidases. PMID:21738475

  16. The Thomsen-Friedenreich antigen and alphaGal-specific human IgG glycoforms: concanavalin A reactivity and relation to survival of cancer patients.

    PubMed

    Kodar, Kristel; Kurtenkov, Oleg; Klaamas, Kersti

    2009-01-01

    Glycan structures of IgG strongly influence the affinity for Fcgamma receptors and antibody effector functions. However, no particular attention has been paid yet to the glycosylation of tumor antigen-specific IgG. The objectives of this study were (i) to investigate the concanavalin A lectin (ConA) reactivity of human anti-Thomsen-Friedenreich (TF) and anti-alphaGal specific IgG in gastric cancer patients and healthy controls and (ii) to evaluate whether the ConA-reactivity of anti-TF and anti-alphaGal specific IgG is associated with the survival rate of patients with cancer. Total IgG was purified from the sera of patients with gastric cancer and healthy blood donors. The anti-TF and anti-alphaGal glycotope specific IgG were detected with ELISA using synthetic saccharide-polyacrylamide conjugates as antigen. In parallel plate, the ConA reactivity of the anti-TF or anti-alphaGal IgG was determined and the ConA index was calculated. Results show that serum anti-TF specific IgG antibodies of patients with cancer contain significantly higher content of ConA positive IgG glycoform compared to IgG of controls. No correlation between the ConA reactivity of anti-TF IgG and anti-alphaGal IgG was observed. High level of anti-TF IgG ConA reactivity was associated with a significantly lower survival rate of patients with gastric cancer. PMID:19860583

  17. Non-Immune Binding of Human IgG to M-Related Proteins Confers Resistance to Phagocytosis of Group A Streptococci in Blood

    PubMed Central

    Courtney, Harry S.; Li, Yi

    2013-01-01

    The non-immune binding of immunoglobulins by bacteria is thought to contribute to the pathogenesis of infections. M-related proteins (Mrp) are group A streptococcal (GAS) receptors for immunoglobulins, but it is not known if this binding has any impact on virulence. To further investigate the binding of immunoglobulins to Mrp, we engineered mutants of an M type 4 strain of GAS by inactivating the genes for mrp, emm, enn, sof, and sfbX and tested these mutants in IgG-binding assays. Inactivation of mrp dramatically decreased the binding of human IgG, whereas inactivation of emm, enn, sof, and sfbx had only minor effects, indicating that Mrp is a major IgG-binding protein. Binding of human immunoglobulins to a purified, recombinant form of Mrp indicated that it selectively binds to the Fc domain of human IgG, but not IgA or IgM and that it preferentially bound subclasses IgG1>IgG4>IgG2>IgG3. Recombinant proteins encompassing different regions of Mrp were engineered and used to map its IgG-binding domain to its A-repeat region and a recombinant protein with 3 A-repeats was a better inhibitor of IgG binding than one with a single A-repeat. A GAS mutant expressing Mrp with an in-frame deletion of DNA encoding the A-repeats had a dramatically reduced ability to bind human IgG and to grow in human blood. Mrp exhibited host specificity in binding IgG; human IgG was the best inhibitor of the binding of IgG followed by pig, horse, monkey, and rabbit IgG. IgG from goat, mouse, rat, cow, donkey, chicken, and guinea pig were poor inhibitors of binding. These findings indicate that Mrp preferentially binds human IgG and that this binding contributes to the ability of GAS to resist phagocytosis and may be a factor in the restriction of GAS infections to the human host. PMID:24205299

  18. Effector functions of a monoclonal aglycosylated mouse IgG2a: binding and activation of complement component C1 and interaction with human monocyte Fc receptor.

    PubMed

    Leatherbarrow, R J; Rademacher, T W; Dwek, R A; Woof, J M; Clark, A; Burton, D R; Richardson, N; Feinstein, A

    1985-04-01

    Aglycosylated monoclonal anti-DNP mouse IgG2a produced in the presence of tunicamycin was compared with the native monoclonal IgG2a with respect to its ability to interact with the first component of complement, C1, and to compete with human IgG for binding to human monocyte Fc receptors. The aglycosylated IgG2a was found to bind subcomponent C1q with an equivalent capacity to the native IgG2a, but the dissociation constant was found to be increased three-fold. When activation of C1 by the glycosylated and aglycosylated IgG2a was compared, the rate of C1 activation by the aglycosylated IgG2a was reduced approximately three-fold. In contrast aglycosylation was accompanied by a large decrease (greater than or equal to 50-fold) in the apparent binding constant of monomeric IgG2a to human monocytes. The data suggest that the aglycosylated IgG2a has a structure which differs in the CH2 domain from the native IgG2a, and that the heterogeneous N-linked oligosaccharides of this monoclonal IgG2a which occur at a conserved position in the CH2 domain play a role in maintaining the integrity of its monocyte-binding site. This lack of monocyte binding may result either from a localized conformational change occurring in a single CH2 domain or from an alteration in the CH2-CH2 cross-domain architecture which is normally structured by a pair of opposing and interacting oligosaccharides. The minimal changes in C1q binding and C1 activation suggest that the oligosaccharides are, at most, indirectly involved in these events. PMID:4033665

  19. Inhibition of platelet-aggregating activity in thrombotic thrombocytopenic purpura plasma by normal adult immunoglobulin G.

    PubMed Central

    Lian, E C; Mui, P T; Siddiqui, F A; Chiu, A Y; Chiu, L L

    1984-01-01

    Plasma from patients with thrombotic thrombocytopenic purpura (TTP) caused the aggregation of autologous and homologous platelets, and effect which was inhibited by normal plasma. IgG purified from seven normal adults at a concentration of 0.7 mg/ml completely inhibited the platelet aggregation induced by plasma obtained from two TTP patients with active disease. The inhibition of platelet aggregation by human adult IgG was concentration dependent, and the inhibitory activity of human IgG was neutralized by rabbit antihuman IgG. Fab fragments inhibited the TTP plasma-induced platelet aggregation as well as intact IgG, whereas Fc fragments had no effect. Platelet aggregation caused by ADP, collagen, epinephrine, or thrombin was not affected by purified human IgG. The prior incubation of IgG with TTP plasma caused a significantly greater reduction of platelet aggregation by TTP plasma than that of IgG and platelet suspension, suggesting that the IgG inhibits TTP plasma-induced platelet aggregation through direct interaction with platelet aggregating factor in TTP plasma. IgG obtained initially from five infants and young children under the age of 4 yr did not possess any inhibitory activity. When one of the children reached 3 yr of age, his IgG inhibited the aggregation induced by one TTP plasma, but not that caused by another plasma. The IgG procured from the same boy at 4 yr of age inhibited the aggregation induced by both TTP plasmas. The IgG purified from the TTP plasma during active disease failed to inhibit the aggregation caused by the same plasma. After recovery, however, the IgG effectively inhibited aggregation. These observations suggest that platelet-aggregating factors present in the TTP plasma are heterogeneous in nature and that the IgG present in the normal adult plasma, which inhibits the TTP plasma-induced platelet aggregation, may be partially responsible for the success of plasma infusion therapy in TTP. Images PMID:6538207

  20. Epitope specificity of rabbit immunoglobulin G (IgG) elicited by pneumococcal type 23F synthetic oligosaccharide- and native polysaccharide-protein conjugate vaccines: comparison with human anti-polysaccharide 23F IgG.

    PubMed Central

    Alonso de Velasco, E; Verheul, A F; van Steijn, A M; Dekker, H A; Feldman, R G; Fernández, I M; Kamerling, J P; Vliegenthart, J F; Verhoef, J; Snippe, H

    1994-01-01

    Streptococcus pneumoniae type 23F capsular polysaccharide (PS23F) consitss of a repeating glycerol-phosphorylated branched tetrasaccharide. The immunogenicities of the following related antigens were investigated: (i) a synthetic trisaccharide comprising the backbone of one repeating unit, (ii) a synthetic tetrasaccharide comprising the complete repeating unit, and (iii) native PS23F (all three conjugated to keyhole limpet hemocyanin [KLH]) and (iv) formalin-killed S. pneumoniae 23F. All antigens except the trisaccharide-KLH conjugate induced relatively high anti-PS23F antibody levels in rabbits. The epitope specificity of such antibodies was then studied by means of an inhibition immunoassay. The alpha(1-->2)-linked L-rhamnose branch was shown to be immunodominant for immunoglobulin G (IgG) induced by tetrasaccharide-KLH, PS23F-KLH, and killed S. pneumoniae 23F: in most sera L-rhamnose totally inhibited the binding of IgG to PS23F. Thus, there appears to be no major difference in epitope specificity between IgG induced by tetrasaccharide-KLH and that induced by antigens containing the polymeric form of PS23F. Human anti-PS23F IgG (either vaccine induced or naturally acquired) had a different epitope specificity: none of the inhibitors used, including L-rhamnose and tetrasaccharide-KLH, exhibited substantial inhibition. These observations suggest that the epitope recognized by human IgG on PS23F is larger than the epitope recognized by rabbit IgG. Both human and rabbit antisera efficiently opsonized type 23F pneumococci, as measured in a phagocytosis assay using human polymorphonuclear leukocytes. PMID:7509318

  1. Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability.

    PubMed

    Edgeworth, Matthew J; Phillips, Jonathan J; Lowe, David C; Kippen, Alistair D; Higazi, Daniel R; Scrivens, James H

    2015-12-01

    Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion-mobility mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins. PMID:26482340

  2. [Monoclonal human immunoglobulin (IgG lambda) with antiethinylestradiol activity, oral contraceptives, and arterial pulmonary thrombosis].

    PubMed

    Beaumont, J L; Lemort, N

    1975-06-23

    In a 36-year-old woman taking an oral contraceptive containing 50 mug of ethinyloestradiol each day, a pulmonary arterial thrombosis and a monoclonal gammapathia were associated. The monoclonal IgI lambda Mai... was prepared. When purified, this IgG lambda binds ethinyloestradiol with strong affinity (Ka= 2.7 times 10(7)M-1) and also 17-beta-oestradiol with a little less affinity (Ka = 0.4 times 10(7)M-1. For those ligands each IgG lambda Mai... molecule has two sites of same affinity and specificity so that a Scatchard plot of the experimental values gives a straight line. It is likely that the antibody sites of the IgG lambda Mai... are the binding sites. These facts support the hypothesis of an immunological mechanism of the thromboembolic disease which may be induced by oral contraceptives. PMID:808320

  3. Glomerular lesions induced in the rabbit by physicochemically altered homologous IgG.

    PubMed Central

    Cavalot, F.; Miyata, M.; Vladutiu, A.; Terranova, V.; Dubiski, S.; Burlingame, R.; Tan, E.; Brentjens, J.; Milgrom, F.; Andres, G.

    1992-01-01

    Immunization of rabbits with physicochemically altered homologous or even autologous IgG induces formation of antibodies combining with IgG of rabbit and of foreign species. Cardiac but not renal lesions were reported in such animals. This study examined the nephritogenic potential of the immune response to cationized or heat-aggregated homologous IgG of b9 or b4 allotype in rabbits of the b4 allotype. Rabbits injected with either b9 or b4 cationized IgG produced antibodies reactive with rabbit and human IgG and with histones; they also developed abnormal glomerular deposits of IgG b4 and C3 corresponding to alterations of the glomerular basement membranes (GBM). Rabbits injected with either b9 or b4 aggregated IgG developed antibodies reactive with rabbit and human IgG and abnormal glomerular deposits of IgG b4 and C3 in the GBM and in the mesangium with subendothelial and mesangial electron-dense deposits. Some rabbits in both groups had proliferative and exudative glomerulonephritis and proteinuria. The results showed that immunization of rabbits with physicochemically altered homologous IgG induces an immune response to rabbit and human IgG and to histones as well as glomerular deposits of autologous IgG and C3 and other glomerular lesions. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 Figure 23 Figure 24 Figure 25 Figure 26 Figure 27 Figure 28 Figure 29 Figure 30 PMID:1546743

  4. Effects of human urine on aggregation of calcium oxalate crystals.

    PubMed

    Springmann, K E; Drach, G W; Gottung, B; Randolph, A D

    1986-01-01

    The importance of aggregation in calcium oxalate urolithiasis, although not fully understood, has long been postulated. Previous investigators of calcium oxalate crystal aggregation have applied static crystallization rather than continuous flow techniques to their studies. We describe the use of a Couette agglomerator in series with our previously reported continuous flow mixed suspension-mixed product removal crystallization system. We compared synthetic urine controls with 5 per cent volume-in-volume human urine additions from normal persons or patients with calcium oxalate stones. There was no significant difference in nucleation, linear crystal growth rate or total crystal mass between normal persons and those with stones. Control nucleation rate was significantly higher than in either human urine addition group. Comparison of aggregator particle size distributions revealed significant differences in aggregation among the control, normal and stone groups. We concluded that urine inhibitors to aggregation are somewhat deficient in patients with stones, resulting in the generation of larger particle masses or eventually stones. PMID:3941471

  5. Multi-Angle Effector Function Analysis of Human Monoclonal IgG Glycovariants

    PubMed Central

    Dashivets, Tetyana; Thomann, Marco; Rueger, Petra; Knaupp, Alexander; Buchner, Johannes; Schlothauer, Tilman

    2015-01-01

    Therapeutic performance of recombinant antibodies relies on two independent mechanisms: antigen recognition and Fc-mediated antibody effector functions. Interaction of Fc-fragment with different FcR triggers antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity and determines longevity of the antibody in serum. In context of therapeutic antibodies FcγRs play the most important role. It has been demonstrated that the Fc-attached sugar moiety is essential for IgG effector functionality, dictates its affinity to individual FcγRs and determines binding to different receptor classes: activating or inhibitory. In this study, we systematically analyze effector functions of monoclonal IgG1 and its eight enzymatically engineered glycosylation variants. The analysis of interaction of glycovariants with FcRs was performed for single, as well as for antigen-bound antibodies and IgGs in a form of immune complex. In addition to functional properties we addressed impact of glycosylation on the structural properties of the tested glycovariants. We demonstrate a clear impact of glycosylation pattern on antibody stability and interaction with different FcγRs. Consistent with previous reports, deglycosylated antibodies failed to bind all Fcγ-receptors, with the exception of high affinity FcγRI. The FcγRII and FcγRIIIa binding activity of IgG1 was observed to depend on the galactosylation level, and hypergalactosylated antibodies demonstrated increased receptor interaction. Sialylation did not decrease the FcγR binding of the tested IgGs; in contrast, sialylation of antibodies improved binding to FcγRIIa and IIb. We demonstrate that glycosylation influences to some extent IgG1 interaction with FcRn. However, independent of glycosylation pattern the interaction of IgG1 with a soluble monomeric target surprisingly resulted in an impaired receptor binding. Here, we demonstrate, that immune complexes (IC), induced by multimeric ligand, compensated for the

  6. Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

    PubMed Central

    van den Bremer, Ewald TJ; Beurskens, Frank J; Voorhorst, Marleen; Engelberts, Patrick J; de Jong, Rob N; van der Boom, Burt G; Cook, Erika M; Lindorfer, Margaret A; Taylor, Ronald P; van Berkel, Patrick HC; Parren, Paul WHI

    2015-01-01

    Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential. PMID:26037225

  7. Three-Dimensional Structure of the Human Myeloma IgG2

    PubMed Central

    Ryazantsev, Sergey; Tischenko, Vladimir; Nguyen, Christopher; Abramov, Vyacheslav; Zav'yalov, Vladimir

    2013-01-01

    Human immunoglobulin G, subclass 2 (hIgG2), plays an important role in immunity to bacterial pathogens and in numerous pathological conditions. However, there is a lack of information regarding the three-dimensional (3D) structure of the hIgG2 molecule. We used electron microscopy (EM), differential scanning microcalorimetry (DSC) and fluorescence for structural analysis of the hIgG2. DSC and fluorescence indicated two types of interaction between CH1 domain of Fab (antigen-binding fragment/subunit) and CH2 domain of Fc (complement fixation fragment/subunit) simultaneously present in the sample: close interaction, which increases the thermostability of both, CH1 and CH2 domains, and weak (or no) interaction, which is typical for most IgGs but not hIgG2. Thermodynamics could not determine if both types of interactions are present within a single molecule. To address this question, EM was used. We employed a single-particle reconstruction and negative staining approach to reveal the three-dimensional structure of the hIgG2. A three-dimensional model of hIgG2 was created at 1.78 nm resolution. The hIgG2 is asymmetrical: one Fab subunit is in close proximity to the upper portion of the Fc subunit (CH2 domain) and the other Fab is distant from Fc. The plane of Fab subunits is nearly perpendicular to Fc. EM structure of the hIgG2 is in good agreement with thermodynamic data: a Fab distant from Fc should exhibit a lower melting temperature while a Fab interacting with Fc should exhibit a higher melting temperature. Both types of Fab subunits exist within one molecule resembling an A/B hIgG2 isoform introduced earlier on physicochemical level by Dillon et al. (2008). In such an arrangement, the access to the upper portion of Fc subunit is partially blocked by a Fab subunit. That might explain for instance why hIgG2 mildly activates complement and binds poorly to Fc receptors. Understanding of the three-dimensional structure of the hIgG2 should lead to better design of

  8. [PHEMA/PEI]-Cu(II) based immobilized metal affinity chromatography cryogels: Application on the separation of IgG from human plasma.

    PubMed

    Bakhshpour, Monireh; Derazshamshir, Ali; Bereli, Nilay; Elkak, Assem; Denizli, Adil

    2016-04-01

    The immobilized metal-affinity chromatography (IMAC) has gained significant interest as a widespread separation and purification tool for therapeutic proteins, nucleic acids and other biological molecules. The enormous potential of IMAC for proteins with natural surface exposed-histidine residues and for recombinant proteins with histidine clusters. Cryogels as monolithic materials have recently been proposed as promising chromatographic adsorbents for the separation of biomolecules in downstream processing. In the present study, IMAC cryogels have been synthesized and utilized for the adsorption and separation of immunoglobulin G (IgG) from IgG solution and whole human plasma. For this purpose, Cu(II)-ions were coupled to poly(hydroxyethyl methacrylate) PHEMA using poly(ethylene imine) (PEI) as the chelating ligand. In this study the cryogels formation optimized by the varied proportion of PEI from 1% to 15% along with different amounts of Cu (II) as chelating metal. The prepared cryogels were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The [PHEMA/PEI]-Cu(II) cryogels were assayed for their capability to bind the human IgG from aqueous solutions. The IMAC cryogels were found to have high affinity toward human IgG. The adsorption of human IgG was investigated onto the PHEMA/PEI cryogels with (10% PEI) and the concentration of Cu (II) varied as 10, 50, 100 and 150 mg/L. The separation of human IgG was achieved in one purification step at pH7.4. The maximum adsorption capacity was observed at the [PHEMA/PEI]-Cu(II) (10% PEI) with 72.28 mg/g of human IgG. The purification efficiency and human IgG purity were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). PMID:26838913

  9. Lonomia obliqua venomous secretion induces human platelet adhesion and aggregation.

    PubMed

    Berger, Markus; Reck, José; Terra, Renata M S; Beys da Silva, Walter O; Santi, Lucélia; Pinto, Antônio F M; Vainstein, Marilene H; Termignoni, Carlos; Guimarães, Jorge A

    2010-10-01

    The caterpillar Lonomia obliqua is a venomous animal that causes numerous accidents, especially in southern Brazil, where it is considered a public health problem. The clinical manifestations include several haemostatic disturbances that lead to a hemorrhagic syndrome. Considering that platelets play a central role in hemostasis, in this work we investigate the effects of L. obliqua venomous secretion upon blood platelets responses in vitro. Results obtained shows that L. obliqua venom directly induces aggregation and ATP secretion in human washed platelets in a dose-dependent manner. Electron microscopy studies clearly showed that the venomous bristle extract was also able to produce direct platelets shape change and adhesion as well as activation and formation of platelet aggregates. Differently from other enzyme inhibitors, the venom-induced platelet aggregation was significatively inhibited by p-bromophenacyl bromide, a specific inhibitor of phospholipases A2. Additional experiments with different pharmacological antagonists indicate that the aggregation response triggered by the venom active components occurs through a calcium-dependent mechanism involving arachidonic acid metabolite(s) of the cyclooxygenase pathway and activation of phosphodiesterase 3A, an enzyme that leads to the consumption of intracellular cAMP content. It was additionally found that L. obliqua-induced platelet aggregation was independent of ADP release. Altogether, these findings are in line with the need for a better understanding of the complex hemorrhagic syndrome resulting from the envenomation caused by L. obliqua caterpillars, and can also give new insights into the management of its clinical profile. PMID:20157842

  10. A Unique Report: Development of Super Anti-Human IgG Monoclone with Optical Density Over Than 3

    PubMed Central

    Aghebati Maleki, Leili; Baradaran, Behzad; Abdolalizadeh, Jalal; Ezzatifar, Fatemeh; Majidi, Jafar

    2013-01-01

    Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity with IgM & IgA by ELISA and confirmed by immunoblotting. The subclasses of the selected mAbs were determined. The best clone was injected intraperitoneally to some pristane-injected mice. Anti-IgG mAb was purified from the animals' ascitic fluid by Ion exchange chromatography and then, mAb was conjugated with HRP. Results: In the present study, over than 50 clones were obtained that 1 clone had optical density over than 3. We named this clone as supermonoclone which was selected for limiting dilution. The result of the immunoblotting, showed sharp band in IgG position and did not show any band in IgM&IgA position. Conclusion: Based on the findings of this study, the conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases. PMID:24312857

  11. Interaction of human IgG chimeric antibodies with the human FcRI and FcRII receptors: requirements for antibody-mediated host cell-target cell interaction.

    PubMed

    Walker, M R; Woof, J M; Brüggemann, M; Jefferis, R; Burton, D R

    1989-04-01

    Chimeric monoclonal antibodies (McAb), specific for the hapten 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP), expressing human IgG1, IgG2, IgG3 and IgG4 subclass constant domains, have been examined for their ability to interact with the human FcRII receptor. Human red blood cells (RBC) sensitized by each of these McAbs have been assayed for their ability to form rosettes with the human histiocytic lymphoma U937 cell line, human B cell line Daudi and erythroblastoid K562 cell line. IgG1 and IgG3 sensitized RBC formed significant rosettes with the FcR- and FcRII+ Daudi and K562 cell lines, the percentage of cells forming rosettes being directly proportional to the degree of sensitization of the RBC. Bromelin treating Daudi cells did not alter this pattern of reactivity, whereas bromelin treated FcRI+ and FcRII+ U937 cells formed significant resettes with IgG1, IgG3 and IgG4 sensitized RBC, demonstrating a difference in the IgG subclass specificity between human FcRI and FcRII. Murine IgG2b anti-NIP sensitized RBC did not form rosettes with any cell line tested; however, RBC sensitized by some members of a panel of murine IgG1 McAb, specific for the glycophorin A molecule, were able to form rosettes with Daudi, U937 and K562 cells. This interaction was enhanced by bromelin treating the Daudi or U937 cells and can be correlated to the disposition of the epitopes recognized, relative to the target cell membrane, those McAbs recognizing epitopes furthest from the RBC surface being most effective in interacting with FcRII. The data are interpreted in terms of a simple model for antibody-mediated cell--cell interaction. PMID:2716734

  12. Human anti-Aβ IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

    PubMed

    Welzel, Alfred T; Williams, Angela D; McWilliams-Koeppen, Helen P; Acero, Luis; Weber, Alfred; Blinder, Veronika; Mably, Alex; Bunk, Sebastian; Hermann, Corinna; Farrell, Michael A; Ehrlich, Hartmut J; Schwarz, Hans P; Walsh, Dominic M; Solomon, Alan; O'Nuallain, Brian

    2012-01-01

    Soluble non-fibrillar assemblies of amyloid-beta (Aβ) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer's disease (AD). Anti-Aβ immunotherapy is a promising and advanced therapeutic strategy, but the precise Aβ species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aβ conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aβ dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aβ extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aβ's conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody's nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aβ monomers and peptide fragments. The antibody's lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aβ NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aβ NAbs are warranted. PMID:23209707

  13. Human Anti-Aβ IgGs Target Conformational Epitopes on Synthetic Dimer Assemblies and the AD Brain-Derived Peptide

    PubMed Central

    Welzel, Alfred T.; Williams, Angela D.; McWilliams-Koeppen, Helen P.; Acero, Luis; Weber, Alfred; Blinder, Veronika; Mably, Alex; Bunk, Sebastian; Hermann, Corinna; Farrell, Michael A.; Ehrlich, Hartmut J.; Schwarz, Hans P.; Walsh, Dominic M.; Solomon, Alan; O’Nuallain, Brian

    2012-01-01

    Soluble non-fibrillar assemblies of amyloid-beta (Aβ) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer’s disease (AD). Anti-Aβ immunotherapy is a promising and advanced therapeutic strategy, but the precise Aβ species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aβ conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aβ dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aβ extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aβ’s conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody’s nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aβ monomers and peptide fragments. The antibody’s lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aβ NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aβ NAbs are warranted. PMID:23209707

  14. Differential antibody isotype reactivity to specific antigens in human lymphatic filariasis: gp15/400 preferentially induces immunoglobulin E (IgE), IgG4, and IgG2.

    PubMed Central

    Yazdanbakhsh, M; Paxton, W A; Brandenburg, A; Van Ree, R; Lens, M; Partono, F; Maizels, R M; Selkirk, M E

    1995-01-01

    Lymphatic filarial infection in humans is associated with a strong skewing of the immune response towards the TH2 arm, with prominent interleukin 4-producing cells and elevated levels of immunoglobulin G4 (IgG4) and IgE antibodies in peripheral blood. To determine how such a generalized TH2 imbalance governs responses to individual parasite antigens, the profiles of isotypes of antibodies to two recombinant proteins of Brugia spp. were studied. One molecule was the C-terminal portion of the filarial heat shock protein 70 (Bpa-26), representative of a cytoplasmic protein, and the second antigen was a single unit of the tandem repeats of a Brugia polypeptide (BpL-4), a secreted product which is prominently exposed to the immune system. Serum samples from 146 individuals resident in areas in which brugian filariasis is endemic were used, and it was found that whereas the levels of IgG1 and IgG3 responses to both Bpa-26 and BpL-4 were high, IgG4 and IgE antibodies to only BpL-4, not to Bpa-26, were prominent. Thus, an antigen which is chronically exposed to the immune system elicited a TH2-dependent isotype switch, as manifested by increased IgG4 and IgE responses. Moreover, IgG4 and IgE responses to BpL-4 showed a strong negative association, suggesting that mediators other than interleukin 4 must be responsible for such differential regulation of these two isotypes. When the data were analyzed as a function of clinical status, a striking association between elevated levels of IgG3 antibodies to Bpa-26 and manifestation of chronic obstructive disease was found; elephantiasis patients showed significantly higher levels of IgG3 antibodies to Bpa-26 than microfilaremics and asymptomatic amicrofilaremics. This indicates that an imbalance of isotypes of antibodies to particular filarial antigens might play a role in the pathogenesis of chronic disease. PMID:7558279

  15. Evaluation of the PANBIO Brucella Immunoglobulin G (IgG) and IgM Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Brucellosis

    PubMed Central

    Araj, George F.; Kattar, Mireille M.; Fattouh, Layla G.; Bajakian, Kayane O.; Kobeissi, Sara A.

    2005-01-01

    PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) were assessed against Brucella standard agglutination tube and Coombs tests. The sensitivities of ELISA IgG and IgM were 91% and 100%, respectively, while the specificity was 100% for both. These ELISAs are simple, rapid, and reliable for the diagnosis of human brucellosis. PMID:16275951

  16. Evaluation of the PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays for diagnosis of human brucellosis.

    PubMed

    Araj, George F; Kattar, Mireille M; Fattouh, Layla G; Bajakian, Kayane O; Kobeissi, Sara A

    2005-11-01

    PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) were assessed against Brucella standard agglutination tube and Coombs tests. The sensitivities of ELISA IgG and IgM were 91% and 100%, respectively, while the specificity was 100% for both. These ELISAs are simple, rapid, and reliable for the diagnosis of human brucellosis. PMID:16275951

  17. An engineered diatom acting like a plasma cell secreting human IgG antibodies with high efficiency

    PubMed Central

    2012-01-01

    Background Although there are many different expression systems for recombinant production of pharmaceutical proteins, many of these suffer from drawbacks such as yield, cost, complexity of purification, and possible contamination with human pathogens. Microalgae have enormous potential for diverse biotechnological applications and currently attract much attention in the biofuel sector. Still underestimated, though, is the idea of using microalgae as solar-fueled expression system for the production of recombinant proteins. Results In this study, we show for the first time that completely assembled and functional human IgG antibodies can not only be expressed to high levels in algal systems, but also secreted very efficiently into the culture medium. We engineered the diatom Phaeodactylum tricornutum to synthesize and secrete a human IgG antibody against the Hepatitis B Virus surface protein. As the diatom P. tricornutum is not known to naturally secrete many endogenous proteins, the secreted antibodies are already very pure making extensive purification steps redundant and production extremely cost efficient. Conclusions Microalgae combine rapid growth rates with all the advantages of eukaryotic expression systems, and offer great potential for solar-powered, low cost production of pharmaceutical proteins. PMID:22970838

  18. Structural Basis for Fc[gamma]RIIa Recognition of Human IgG and Formation of Inflammatory Signaling Complexes

    SciTech Connect

    Ramsland, Paul A.; Farrugia, William; Bradford, Tessa M.; Sardjono, Caroline Tan; Esparon, Sandra; Trist, Halina M.; Powell, Maree S.; Tan, Peck Szee; Cendron, Angela C.; Wines, Bruce D.; Scott, Andrew M.; Hogarth, P. Mark

    2011-09-20

    The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. Fc{gamma}RIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of Fc{gamma}RIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of Fc{gamma}RIIa (Fc{gamma}RIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence Fc{gamma}RIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for Fc{gamma}RIIa (IV.3), Fc{gamma}RIIb (X63-21), and a pan Fc{gamma}RII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of Fc{gamma}RIIa and Fc{gamma}RIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of Fc{gamma}RIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.

  19. IgG and IgA with potential microbial-binding activity are expressed by normal human skin epidermal cells.

    PubMed

    Jiang, Dongyang; Ge, Jing; Liao, Qinyuan; Ma, Junfan; Liu, Yang; Huang, Jing; Wang, Chong; Xu, Weiyan; Zheng, Jie; Shao, Wenwei; Lee, Gregory; Qiu, Xiaoyan

    2015-01-01

    The innate immune system of the skin is thought to depend largely on a multi-layered mechanical barrier supplemented by epidermis-derived antimicrobial peptides. To date, there are no reports of antimicrobial antibody secretion by the epidermis. In this study, we report the expression of functional immunoglobulin G (IgG) and immunoglobulin A (IgA), previously thought to be only produced by B cells, in normal human epidermal cells and the human keratinocyte line HaCaT. While B cells express a fully diverse Ig, epidermal cell-expressed IgG or IgA showed one or two conservative VHDJH rearrangements in each individual. These unique VDJ rearrangements in epidermal cells were found neither in the B cell-derived Ig VDJ databases published by others nor in our positive controls. IgG and IgA from epidermal cells of the same individual had different VDJ rearrangement patterns. IgG was found primarily in prickle cells, and IgA was mainly detected in basal cells. Both epidermal cell-derived IgG and IgA showed potential antibody activity by binding pathogens like Staphylococcus aureus, the most common pathogenic skin bacteria, but the microbial-binding profile was different. Our data indicates that normal human epidermal cells spontaneously express IgG and IgA, and we speculate that these Igs participate in skin innate immunity. PMID:25625513

  20. Small-Angle X-ray Scattering Screening Complements Conventional Biophysical Analysis: Comparative Structural and Biophysical Analysis of Monoclonal Antibodies IgG1, IgG2, and IgG4

    PubMed Central

    Tian, Xinsheng; Langkilde, Annette E; Thorolfsson, Matthias; Rasmussen, Hanne B; Vestergaard, Bente

    2014-01-01

    A crucial step in the development of therapeutic monoclonal antibodies is the selection of robust pharmaceutical candidates and screening of efficacious protein formulations to increase the resistance toward physicochemical degradation and aggregation during processing and storage. Here, we introduce small-angle X-ray scattering (SAXS) to characterize antibody solution behavior, which strongly complements conventional biophysical analysis. First, we apply a variety of conventional biophysical techniques for the evaluation of structural, conformational, and colloidal stability and report a systematic comparison between designed humanized IgG1, IgG2, and IgG4 with identical variable regions. Then, the high information content of SAXS data enables sensitive detection of structural differences between three IgG subclasses at neutral pH and rapid formation of dimers of IgG2 and IgG4 at low pH. We reveal subclass-specific variation in intermolecular repulsion already at low and medium protein concentrations, which explains the observed improved stability of IgG1 with respect to aggregation. We show how excipients dramatically influence such repulsive effects, hence demonstrating the potential application of extensive SAXS screening in antibody selection, eventual engineering, and formulation development. PMID:24700358

  1. Protective role of mouse IgG1 in cryoglobulinaemia; insights from an animal model and relevance to human pathology.

    PubMed

    Chemouny, Jonathan Maurice; Hurtado-Nedelec, Margarita; Flament, Héloïse; Ben Mkaddem, Sanae; Daugas, Eric; Vrtovsnik, François; Berthelot, Laureline; Monteiro, Renato C

    2016-08-01

    Strait et al. described a novel mouse model of cryoglobulinaemia by challenging mice deficient in the immunoglobulin (Ig)G1 subclass (γ1(-) mice) with goat anti-mouse IgD [5]. The phenotype of wild-type mice was not remarkable, whereas γ1(-) mice developed IgG3 anti-goat IgG cryoglobulins as well as severe and lethal glomerulonephritis. Renal phenotype could not be rescued in γ1(-) mice by the deletion of C3, fragment crystalline γ receptor (FcγR) or J chain. On the other hand, early injection of IgG1, IgG2a or IgG2b inhibited the pathogenic effects of IgG3 in an antigen-dependent manner even in the absence of the FcγRIIb, an anti-inflammatory receptor. The authors concluded that the pathogenic role of IgG3 and the protective characteristic of IgG1 in this model were not explained by their abilities to bind to FcRs or effector molecules but are rather due to structural discrepancies enhancing the precipitation properties/solubility of IgG3/IgG1-containing immune complexes. The present article aims to discuss the current knowledge on IgG biology and the properties of IgGs explaining their differential propensity to acquire cryoglobulin activity. PMID:26410885

  2. Direct cellular delivery of human proteasomes to delay tau aggregation.

    PubMed

    Han, Dong Hoon; Na, Hee-Kyung; Choi, Won Hoon; Lee, Jung Hoon; Kim, Yun Kyung; Won, Cheolhee; Lee, Seung-Han; Kim, Kwang Pyo; Kuret, Jeff; Min, Dal-Hee; Lee, Min Jae

    2014-01-01

    The 26S proteasome is the primary machinery that degrades ubiquitin (Ub)-conjugated proteins, including many proteotoxic proteins implicated in neurodegeneraton. It has been suggested that the elevation of proteasomal activity is tolerable to cells and may be beneficial to prevent the accumulation of protein aggregates. Here we show that purified proteasomes can be directly transported into cells through mesoporous silica nanoparticle-mediated endocytosis. Proteasomes that are loaded onto nanoparticles through non-covalent interactions between polyhistidine tags and nickel ions fully retain their proteolytic activity. Cells treated with exogenous proteasomes are more efficient in degrading overexpressed human tau than endogenous proteasomal substrates, resulting in decreased levels of tau aggregates. Moreover, exogenous proteasome delivery significantly promotes cell survival against proteotoxic stress caused by tau and reactive oxygen species. These data demonstrate that increasing cellular proteasome activity through the direct delivery of purified proteasomes may be an effective strategy for reducing cellular levels of proteotoxic proteins. PMID:25476420

  3. Human IgG1 Responses to Surface Localised Schistosoma mansoni Ly6 Family Members Drop following Praziquantel Treatment

    PubMed Central

    Chalmers, Iain W.; Fitzsimmons, Colin M.; Brown, Martha; Pierrot, Christine; Jones, Frances M.; Wawrzyniak, Jakub M.; Fernandez-Fuentes, Narcis; Tukahebwa, Edridah M.; Dunne, David W.; Khalife, Jamal; Hoffmann, Karl F.

    2015-01-01

    Background The heptalaminate-covered, syncytial tegument is an important anatomical adaptation that enables schistosome parasites to maintain long-term, intravascular residence in definitive hosts. Investigation of the proteins present in this surface layer and the immune responses elicited by them during infection is crucial to our understanding of host/parasite interactions. Recent studies have revealed a number of novel tegumental surface proteins including three (SmCD59a, SmCD59b and Sm29) containing uPAR/Ly6 domains (renamed SmLy6A SmLy6B and SmLy6D in this study). While vaccination with SmLy6A (SmCD59a) and SmLy6D (Sm29) induces protective immunity in experimental models, human immunoglobulin responses to representative SmLy6 family members have yet to be thoroughly explored. Methodology/Principal Findings Using a PSI-BLAST-based search, we present a comprehensive reanalysis of the Schistosoma mansoni Ly6 family (SmLy6A-K). Our examination extends the number of members to eleven (including three novel proteins) and provides strong evidence that the previously identified vaccine candidate Sm29 (renamed SmLy6D) is a unique double uPAR/Ly6 domain-containing representative. Presence of canonical cysteine residues, signal peptides and GPI-anchor sites strongly suggest that all SmLy6 proteins are cell surface-bound. To provide evidence that SmLy6 members are immunogenic in human populations, we report IgG1 (as well as IgG4 and IgE) responses against two surface-bound representatives (SmLy6A and SmLy6B) within a cohort of S. mansoni-infected Ugandan males before and after praziquantel treatment. While pre-treatment IgG1 prevalence for SmLy6A and SmLy6B differs amongst the studied population (7.4% and 25.3% of the cohort, respectively), these values are both higher than IgG1 prevalence (2.7%) for a sub-surface tegumental antigen, SmTAL1. Further, post-treatment IgG1 levels against surface-associated SmLy6A and SmLy6B significantly drop (p = 0.020 and p < 0

  4. Goat anti-rabbit IgG conjugated fluorescent dye-doped silica nanoparticles for human breast carcinoma cell recognition.

    PubMed

    Chen, Min-Yan; Chen, Ze-Zhong; Wu, Ling-Ling; Tang, Hong-Wu; Pang, Dai-Wen

    2013-11-12

    We report an indirect method for cancer cell recognition using photostable fluorescent silica nanoprobes as biological labels. The dye-doped fluorescent silica nanoparticles were synthesized using the water-in-oil (W/O) reverse microemulsion method. The silica matrix was produced by the controlled hydrolysis of tetraethylorthosilicate (TEOS) in water nanodroplets with the initiation of ammonia (NH3·H2O). Fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate conjugated with dextran (RBITC-Dextran) was doped in silica nanoparticles (NPs) with a size of 60 ± 5 nm as a fluorescent signal element by covalent bonding and steric hindrance, respectively. The secondary antibody, goat anti-rabbit IgG, was conjugated on the surface of the PEG-terminated modified FITC-doped or RBITC-Dextran-doped silica nanoparticles (PFSiNPs or PBSiNPs) by covalent binding to the PEG linkers using the cyanogen bromide method. The concentrations of goat anti-rabbit IgG covering the nanoprobes were quantified via the Bradford method. In the proof-of-concept experiment, an epithelial cell adhesion molecule (EpCAM) on the human breast cancer SK-Br-3 cell surface was used as the tumor marker, and the nanoparticle functionalized with rabbit anti-EpCAM antibody was employed as the nanoprobe for cancer cell recognition. Compared with fluorescent dye labeled IgG (FITC-IgG and RBITC-IgG), the designed nanoprobes display dramatically increased stability of fluorescence as well as photostability under continuous irradiation. PMID:24179992

  5. Crystal structure of Streptococcus pyogenes EndoS, an immunomodulatory endoglycosidase specific for human IgG antibodies

    PubMed Central

    Trastoy, Beatriz; Lomino, Joseph V.; Pierce, Brian G.; Carter, Lester G.; Günther, Sebastian; Giddens, John P.; Snyder, Greg A.; Weiss, Thomas M.; Weng, Zhiping; Wang, Lai-Xi; Sundberg, Eric J.

    2014-01-01

    To evade host immune mechanisms, many bacteria secrete immunomodulatory enzymes. Streptococcus pyogenes, one of the most common human pathogens, secretes a large endoglycosidase, EndoS, which removes carbohydrates in a highly specific manner from IgG antibodies. This modification renders antibodies incapable of eliciting host effector functions through either complement or Fc γ receptors, providing the bacteria with a survival advantage. On account of this antibody-specific modifying activity, EndoS is being developed as a promising injectable therapeutic for autoimmune diseases that rely on autoantibodies. Additionally, EndoS is a key enzyme used in the chemoenzymatic synthesis of homogenously glycosylated antibodies with tailored Fc γ receptor-mediated effector functions. Despite the tremendous utility of this enzyme, the molecular basis of EndoS specificity for, and processing of, IgG antibodies has remained poorly understood. Here, we report the X-ray crystal structure of EndoS and provide a model of its encounter complex with its substrate, the IgG1 Fc domain. We show that EndoS is composed of five distinct protein domains, including glycosidase, leucine-rich repeat, hybrid Ig, carbohydrate binding module, and three-helix bundle domains, arranged in a distinctive V-shaped conformation. Our data suggest that the substrate enters the concave interior of the enzyme structure, is held in place by the carbohydrate binding module, and that concerted conformational changes in both enzyme and substrate are required for subsequent antibody deglycosylation. The EndoS structure presented here provides a framework from which novel endoglycosidases could be engineered for additional clinical and biotechnological applications. PMID:24753590

  6. Characterization of the basic charge variants of a human IgG1

    PubMed Central

    Lu, Franklin; Derfus, Gayle; Kluck, Brian; Nogal, Bartek; Emery, Craig; Summers, Christie; Zheng, Kai; Bayer, Robert; Amanullah, Ashraf

    2011-01-01

    We report a case study of an IgG1 with a unique basic charge variant profile caused by C-terminal proline amidation on either one or two heavy chains. The proline amidation was sensitive to copper ion concentration in the production media during cell culture: the higher the Cu2+ ion concentration, the higher the level of proline amidation detected. This conclusion was supported by the analysis of samples that revealed direct correlation between the proline amidation level observed from peptide maps and the level of basic peaks measured by imaged capillary isoelectric focusing and a pH gradient ion-exchange chromatography method. The importance of these observations to therapeutic antibody production is discussed. PMID:22123059

  7. Evaluation of nanoparticle aggregation in human blood serum.

    PubMed

    Rausch, Kristin; Reuter, Anika; Fischer, Karl; Schmidt, Manfred

    2010-11-01

    In a certain stage of development, the performance of nanoparticle- or polymer-drug conjugates is tested "in vivo", that is, in mice or rats. Besides pharmaceutical and chemical characterization, the structural characterization of such drug carrier systems in terms of size, size distribution, and shape is typically performed in physiological salt solution prior to animal tests. The present work introduces a simple method based on dynamic light scattering to monitor the particle size in blood serum. Utilizing a model system of pegylated poly-l-lysines (PLL-g-PEOx) of various degrees of pegylation, x, it is demonstrated that large aggregates may form in human serum solution that are not observed in isotonic salt solution. Aggregates of a few hundred nanometers in size were found in mixtures of serum solution and PLL-g-PEOx with degrees of pegylation <10%, whereas no aggregates are being observed if the degree of pegylation exceeds 20%. The described method may have the potential to become an easy and routine test for drug carrier systems prior to animal applications. PMID:20961117

  8. Membrane Permeation Induced by Aggregates of Human Islet Amyloid Polypeptides

    PubMed Central

    Poojari, Chetan; Xiao, Dequan; Batista, Victor S.; Strodel, Birgit

    2013-01-01

    Several neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases as well as nonneuropathic diseases such as type II diabetes and atrial amyloidosis are associated with aggregation of amyloid polypeptides into fibrillar structures, or plaques. In this study, we use molecular dynamics simulations to test the stability and orientation of membrane-embedded aggregates of the human islet amyloid polypeptide (hIAPP) implicated in type II diabetes. We find that in both monolayers and bilayers of dipalmitoylphosphatidylglycerol (DPPG) hIAPP trimers and tetramers remain inside the membranes and preserve their β-sheet secondary structure. Lipid bilayer-inserted hIAPP trimers and tetramers orient inside DPPG at 60° relative to the membrane/water interface and lead to water permeation and Na+ intrusion, consistent with ion-toxicity in islet β-cells. In particular, hIAPP trimers form a water-filled β-sandwich that induce water permeability comparable with channel-forming proteins, such as aquaporins and gramicidin-A. The predicted disruptive orientation is consistent with the amphiphilic properties of the hIAPP aggregates and could be probed by chiral sum frequency generation (SFG) spectroscopy, as predicted by the simulated SFG spectra. PMID:24268144

  9. Snake venomics of monocled cobra (Naja kaouthia) and investigation of human IgG response against venom toxins.

    PubMed

    Laustsen, Andreas H; Gutiérrez, José María; Lohse, Brian; Rasmussen, Arne R; Fernández, Julián; Milbo, Christina; Lomonte, Bruno

    2015-06-01

    The venom proteome of the monocled cobra, Naja kaouthia, from Thailand, was characterized by RP-HPLC, SDS-PAGE, and MALDI-TOF-TOF analyses, yielding 38 different proteins that were either identified or assigned to families. Estimation of relative protein abundances revealed that venom is dominated by three-finger toxins (77.5%; including 24.3% cytotoxins and 53.2% neurotoxins) and phospholipases A2 (13.5%). It also contains lower proportions of components belonging to nerve growth factor, ohanin/vespryn, cysteine-rich secretory protein, C-type lectin/lectin-like, nucleotidase, phosphodiesterase, metalloproteinase, l-amino acid oxidase, cobra venom factor, and cytidyltransferase protein families. Small amounts of three nucleosides were also evidenced: adenosine, guanosine, and inosine. The most relevant lethal components, categorized by means of a 'toxicity score', were α-neurotoxins, followed by cytotoxins/cardiotoxins. IgGs isolated from a person who had repeatedly self-immunized with a variety of snake venoms were immunoprofiled by ELISA against all venom fractions. Stronger responses against larger toxins, but lower against the most critical α-neurotoxins were obtained. As expected, no neutralization potential against N. kaouthia venom was therefore detected. Combined, our results display a high level of venom complexity, unveil the most relevant toxins to be neutralized, and provide prospects of discovering human IgGs with toxin neutralizing abilities through use of phage display screening. PMID:25771242

  10. Localization of the binding site for the human high-affinity Fc receptor on IgG.

    PubMed

    Duncan, A R; Woof, J M; Partridge, L J; Burton, D R; Winter, G

    1988-04-01

    A major pathway in the clearance of pathogens involves the coating of the pathogen with specific antibodies, and the binding of the antibody Fc region to cell receptors. This can trigger engulfment of the pathogen by phagocytes or lysis by killer cells. By oligonucleotide site-directed mutagenesis we have engineered a single amino acid change in a mouse IgG2b antibody (Glu 235----Leu) which now enables the antibody to bind to the FcRI (high affinity) receptor on human monocytes with a 100-fold improvement in affinity. This indicates that Leu 235 is a major determinant in the binding of antibody to FcRI and that the receptor may interact directly with the region linking the CH2 domain to the hinge. Tailoring the affinity of antibodies for cell receptors could help dissect their role in clearing pathogen. PMID:2965792

  11. Repertoire of Epitopes Recognized by Serum IgG from Humans Vaccinated with Herpes Simplex Virus 2 Glycoprotein D

    PubMed Central

    Huang, Zhen-Yu; Cairns, Tina M.; Gallagher, John R.; Lou, Huan; Ponce-de-Leon, Manuel; Belshe, Robert B.; Eisenberg, Roselyn J.; Cohen, Gary H.

    2014-01-01

    ABSTRACT The results of a clinical trial of a subunit vaccine against genital herpes were recently reported (R. B. Belshe, P. A. Leone, D. I. Bernstein, A. Wald, M. J. Levin, J. T. Stapleton, I. Gorfinkel, R. L. Morrow, M. G. Ewell, A. Stokes-Riner, G. Dubin, T. C. Heineman, J. M. Schulte, C. D. Deal, N. Engl. J. Med. 366:34–43, 2012, doi:10.1056/NEJMoa1103151). The vaccine consisted of a soluble form of herpes simplex virus 2 (HSV-2) glycoprotein D (gD2) with adjuvant. The goal of the current study was to examine the composition of the humoral response to gD2 within a selected subset of vaccinated individuals. Serum samples from 30 vaccine recipients were selected based upon relative enzyme-linked immunosorbent assay (ELISA) titers against gD2; 10 samples had high titers, 10 had medium titers, and the remaining 10 had low ELISA titers. We employed a novel, biosensor-based monoclonal antibody (MAb)-blocking assay to determine whether gD2 vaccination elicited IgG responses against epitopes overlapping those of well-characterized MAbs. Importantly, IgGs from the majority of gD2-immunized subjects competed for gD binding with four antigenically distinct virus-neutralizing MAbs (MC2, MC5, MC23, and DL11). Screening of patient IgGs against overlapping peptides spanning the gD2 ectodomain revealed that about half of the samples contained antibodies against linear epitopes within the N and C termini of gD2. We found that the virus-neutralizing abilities of the 10 most potent samples correlated with overall gD-binding activity and to an even greater extent with the combined content of IgGs against the epitopes of MAbs MC2, MC5, MC23, and DL11. This suggests that optimal virus-neutralizing activity is achieved by strong and balanced responses to the four major discontinuous neutralizing epitopes of gD2. IMPORTANCE Several herpes simplex virus 2 (HSV-2) subunit vaccine studies have been conducted in human subjects using a recombinant form of HSV-2 glycoprotein D (gD2

  12. Cloning of Human IgG Fc cDNA and Expression of Whole Human Anti-HBsAg Antibody in CHO Cells.

    PubMed

    Tong, Yi-Gang; Xu, Jing; Liu, Guo-Qi; Zhang, Yong-Guo; Cheng, Wan-Rong; Liu, Shu-Ling; Wang, Hai-Tao

    2000-01-01

    Messenger RNA was extracted from human peripheral lymphocytes and first strand cDNA was prepared by reverse-transciption. The cDNA of Fc fragment of human IgG1 was then obtained by PCR and was cloned into the pGEM T-vector. The DNA sequences encoding signal peptides of both light and heavy chains were synthesized and cloned respectively. For construction of the light chain expression plasmid, the light chain signal sequence was linked with the light chain variable and constant regions (VL-CL) which had been cloned previously by screening of phage display libraries with HBsAg. The resulting full-lenth light chain sequence was then inserted into pcDNA3.1, a mammalian expression vector. For construction of the heavy chain expression plasmid, the heavy chain signal sequence, the variable region, the first constant region (VH-CH1, cloned previously by screening of phage display libraries with HBsAg) and Fc fragment sequence were ligated to form a full-length heavy chain ORF, which was then cloned into another mammalian expression vector, pCI-DHFR1. CHO(dhfr(-)) cells were cotransfected with the above light and heavy chain expression plasmids, and cell clones expressing human anti-HBsAg antibodies were selected by G418 and methotrexate (MTX). The recombinant human antibodies were purified with protein L affinity chromatography from the cell culture medium. As human serum IgG, the recombinant IgG exhibited only one band with a molecular weight of more than 100 kD in non-reducing SDS-PAGE in reducing SDS-PAGE, however, it turned out to be two bands of approximately 50 kD and 25 kD respectively. Western-blot analysis demonstrated that the whole IgG in the non-reducing SDS-PAGE, and the heavy chain in the reducing SDS-PAGE both reacted with goat anti-human Fc antiserum. PMID:12075430

  13. Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen: Functionality and stability studies.

    PubMed

    Klaus, Tomasz; Bzowska, Monika; Kulesza, Małgorzata; Kabat, Agnieszka Martyna; Jemioła-Rzemińska, Małgorzata; Czaplicki, Dominik; Makuch, Krzysztof; Jucha, Jarosław; Karabasz, Alicja; Bereta, Joanna

    2016-01-01

    Mouse immunoglobulins M (IgMs) that recognize human blood group antigens induce haemagglutination and are used worldwide for diagnostic blood typing. Contrary to the current belief that IgGs are too small to simultaneously bind antigens on two different erythrocytes, we obtained agglutinating mouse IgG3 that recognized antigen B of the human ABO blood group system. Mouse IgG3 is an intriguing isotype that has the ability to form Fc-dependent oligomers. However, F(ab')2 fragments of the IgG3 were sufficient to agglutinate type B red blood cells; therefore, IgG3-triggered agglutination did not require oligomerization. Molecular modelling indicated that mouse IgG3 has a larger range of Fab arms than other mouse IgG subclasses and that the unique properties of mouse IgG3 are likely due to the structure of its hinge region. With a focus on applications in diagnostics, we compared the stability of IgG3 and two IgMs in formulated blood typing reagents using an accelerated storage approach and differential scanning calorimetry. IgG3 was much more stable than IgMs. Interestingly, the rapid decrease in IgM activity was caused by aggregation of the molecules and a previously unknown posttranslational proteolytic processing of the μ heavy chain. Our data point to mouse IgG3 as a potent diagnostic tool. PMID:27484487

  14. Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen: Functionality and stability studies

    PubMed Central

    Klaus, Tomasz; Bzowska, Monika; Kulesza, Małgorzata; Kabat, Agnieszka Martyna; Jemioła-Rzemińska, Małgorzata; Czaplicki, Dominik; Makuch, Krzysztof; Jucha, Jarosław; Karabasz, Alicja; Bereta, Joanna

    2016-01-01

    Mouse immunoglobulins M (IgMs) that recognize human blood group antigens induce haemagglutination and are used worldwide for diagnostic blood typing. Contrary to the current belief that IgGs are too small to simultaneously bind antigens on two different erythrocytes, we obtained agglutinating mouse IgG3 that recognized antigen B of the human ABO blood group system. Mouse IgG3 is an intriguing isotype that has the ability to form Fc-dependent oligomers. However, F(ab′)2 fragments of the IgG3 were sufficient to agglutinate type B red blood cells; therefore, IgG3-triggered agglutination did not require oligomerization. Molecular modelling indicated that mouse IgG3 has a larger range of Fab arms than other mouse IgG subclasses and that the unique properties of mouse IgG3 are likely due to the structure of its hinge region. With a focus on applications in diagnostics, we compared the stability of IgG3 and two IgMs in formulated blood typing reagents using an accelerated storage approach and differential scanning calorimetry. IgG3 was much more stable than IgMs. Interestingly, the rapid decrease in IgM activity was caused by aggregation of the molecules and a previously unknown posttranslational proteolytic processing of the μ heavy chain. Our data point to mouse IgG3 as a potent diagnostic tool. PMID:27484487

  15. An Enhanced Pre- and Postnatal Development Study in Cynomolgus Monkeys with Tabalumab: A Human IgG4 Monoclonal Antibody.

    PubMed

    Breslin, William J; Hilbish, Kim G; Martin, Jennifer A; Halstead, Carolyn A; Newcomb, Deanna L; Chellman, Gary J

    2015-06-01

    Tabalumab, a human IgG4 monoclonal antibody (mAb) with neutralizing activity against both soluble and membrane B-cell activating factor (BAFF), has been under development for the treatment of autoimmune diseases. The purpose of this study was to determine the potential adverse effects of maternal tabalumab exposure on pregnancy, parturition, and lactation of the mothers and on the growth, viability, and development of the offspring through postnatal day (PND) 204. Tabalumab was administered by subcutaneous injection to presumed pregnant cynomolgus monkeys (16-19 per group) every 2 weeks from gestation day (GD) 20 to 22 until parturition at doses of 0, 0.3, or 30 mg/kg. Evaluations in mothers and infants included clinical signs, body weight, toxicokinetics, blood lymphocyte phenotyping, T-cell-dependent antibody response (infants only), antitherapeutic antibody (ATA), organ weights (infants only), and gross and microscopic histopathology. Infants were also examined for external and visceral morphologic and neurobehavioral development. There were no adverse tabalumab-related effects on maternal or infant endpoints. An expected pharmacological decrease in peripheral blood B-lymphocytes occurred in adults and infants; however, B-cell recovery was evident by PND154 in adults and infants at 0.3 mg/kg and by PND204 in infants at 30 mg/kg. At 30 mg/kg, a reduced IgM antibody response to T-cell-dependent antigen keyhole limpet hemocyanin (KLH) was observed following primary immunization. Following secondary KLH immunization, all infants in both dose groups mounted anti-KLH IgM and IgG antibody responses similar to control. Placental and mammary transfer of tabalumab was demonstrated. In conclusion, the no-observed-adverse-effect level for maternal and developmental toxicity was 30 mg/kg, the highest dose tested. Exposures at 30 mg/kg provide a margin of safety of 16× the anticipated clinical exposure. PMID:26195230

  16. QUALITATIVE MEASUREMENTS OF IGE AND IGG IN HUMAN ASTHMATIC SERUM FOR MOLD REACTIVITY

    EPA Science Inventory

    Rational: Molds have the ability to induce allergic asthma-like responses in mouse models; however, their role in human disease is unclear. This study was to develop a screening tool for reactivity of human sera to mold extracts by using a minimal amount of sera for a qual...

  17. Highly Immunoreactive IgG Antibodies Directed against a Set of Twenty Human Proteins in the Sera of Patients with Amyotrophic Lateral Sclerosis Identified by Protein Array

    PubMed Central

    May, Caroline; Nordhoff, Eckhard; Casjens, Swaantje; Turewicz, Michael; Eisenacher, Martin; Gold, Ralf; Brüning, Thomas; Pesch, Beate; Stephan, Christian; Woitalla, Dirk; Penke, Botond; Janáky, Tamás; Virók, Dezső; Siklós, László

    2014-01-01

    Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disorder, is characterized by the progressive and selective loss of upper and lower motor neurons. Diagnosis of this disorder is based on clinical assessment, and the average survival time is less than 3 years. Injections of IgG from ALS patients into mice are known to specifically mark motor neurons. Moreover, IgG has been found in upper and lower motor neurons in ALS patients. These results led us to perform a case-control study using human protein microarrays to identify the antibody profiles of serum samples from 20 ALS patients and 20 healthy controls. We demonstrated high levels of 20 IgG antibodies that distinguished the patients from the controls. These findings suggest that a panel of antibodies may serve as a potential diagnostic biomarker for ALS. PMID:24586901

  18. Uncovering the Mechanism of Aggregation of Human Transthyretin*

    PubMed Central

    Saelices, Lorena; Johnson, Lisa M.; Liang, Wilson Y.; Sawaya, Michael R.; Cascio, Duilio; Ruchala, Piotr; Whitelegge, Julian; Jiang, Lin; Riek, Roland; Eisenberg, David S.

    2015-01-01

    The tetrameric thyroxine transport protein transthyretin (TTR) forms amyloid fibrils upon dissociation and monomer unfolding. The aggregation of transthyretin has been reported as the cause of the life-threatening transthyretin amyloidosis. The standard treatment of familial cases of TTR amyloidosis has been liver transplantation. Although aggregation-preventing strategies involving ligands are known, understanding the mechanism of TTR aggregation can lead to additional inhibition approaches. Several models of TTR amyloid fibrils have been proposed, but the segments that drive aggregation of the protein have remained unknown. Here we identify β-strands F and H as necessary for TTR aggregation. Based on the crystal structures of these segments, we designed two non-natural peptide inhibitors that block aggregation. This work provides the first characterization of peptide inhibitors for TTR aggregation, establishing a novel therapeutic strategy. PMID:26459562

  19. Separation of human IgG fragments using copper, nickel, zinc, and cobalt chelated to CM-Asp-agarose by positive and negative chromatography.

    PubMed

    Mourão, Cecília Alves; Carmignotto, Gabriela Pannunzio; Bueno, Sonia Maria Alves

    2016-04-01

    This study evaluated the feasibility of using immobilized metal-ion affinity chromatography (IMAC) for separation of human Fab fragments using four different transition metal ions copper, nickel, zinc, and cobalt chelated to CM-Asp (carboxymethylaspartate) immobilized on the agarose gel. The Fab and Fc fragments (from human IgG digested with papain) interacted differently with the chelates studied, depending on the adsorption buffer system. The interaction between chelate and Fc fragment is predominantly based on the coordination bonds using adsorption buffer containing NaCl. Negative chromatography was performed on Cu(II)-CM-Asp-agarose obtaining 2.9mg of Fab per mL of adsorbent in nonretained fractions (Fc fragment-free without uncleaved IgG). The adsorption of Fab fragments is governed by electrostatic forces in the absence of NaCl in the adsorption buffer. High selectivity was achieved on Co(II)-CM-Asp-agarose and 5.7mg of Fab per mL of adsorbent was obtained in eluted fractions without Fc fragments, although having uncleaved IgG. The results showed that chromatography on transition metal ions chetated to CM-Asp-agarose is a promising approach to separation of Fab fragments from papain-digested human IgG solution. PMID:26974869

  20. Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG

    PubMed Central

    Fischer, Nicolas; Elson, Greg; Magistrelli, Giovanni; Dheilly, Elie; Fouque, Nicolas; Laurendon, Amélie; Gueneau, Franck; Ravn, Ulla; Depoisier, Jean-François; Moine, Valery; Raimondi, Sylvain; Malinge, Pauline; Di Grazia, Laura; Rousseau, François; Poitevin, Yves; Calloud, Sébastien; Cayatte, Pierre-Alexis; Alcoz, Mathias; Pontini, Guillemette; Fagète, Séverine; Broyer, Lucile; Corbier, Marie; Schrag, Delphine; Didelot, Gérard; Bosson, Nicolas; Costes, Nessie; Cons, Laura; Buatois, Vanessa; Johnson, Zoe; Ferlin, Walter; Masternak, Krzysztof; Kosco-Vilbois, Marie

    2015-01-01

    Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies. PMID:25672245

  1. Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG.

    PubMed

    Fischer, Nicolas; Elson, Greg; Magistrelli, Giovanni; Dheilly, Elie; Fouque, Nicolas; Laurendon, Amélie; Gueneau, Franck; Ravn, Ulla; Depoisier, Jean-François; Moine, Valery; Raimondi, Sylvain; Malinge, Pauline; Di Grazia, Laura; Rousseau, François; Poitevin, Yves; Calloud, Sébastien; Cayatte, Pierre-Alexis; Alcoz, Mathias; Pontini, Guillemette; Fagète, Séverine; Broyer, Lucile; Corbier, Marie; Schrag, Delphine; Didelot, Gérard; Bosson, Nicolas; Costes, Nessie; Cons, Laura; Buatois, Vanessa; Johnson, Zoe; Ferlin, Walter; Masternak, Krzysztof; Kosco-Vilbois, Marie

    2015-01-01

    Bispecific antibodies enable unique therapeutic approaches but it remains a challenge to produce them at the industrial scale, and the modifications introduced to achieve bispecificity often have an impact on stability and risk of immunogenicity. Here we describe a fully human bispecific IgG devoid of any modification, which can be produced at the industrial scale, using a platform process. This format, referred to as a κλ-body, is assembled by co-expressing one heavy chain and two different light chains, one κ and one λ. Using ten different targets, we demonstrate that light chains can play a dominant role in mediating specificity and high affinity. The κλ-bodies support multiple modes of action, and their stability and pharmacokinetic properties are indistinguishable from therapeutic antibodies. Thus, the κλ-body represents a unique, fully human format that exploits light-chain variable domains for antigen binding and light-chain constant domains for robust downstream processing, to realize the potential of bispecific antibodies. PMID:25672245

  2. Fusion Protein Comprising Factor H Domains 6 and 7 and Human IgG1 Fc as an Antibacterial Immunotherapeutic

    PubMed Central

    Shaughnessy, Jutamas; Vu, David M.; Punjabi, Rahi; Serra-Pladevall, Judit; DeOliveira, Rosane B.; Granoff, Dan M.

    2014-01-01

    The emergence of antimicrobial resistance among several medically important pathogens represents a serious threat to human health globally and necessitates the development of novel therapeutics. Complement forms a key arm of innate immune defenses against invading pathogens. A mechanism of complement evasion employed by many pathogens is binding of complement inhibitors, including factor H (FH), a key downregulator of the alternative pathway. Most FH-binding bacteria engage FH through regions in FH spanned by domains 6 and 7 and/or 18 through 20. We created a chimeric protein that comprised human FH domains 6 and 7 fused to human IgG1 Fc (FH6,7/HuFc) and tested its activity as an immunotherapeutic against Neisseria meningitidis, which binds FH through domains 6 and 7. FH6,7/HuFc bound to meningococci and effectively blocked FH binding to bacteria. FH6,7/HuFc enhanced human C3 and C4 deposition and facilitated complement-mediated killing in a dose-responsive manner; complement activation and killing were classical pathway dependent. To investigate in vivo efficacy, infant Wistar rats were treated intraperitoneally (IP) with different doses of FH6,7/HuFc and challenged 2 h later with serogroup C strain 4243 given IP. At 8 to 9 h after the challenge, the FH6,7/HuFc-treated rats had >100-fold fewer CFU per ml of blood than control animals pretreated with phosphate-buffered saline (PBS) or FH18–20/HuFc, which does not bind to meningococci (P < 0.0001). These data provide proof of concept of the utility of FH/Fc fusion proteins as anti-infective immunotherapeutics. Because many microbes share a common binding region(s) in FH, FH/Fc chimeric proteins may be a promising candidate for adjunctive therapy against drug-resistant pathogens. PMID:25143339

  3. Aggregation of Human Eyelid Adipose-derived Stem Cells by Human Body Fluids

    PubMed Central

    Song, Yeonhwa; Yun, Sujin; Yang, Hye Jin; Yoon, A Young; Kim, Haekwon

    2012-01-01

    Fetal bovine serum (FBS) is the most frequently used serum for the cultivation of mammalian cells. However, since animal-derived materials might not be appropriate due to safety issues, allogeneic human serum (HS) has been used to replace FBS, particularly for the culture of human cells. While there has been a debate about the advantages of HS, its precise effect on human adult stem cells have not been clarified. The present study aimed to investigate the effect of HS on the human eyelid adipose stem cells (HEACs) in vitro. When HEACs were cultivated in a medium containing 10% HS, many cells moved into several spots and aggregated there. The phenomenon was observed as early as 9 days following 10% HS treatment, and 12 days following 5% HS plus 5% FBS treatment. However, the aggregation was never observed when the same cells were cultivated with 10% FBS or bovine serum albumin. To examine whether cell density might affect the aggregation, cells were seeded with different densities on 12-well dish. Until the beginning of aggregation, cells seeded at low densities exhibited the longest culture period of 16 days whereas cells seeded at high densities showed the shortest period of 9 days to form aggregation. The number of cells was 15.1±0.2×104 as the least for the low density group, and 29.3±2.8×104 as the greatest for the high density group. When human cord blood serum or normal bovine serum was examined for the same effect on HEACs, interestingly, cord blood serum induced the aggregation of cells whereas bovine serum treatment has never induced. When cells were cultivated with 10% HS for 9 days, they were obtained and analyzed by RT-PCR. Compared to FBS-cultivated HEACs, HS-cultivated HEACs did not express VIM, and less expressed GATA4, PALLD. On the other hand, HS-cultivated HEACs expressed MAP2 more than FBS-cultivated HEACs. In conclusion, human adult stem cells could move and form aggregates by the treatment with human body fluids. PMID:25949109

  4. Identification of cyclic peptides able to mimic the functional epitope of IgG1-Fc for human FcγRI

    PubMed Central

    Bonetto, Stephane; Spadola, Loredana; Buchanan, Andrew G.; Jermutus, Lutz; Lund, John

    2009-01-01

    Identification of short, structured peptides able to mimic potently protein-protein interfaces remains a challenge in drug discovery. We report here the use of a naive cyclic peptide phage display library to identify peptide ligands able to recognize and mimic IgG1-Fc functions with FcγRI. Selection by competing off binders to FcγRI with IgG1 allowed the isolation of a family of peptides sharing the common consensus sequence TX2CXXθPXLLGCΦXE (θ represents a hydrophobic residue, Φ is usually an acidic residue, and X is any residue) and able to inhibit IgG1 binding to FcγRI. In soluble form, these peptides antagonize superoxide generation mediated by IgG1. In complexed form, they trigger phagocytosis and a superoxide burst. Unlike IgG, these peptides are strictly FcγRI-specific among the FcγRs. Molecular modeling studies suggest that these peptides can adopt 2 distinct and complementary conformers, each able to mimic the discontinuous interface contacts constituted by the Cγ2-A and -B chains of Fc for FcγRI. In addition, by covalent homodimerization, we engineered a synthetic bivalent 37-mer peptide that retains the ability to trigger effector functions. We demonstrate here that it is feasible to maintain IgG-Fc function within a small structured peptide. These peptides represent a new format for modulation of effector functions.—Bonetto, S., Spadola, L., Buchanan, A. G., Jermutus, L. Lund, J. Identification of cyclic peptides able to mimic the functional epitope of IgG1-Fc for human FcγRI. PMID:18957574

  5. A comparison of the effects of chlorpromazine and more selective histamine and 5-hydroxytryptamine antagonists on human IgG synthesis in vitro.

    PubMed

    Martinez, F; Coleman, J W

    1990-01-01

    We have shown previously that chlorpromazine, a drug associated with immunological abnormalities in vivo, significantly potentiates pokeweed mitogen (PWM)-stimulated IgG synthesis by human peripheral blood mononuclear cells (PBMC) in culture. Chlorpromazine is a pharmacological antagonist of histamine and 5-hydroxytryptamine (5-HT) and thus may exert its immune-enhancing effects by competing with these amines for their respective receptors, which are known to be present on lymphocytes. In this report we show that histamine and 5-HT are present at micromolar concentrations in PBMC cultures. To examine the role of histamine and 5-HT in chlorpromazine-induced enhancement of IgG synthesis we incubated PWM-treated cells with a range of selective histamine and 5-HT antagonists, and with the amines added to cultures either alone or in combination with chlorpromazine. The H1 antagonists mepyramine and promethazine and the H2 antagonist cimetidine had no significant effect on IgG synthesis. The combined 5-HT1/5-HT2 antagonists methysergide and methiothepin also failed to modulate synthesis. Neither histamine nor 5-HT at concentrations up to 100 microM modulated IgG synthesis, nor did they abrogate the enhancement of IgG synthesis induced by chlorpromazine. We conclude that the modulation of IgG synthesis in vitro by chlorpromazine cannot be attributed to an interaction of this drug with lymphocyte receptors for histamine and 5-HT. Other possibilities for the mechanism of action of this drug on immune function are discussed. PMID:2329012

  6. Plasmacytoid Dendritic Cell Activation and IFN-α Production Are Prominent Features of Murine Autoimmune Pancreatitis and Human IgG4-Related Autoimmune Pancreatitis.

    PubMed

    Arai, Yasuyuki; Yamashita, Kouhei; Kuriyama, Katsutoshi; Shiokawa, Masahiro; Kodama, Yuzo; Sakurai, Toshiharu; Mizugishi, Kiyomi; Uchida, Kazushige; Kadowaki, Norimitsu; Takaori-Kondo, Akifumi; Kudo, Masatoshi; Okazaki, Kazuichi; Strober, Warren; Chiba, Tsutomu; Watanabe, Tomohiro

    2015-10-01

    The abnormal immune response accompanying IgG4-related autoimmune pancreatitis (AIP) is presently unclear. In this study, we examined the role of plasmacytoid dendritic cell (pDC) activation and IFN-α production in this disease as well as in a murine model of AIP (MRL/Mp mice treated with polyinosinic-polycytidylic acid). We found that the development of AIP in treated MRL/Mp mice occurred in parallel with pancreatic accumulation of pDCs producing IFN-α, and with pDC depletion and IFN-α-blocking studies, we showed that such accumulation was necessary for AIP induction. In addition, we found that the pancreas of treated MRL/Mp mice contained neutrophil extracellular traps (NETs) shown previously to stimulate pDCs to produce IFN-α. Consistent with these findings, we found that patients with IgG4-related AIP also exhibited pancreatic tissue localization of IFN-α-expressing pDCs and had significantly higher serum IFN-α levels than healthy controls. In addition, the inflamed pancreas of these patients but not controls also contained NETs that were shown to be capable of pDC activation. More importantly, patient pDCs cultured in the presence of NETs produced greatly increased levels of IFN-α and induced control B cells to produce IgG4 (but not IgG1) as compared with control pDCs. These data suggest that pDC activation and production of IFN-α is a major cause of murine AIP; in addition, the increased pDC production of IFN-α and its relation to IgG4 production observed in IgG4-related AIP suggest that this mechanism also plays a role in the human disease. PMID:26297761

  7. High Human Cytomegalovirus IgG Level is Associated with Increased Incidence of Diabetic Atherosclerosis in Type 2 Diabetes Mellitus Patients

    PubMed Central

    Zhang, Jun; Liu, Yuan-yuan; Sun, Hui-ling; Li, Shan; Xiong, Hai-rong; Yang, Zhan-qiu; Xiang, Guang-da; Jiang, Xiao-jing

    2015-01-01

    Background At present, whether human cytomegalovirus (HCMV) infection is associated with type 2 diabetes mellitus (T2DM) is debatable. The effect of active HCMV infection on glucose regulation has been poorly studied. Although HCMV infection is correlated with atherosclerosis in cardiovascular disease, the role of HCMV infection in the development of diabetic atherosclerosis in T2DM is unclear and is usually neglected by endocrinologists. The aim of this study was to assess the effects of HCMV infection on glucose regulation and the development of diabetic atherosclerosis in T2DM patients. Material/Methods A total of 222 hospitalized T2DM patients were enrolled. Nested polymerase chain reactions were used to detect HCMV DNA extracted from peripheral blood leukocytes. Quantitative real-time PCR was used to determine viral load. HCMV IgG antibody concentrations were analyzed by chemiluminescence immunoassay. Results HCMV active infection, viral load, and HCMV IgG titers were not correlated with glucose regulation. Binary logistic regression demonstrated that the highest quartile of HCMV IgG concentration (>500 U/ml) was correlated with the incidence of diabetic atherosclerosis (OR: 8.0, 95%CI: 2.3–27.2), and that titer >127U/ml of HCMV IgG is an independent predictor for the development of diabetic atherosclerosis in T2DM patients (OR: 4.6, 95%CI: 1.9–11.3) after adjustment for all potential confounding factors. Conclusions Active HCMV infection is unlikely to influence glucose regulation in T2DM. However, HCMV IgG titers are associated with the incidence of diabetic atherosclerosis, and titer >127U/ml of HCMV IgG might be an independent risk factor for the development of diabetic atherosclerosis in T2DM patients. PMID:26717490

  8. Structural basis for binding of human IgG1 to its high-affinity human receptor FcγRI

    PubMed Central

    Kiyoshi, Masato; Caaveiro, Jose M.M.; Kawai, Takeaki; Tashiro, Shinya; Ide, Teruhiko; Asaoka, Yoshiharu; Hatayama, Kouta; Tsumoto, Kouhei

    2015-01-01

    Cell-surface Fcγ receptors mediate innate and adaptive immune responses. Human Fcγ receptor I (hFcγRI) binds IgGs with high affinity and is the only Fcγ receptor that can effectively capture monomeric IgGs. However, the molecular basis of hFcγRI's interaction with Fc has not been determined, limiting our understanding of this major immune receptor. Here we report the crystal structure of a complex between hFcγRI and human Fc, at 1.80 Å resolution, revealing an unique hydrophobic pocket at the surface of hFcγRI perfectly suited for residue Leu235 of Fc, which explains the high affinity of this complex. Structural, kinetic and thermodynamic data demonstrate that the binding mechanism is governed by a combination of non-covalent interactions, bridging water molecules and the dynamic features of Fc. In addition, the hinge region of hFcγRI-bound Fc adopts a straight conformation, potentially orienting the Fab moiety. These findings will stimulate the development of novel therapeutic strategies involving hFcγRI. PMID:25925696

  9. Neutrophils extracellular traps damage Naegleria fowleri trophozoites opsonized with human IgG.

    PubMed

    Contis-Montes de Oca, A; Carrasco-Yépez, M; Campos-Rodríguez, R; Pacheco-Yépez, J; Bonilla-Lemus, P; Pérez-López, J; Rojas-Hernández, S

    2016-08-01

    Naegleria fowleri infects humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N. fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. NETs are composed of nuclear DNA combined with histones and antibacterial proteins, and these structures are released from the cell to direct its antimicrobial attack. In this work, we evaluate the capacity of N. fowleri to induce the liberation of NETs by human PMN cells. Neutrophils were cocultured with unopsonized or IgG-opsonized N. fowleri trophozoites. DNA, histone, myeloperoxidase (MPO) and neutrophil elastase (NE) were stained, and the formation of NETs was evaluated by confocal microscopy and by quantifying the levels of extracellular DNA. Our results showed N. fowleri induce the liberation of NETs including release of MPO and NE by human PMN cells as exposure interaction time is increased, but N. fowleri trophozoites evaded killing. However, when trophozoites were opsonized, they were susceptible to the neutrophils activity. Therefore, our study suggests that antibody-mediated PMNs activation through NET formation may be crucial for antimicrobial responses against N. fowleri. PMID:27189133

  10. Use of immunoblot technique for detection of human IgE and IgG antibodies to individual silk proteins.

    PubMed

    Dewair, M; Baur, X; Ziegler, K

    1985-10-01

    Allergenic proteins were extracted from one silk batch that was imported to be used as filling material for bed mattresses and rugs. IgE and IgG antibodies to the extracted silk proteins were measured by RAST in sera of nine silk-sensitive persons as well as in sera of healthy control donors. Silk proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into 12 polypeptides of molecular weights between 14 and 70 kilodaltons. By means of the immunoblot technique, IgE and IgG antibodies to the individual silk polypeptides could be detected. Sera of silk-sensitive persons contained high titers of IgE and low titers of IgG antibodies to the separated silk polypeptides. Sera of control donors contained low IgG antibody titers to a limited number of these polypeptides. PMID:4056241

  11. The different effector function capabilities of the seven equine IgG subclasses have implications for vaccine strategies.

    PubMed

    Lewis, Melanie J; Wagner, Bettina; Woof, Jenny M

    2008-02-01

    Recombinant versions of the seven equine IgG subclasses were expressed in CHO cells. All assembled into intact immunoglobulins stabilised by disulphide bridges, although, reminiscent of human IgG4, a small proportion of equine IgG4 and IgG7 were held together by non-covalent bonds alone. All seven IgGs were N-glycosylated. In addition IgG3 appeared to be O-glycosylated and could bind the lectin jacalin. Staphylococcal protein A displayed weak binding for the equine IgGs in the order: IgG1>IgG3>IgG4>IgG7>IgG2=IgG5>IgG6. Streptococcal protein G bound strongly to IgG1, IgG4 and IgG7, moderately to IgG3, weakly to IgG2 and IgG6, and not at all to IgG5. Analysis of antibody effector functions revealed that IgG1, IgG3, IgG4, IgG5 and IgG7, but not IgG2 and IgG6, were able to elicit a strong respiratory burst from equine peripheral blood leukocytes, predicting that the former five IgG subclasses are able to interact with Fc receptors on effector cells. IgG1, IgG3, IgG4 and IgG7, but not IgG2, IgG5 and IgG6, were able to bind complement C1q and activate complement via the classical pathway. The differential effector function capabilities of the subclasses suggest that, for maximum efficacy, equine vaccine strategies should seek to elicit antibody responses of the IgG1, IgG3, IgG4, and IgG7 subclasses. PMID:17669496

  12. The Effect of Ovine Secreted Soluble Factors on Human Dermal Papilla Cell Aggregation

    PubMed Central

    Sari, Agnes Rosarina Prita; Rufaut, Nicholas Wolfgang; Jones, Leslie Norman; Sinclair, Rodney Daniel

    2016-01-01

    Context: In androgenetic alopecia, follicular miniaturization and dynamic changes to the hair cycle produce patterned baldness. The most effective treatment for baldness is hair transplantation surgery. The major limitation to hair transplantation is the availability of donor hair from the relatively unaffected occipital scalp. Hair induction with in vitro expansion of donor follicle populations has the potential to overcome this. The major obstacle to this is that in vitro expansion of human dermal papilla cell (DPC) colonies is associated with irreversible loss of aggregative behavior and hair follicle-inductive potential. In contrast, cultured ovine DPCs maintain these properties after extensive proliferation. Aims: To determine whether aggregating ovine DPC secrete factors that enhance the aggregative behavior or inductive potential of human DPC. Subjects and Methods: Fluorescently-labelled ovine DPC were mixed in culture with human DPC at passage number seven-nine, which had lost their aggregative behavior. The effects of different culture substrates and medium compositions on aggregative behavior were determined. Ovine and human papilla cells were co-cultured, separated by a permeable membrane to determine whether the ovine cells secrete soluble factors that affect human papilla cells. Results: In direct co-culture experiments, well-formed aggregates were produced by 90:10 human:ovine and 50:50 human:ovine DPC mixtures. In contrast, unmixed human DPC remained in a monolayer state after 18 days. Both human and ovine DPC had a higher tendency to aggregate in medium containing 20% (v/v) lamb serum (LS) compared to 10% (v/v) fetal calf serum (FCS). In co-culture experiments separated with permeable membrane, the human DPC aggregates were bigger and more rapidly formed with the addition of ovine secreted soluble factors. Conclusions: Soluble factors secreted by ovine DPC and present in LS increase the aggregative behavior of human DPC. These molecules might

  13. Inhibition of low- and high-threshold Ca2+ channels of human neuroblastoma IMR32 cells by Lambert-Eaton myasthenic syndrome (LEMS) IgGs.

    PubMed

    Grassi, C; Magnelli, V; Carabelli, V; Sher, E; Carbone, E

    1994-11-01

    IgGs from two LEMS patients applied to human neuroblastoma IMR32 cells reduced the density of low- (LVA; T) and high-threshold (HVA; L and N) Ba2+ currents by different percentages: 36% (LVA) and 56% (HVA) for one and 48% and 45% for the other. A pharmacological assay of IgGs action based on the block of L-type channel by nifedipine and on the delayed activation of N-type channel by noradrenaline, indicated a preferential inhibition of the N-type current in IMR32 cells (55% and 47% for the two patients). The L-type current, contributing to approximately one-third of the total, was also depressed by LEMS IgGs but to a minor degree (49% and 30%). Except for an increase of single N-type channel inactivation, LEMS antibodies preserved the elementary properties of single HVA channels, suggesting that the macroscopic current reduction after IgGs treatment is likely due to a decrease in the number of active HVA Ca2+ channels. PMID:7898770

  14. Chimeric Anti-CD14 IGG2/4 Hybrid Antibodies for Therapeutic Intervention in Pig and Human Models of Inflammation

    PubMed Central

    Lau, Corinna; Gunnarsen, Kristin S.; Høydahl, Lene S.; Andersen, Jan Terje; Berntzen, Gøril; Pharo, Anne; Lindstad, Julie K.; Ludviksen, Judith K.; Brekke, Ole-Lars; Barratt-Due, Andreas; Nielsen, Erik Waage; Stokes, Christopher R.; Espevik, Terje; Sandlie, Inger

    2013-01-01

    CD14 is a key recognition molecule of innate immune responses, interacting with several TLRs. TLR signaling cross-talks extensively with the complement system, and combined CD14 and complement inhibition has been proved effective in attenuating inflammatory responses. Pig models of human diseases have emerged as valuable tools to study therapeutic intervention, but suitable neutralizing Abs are rare. Undesired Fc-mediated functions, such as platelet activation and IL-8 release induced by the porcine CD14-specific clone Mil2, limit further studies. Therefore, an inert human IgG2/IgG4 hybrid C region was chosen for an rMil2. As revealed in ex vivo and in vivo pig experiments, rMil2 inhibited the CD14-mediated proinflammatory cytokine response similar to the original clone, but lacked the undesired Fc-effects, and inflammation was attenuated further by simultaneous complement inhibition. Moreover, rMil2 bound porcine FcRn, a regulator of t1/2 and biodistribution. Thus, rMil2, particularly combined with complement inhibitors, should be well suited for in vivo studies using porcine models of diseases, such as sepsis and ischemia-reperfusion injury. Similarly, the recombinant anti-human CD14 IgG2/4 Ab, r18D11, was generated with greatly reduced Fc-mediated effects and preserved inhibitory function ex vivo. Such Abs might be drug candidates for the treatment of innate immunity-mediated human diseases. PMID:24062486

  15. Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal importance of S254 and Y436 in binding human neonatal Fc receptor.

    PubMed

    Burvenich, Ingrid J G; Farrugia, William; Lee, Fook T; Catimel, Bruno; Liu, Zhanqi; Makris, Dahna; Cao, Diana; O'Keefe, Graeme J; Brechbiel, Martin W; King, Dylan; Spirkoska, Violeta; Allan, Laura C; Ramsland, Paul A; Scott, Andrew M

    2016-01-01

    IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')2 (t1/2β, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')2, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t1/2β, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t1/2β, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic. PMID:27030023

  16. Single-Molecule Imaging of Individual Amyloid Protein Aggregates in Human Biofluids

    PubMed Central

    2016-01-01

    The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β. In addition, we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the average aggregate concentration in CSF from Parkinson’s disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders. PMID:26800462

  17. Anti-GaL IgG antibodies in sera of newborn humans and baboons and its significance in pig xenotransplantation.

    PubMed

    Minanov, O P; Itescu, S; Neethling, F A; Morgenthau, A S; Kwiatkowski, P; Cooper, D K; Michler, R E

    1997-01-27

    We have previously demonstrated that hyperacute rejection does not occur in a pig-to-newborn baboon heart transplant model, presumably because of low levels of cytotoxic antipig antibodies present in the serum of newborn baboons. Cytotoxic antipig antibodies are primarily directed to alpha-1,3-galactosyl (alpha Gal) residues on endothelial cell surface structures Twenty-one full-term humans and 5 full-term baboons were tested for complement mediated lysis (CML) of pig kidney (PK-15) cells and anti-alpha Gal activity with an ELISA using BSA-conjugated alpha Gal residues as target. To evaluate the significance of the anti-alpha Gal titers in vivo 5 newborn baboons underwent heterotopic pig cardiac xenotransplantation. Six of 21 human samples and 1 of 5 baboon samples demonstrated significant cytotoxicity to PK-15 cells. Twelve of 21 newborn humans had anti-alpha Gal IgG antibodies at titers of 1:80 or greater. None of the samples had anti-alpha Gal IgM. In newborn baboons, 1 of 5 sera had anti-alpha Gal IgG antibodies at titers greater than 1:80 and none of these samples had anti-alpha Gal IgM. Xenografts survived for an average of 3.6 days, even in the baboon with high anti-alpha Gal IgG titers. Analysis of the explanted grafts showed minimal evidence of complement-mediated hyperacute rejection (HAR), but prominent mononuclear cell infiltrates. In serum tested posttransplant there was an induced anti-alpha Gal response with cytotoxicity against PK-15 cells. These results show that anti-alpha Gal IgM is absent in newborn human and baboon sera, allowing pig grafts to avoid HAR. However, the presence of anti-alpha Gal IgG may be associated with mononuclear cell infiltration of the xenograft and its subsequent rejection. PMID:9020315

  18. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    SciTech Connect

    Berger, Christian; Madshus, Inger Helene; Stang, Espen

    2012-12-10

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: Black-Right-Pointing-Pointer Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. Black-Right-Pointing-Pointer Antibody combination causes internalization of EGFR by macropinocytosis. Black-Right-Pointing-Pointer Antibody-induced internalization of EGFR is independent of EGFR kinase activity. Black-Right-Pointing-Pointer Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  19. Isoelectric focusing-affinity immunoblot analysis of mouse monoclonal antibodies to the four human IgG subclasses

    NASA Technical Reports Server (NTRS)

    Hamilton, Robert G.; Roebber, Marianne; Rodkey, L. Scott; Reimer, Charles B.

    1987-01-01

    Isoelectric focusing (IEF)/affinity immunoblotting and enzyme-linked immunosorbent assay (ELISA) were used for parallel analysis of murine monoclonal antihuman IgG-subclass antisera (MoAbs). Coomassie Blue-stained protein bands in the pH region 5.5-8.0 were shown to be murine IgG by direct blotting onto nitrocellulose followed by detection with conjugated antimouse IgG. Use of IgG myeloma antigen-coated nitrocellulose in the IEF-affinity immunoblot allowed detection of the charge microheterogeneity of MoAbs. The MoAb group contained one to five major dense bands flanked by up to four minor fainter bands, all with pIs ranging from 6.1 to 7.8. Semiquantitative estimates of binding specificity in the IEF-affinity blot compared well with cross-reactivity data obtained from a quantitative ELISA.

  20. 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

  1. Mobile phone usage in complex urban systems: a space-time, aggregated human activity study

    NASA Astrophysics Data System (ADS)

    Tranos, Emmanouil; Nijkamp, Peter

    2015-04-01

    The present study aims to demonstrate the importance of digital data for investigating space-time dynamics of aggregated human activity in urban systems. Such dynamics can be monitored and modelled using data from mobile phone operators regarding mobile telephone usage. Using such an extensive dataset from the city of Amsterdam, this paper introduces space-time explanatory models of aggregated human activity patterns. Various modelling experiments and results are presented, which demonstrate that mobile telephone data are a good proxy of the space-time dynamics of aggregated human activity in the city.

  2. A Human Monoclonal IgG That Binds Aβ Assemblies and Diverse Amyloids Exhibits Anti-Amyloid Activities In Vitro and In Vivo

    PubMed Central

    O'Nuallain, Brian; Puligedda, Rama Devudu; Ondrejcak, Tomas; Adekar, Sharad P.; Chen, Cindy; Cruz, Pedro E.; Rosario, Awilda M.; Macy, Sallie; Mably, Alexandra J.; Walsh, Dominic M.; Vidal, Ruben; Solomon, Alan; Brown, Daniel; Rowan, Michael J.; Golde, Todd E.

    2015-01-01

    Alzheimer's disease (AD) and familial Danish dementia (FDD) are degenerative neurological diseases characterized by amyloid pathology. Normal human sera contain IgG antibodies that specifically bind diverse preamyloid and amyloid proteins and have shown therapeutic potential in vitro and in vivo. We cloned one of these antibodies, 3H3, from memory B cells of a healthy individual using a hybridoma method. 3H3 is an affinity-matured IgG that binds a pan-amyloid epitope, recognizing both Aβ and λ Ig light chain (LC) amyloids, which are associated with AD and primary amyloidosis, respectively. The pan-amyloid-binding properties of 3H3 were demonstrated using ELISA, immunohistochemical studies, and competition binding assays. Functional studies showed that 3H3 inhibits both Aβ and LC amyloid formation in vitro and abrogates disruption of hippocampal synaptic plasticity by AD-patient-derived soluble Aβ in vivo. A 3H3 single-chain variable fragment (scFv) retained the binding specificity of the 3H3 IgG and, when expressed in the brains of transgenic mice using an adeno-associated virus (AAV) vector, decreased parenchymal Aβ amyloid deposition in TgCRND8 mice and ADan (Danish Amyloid) cerebral amyloid angiopathy in the mouse model of FDD. These data indicate that naturally occurring human IgGs can recognize a conformational, amyloid-specific epitope and have potent anti-amyloid activities, providing a rationale to test their potential as antibody therapeutics for diverse neurological and other amyloid diseases. PMID:25904780

  3. Effective therapy of human lymphoma xenografts with a novel recombinant ribonuclease/anti-CD74 humanized IgG4 antibody immunotoxin.

    PubMed

    Chang, Chien-Hsing; Sapra, Puja; Vanama, Sailaja S; Hansen, Hans J; Horak, Ivan D; Goldenberg, David M

    2005-12-15

    Ranpirnase (Rap) is a cytotoxic ribonuclease (RNase) isolated from frog oocytes. Here we describe high antitumor activity of a novel immunotoxin, 2L-Rap-hLL1-gamma4P, composed of 2 Rap molecules, each fused to the N terminus of the light chain of hLL1, an internalizing anti-CD74 humanized antibody. To reduce unwanted side effects, the constant region of hLL1 was changed from gamma1 to gamma4 and further to gamma4P by replacing serine228 to proline to prevent the formation of a half immunoglobulin G (IgG) common for IgG4. In vitro, 2L-Rap-hLL1-gamma4P retained RNase activity, specific binding to CD74, and was significantly more potent against CD74+ cell lines (Daudi, Raji, and MC/CAR) than naked hLL1. In vivo, the pharmacokinetic profile of 2L-Rap-hLL1-gamma4P was similar to that of naked hLL1. The maximum tolerated dose of 2L-Rap-hLL1-gamma4P in severe combined immunodeficient mice (SCID) or BALB/c mice was 50 microg per mouse. In Raji and Daudi Burkitt lymphoma xenograft models, treatment with a single 5 to 50 microg dose of 2L-Rap-hLL1-gamma4P, given as early or delayed treatment, resulted in cures of most animals. Treatment with 2L-Rap-hLL1-gamma4P was significantly better than all controls, including saline, naked hLL1, and nonspecific immunotoxin. In conclusion, 2L-Rap-hLL1-gamma4P demonstrated excellent in vitro and in vivo efficacy and thus merits further consideration as a therapeutic for CD74+ tumors. PMID:16109781

  4. Modulation of Single-Cell IgG Secretion Frequency and Rates in Human Memory B Cells by CpG DNA, CD40L, IL-21 and Cell Division∥

    PubMed Central

    Henn, Alicia D.; Rebhahn, Jonathan; Brown, Miguel A.; Murphy, Alison J.; Coca, Mircea N.; Hyrien, Ollivier; Pellegrin, Tina; Mosmann, Tim; Zand, Martin S.

    2009-01-01

    During the recall response by CD27+ IgG class switched human memory B cells, total IgG secreted is a function of (1) the number of IgG secreting cells (IgG-SC) and (2) the secretion rate of each cell. Here we report the quantitative ELISPOT method (qELISPOT) for simultaneous estimation of single cell IgG secretion rates and secreting cell frequencies in human B cell populations. We found that CD27+ IgMneg memory B cells activated with CpG and cytokines had considerable heterogeneity in the IgG secretion rates, with two major secretion rate subpopulations. B cell receptor cross-linking reduced the frequency of cells with high per-cell IgG secretion rates, with a parallel decrease in CD27hi B cell blasts. Increased cell death may account for the BCR-stimulated reduction in high-rate IgG-SC CD27hi B cell blasts. In contrast, the addition of IL-21 to CD40L +IL-4 activated human memory B cells induced a high-rate IgG-SC population in B cells with otherwise low per-cell IgG secretion rates. The profiles of human B cell IgG secretion rates followed the same biphasic distribution and range irrespective of division class. This, along with the presence of non-IgG-producing, dividing B cells in CpG+ck-activated B memory B cell populations, is suggestive of an “On/Off switch” regulating IgG secretion. Finally, these data support a mixture model of IgG secretion in which IgG secreted over time is modulated by the frequency of IgG secreting cells and the distribution of their IgG secretion rates. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this

  5. Leishmania mexicana Infection Induces IgG to Parasite Surface Glycoinositol Phospholipids that Can Induce IL-10 in Mice and Humans

    PubMed Central

    Buxbaum, Laurence U.

    2013-01-01

    Infection with the intracellular protozoan parasite Leishmania mexicana causes chronic disease in C57BL/6 mice, in which cutaneous lesions persist for many months with high parasite burdens (107–108 parasites). This chronic disease process requires host IL-10 and FcγRIII. When Leishmania amastigotes are released from cells, surface-bound IgG can induce IL-10 and suppress IL-12 production from macrophages. These changes decrease IFN-γ from T cells and nitric oxide production in infected cells, which are both required for Leishmania control. However, antibodies targets and the kinetics of antibody production are unknown. Several groups have been unsuccessful in identifying amastigote surface proteins that bind IgG. We now show that glycoinositol phospholipids (GIPLs) of L. mexicana are recognized by mouse IgG1 by 6 weeks of infection, with a rapid increase between 12 and 16 weeks, consistent with the timing of chronic disease in C57BL/6 mice vs. healing in FcγRIII-deficient mice. A single prominent spot on TLC is recognized by IgG, and the glycolipid is a glycosyl phosphatidylinositol containing a branched mannose structure. We show that the lipid structure of the GIPL (the sn-2 fatty acid) is required for antibody recognition. This GIPL is abundant in L. mexicana amastigotes, rare in stationary-phase promastigotes, and absent in L. major, consistent with a role for antibodies to GIPLs in chronic disease. A mouse monoclonal anti-GIPL IgG recognizes GIPLs on the parasite surface, and induces IL-10 from macrophages. The current work also extends this mouse analysis to humans, finding that L. mexicana-infected humans with localized and diffuse cutaneous leishmaniasis have antibodies that recognize GIPLs, can bind to the surface of amastigotes, and can induce IL-10 from human monocytes. Further characterization of the target glycolipids will have important implications for drug and vaccine development and will elucidate the poorly understood role of glycolipids in

  6. Arachidonate metabolism, 5-hydroxytryptamine release and aggregation in human platelets activated by palmitaldehyde acetal phosphatidic acid.

    PubMed Central

    Brammer, J. P.; Maguire, M. H.

    1984-01-01

    Palmitaldehyde acetal phosphatidic acid ( PGAP ) caused dose-dependent aggregation of human platelets resuspended in modified Tyrode medium, with a threshold concentration of 0.5-1 microM and an EC50 of 4 microM. Concentrations of PGAP which elicited biphasic irreversible aggregation concomitantly induced formation of 1.02 +/- 0.029 nmol (mean +/- s.e. mean) of malondialdehyde (MDA) per 10(9) platelets and caused release of 58 +/- 2.8% of platelet [14C]-5-hydroxytryptamine ([14C]-5-HT) from prelabelled platelets; no MDA formation or [14C]-5-HT release occurred at lower doses of PGAP which elicited only monophasic reversible aggregation. Adenosine 5'-pyrophosphate (ADP)-induced platelet activation resulted in formation of 0.344 +/- 0.004 nmol of MDA per 10(9) platelets in association with irreversible aggregation and 49.1 +/- 1% release of [14C]-5-HT. Mepacrine, a phospholipase A2 inhibitor, at 2.5 microM reduced PGAP -induced MDA formation and [14C]-5-HT release by the resuspended platelets without affecting irreversible aggregation; higher concentrations of mepacrine abolished all three responses. Chlorpromazine, a calmodulin antagonist, similarly inhibited PGAP -induced MDA formation and irreversible aggregation, and at 100 microM abolished monophasic aggregation. The cyclo-oxygenase inhibitor indomethacin caused a concentration-dependent reduction of PGAP -induced MDA formation by resuspended human platelets without significantly inhibiting [14C]-5-HT release or irreversible aggregation; concentrations (greater than or equal to 1.75 microM) which inhibited MDA formation by more than 94% abolished [14C]-5-HT release, and converted second phase irreversible aggregation to an extensive reversible response. 2-Methylthioadenosine 5'-phosphate (2 methylthio-AMP), an ADP antagonist, inhibited PGAP -induced MDA formation, [14C]-5-HT release and second phase aggregation in the human platelet suspensions in a parallel, concentration-dependent manner; at 9.4 microM 2

  7. Detection of Human Papillomavirus 16-Specific IgG and IgM Antibodies in Patient Sera: A Potential Indicator of Oral Squamous Cell Carcinoma Risk Factor.

    PubMed

    Kerishnan, Jesinda P; Gopinath, Subash C B; Kai, Sia Bik; Tang, Thean-Hock; Ng, Helen Lee-Ching; Rahman, Zainal Ariff Abdul; Hashim, Uda; Chen, Yeng

    2016-01-01

    The association between human papillomavirus type 16 (HPV16) and oral cancer has been widely reported. However, detecting anti-HPV antibodies in patient sera to determine risk for oral squamous cell carcinoma (OSCC) has not been well studied. In the present investigation, a total of 206 OSCC serum samples from the Malaysian Oral Cancer Database & Tissue Bank System, with 134 control serum samples, were analyzed by enzyme-linked immunosorbant assay (ELISA) to detect HPV16-specific IgG and IgM antibodies. In addition, nested PCR analysis using comprehensive consensus primers (PGMY09/11 and GP5(+)/6(+)) was used to confirm the presence of HPV. Furthermore, we have evaluated the association of various additional causal factors (e.g., smoking, alcohol consumption, and betel quid chewing) in HPV-infected OSCC patients. Statistical analysis of the Malaysian population indicated that OSCC was more prevalent in female Indian patients that practices betel quid chewing. ELISA revealed that HPV16 IgG, which demonstrates past exposure, could be detected in 197 (95.6%) OSCC patients and HPV16-specific IgM was found in a total of 42 (20.4%) OSCC patients, indicating current exposure. Taken together, our study suggest that HPV infection may play a significant role in OSCC (OR: 13.6; 95% CI: 3.89-47.51) and HPV16-specific IgG and IgM antibodies could represent a significant indicator of risk factors in OSCC patients. PMID:27279791

  8. Detection of Human Papillomavirus 16-Specific IgG and IgM Antibodies in Patient Sera: A Potential Indicator of Oral Squamous Cell Carcinoma Risk Factor

    PubMed Central

    Kerishnan, Jesinda P.; Gopinath, Subash C.B.; Kai, Sia Bik; Tang, Thean-Hock; Ng, Helen Lee-Ching; Rahman, Zainal Ariff Abdul; Hashim, Uda; Chen, Yeng

    2016-01-01

    The association between human papillomavirus type 16 (HPV16) and oral cancer has been widely reported. However, detecting anti-HPV antibodies in patient sera to determine risk for oral squamous cell carcinoma (OSCC) has not been well studied. In the present investigation, a total of 206 OSCC serum samples from the Malaysian Oral Cancer Database & Tissue Bank System, with 134 control serum samples, were analyzed by enzyme-linked immunosorbant assay (ELISA) to detect HPV16-specific IgG and IgM antibodies. In addition, nested PCR analysis using comprehensive consensus primers (PGMY09/11 and GP5+/6+) was used to confirm the presence of HPV. Furthermore, we have evaluated the association of various additional causal factors (e.g., smoking, alcohol consumption, and betel quid chewing) in HPV-infected OSCC patients. Statistical analysis of the Malaysian population indicated that OSCC was more prevalent in female Indian patients that practices betel quid chewing. ELISA revealed that HPV16 IgG, which demonstrates past exposure, could be detected in 197 (95.6%) OSCC patients and HPV16-specific IgM was found in a total of 42 (20.4%) OSCC patients, indicating current exposure. Taken together, our study suggest that HPV infection may play a significant role in OSCC (OR: 13.6; 95% CI: 3.89-47.51) and HPV16-specific IgG and IgM antibodies could represent a significant indicator of risk factors in OSCC patients. PMID:27279791

  9. Using Human iPSC-Derived Neurons to Model TAU Aggregation

    PubMed Central

    Verheyen, An; Diels, Annick; Dijkmans, Joyce; Oyelami, Tutu; Meneghello, Giulia; Mertens, Liesbeth; Versweyveld, Sofie; Borgers, Marianne; Buist, Arjan; Peeters, Pieter; Cik, Miroslav

    2015-01-01

    Alzheimer’s disease and frontotemporal dementia are amongst the most common forms of dementia characterized by the formation and deposition of abnormal TAU in the brain. In order to develop a translational human TAU aggregation model suitable for screening, we transduced TAU harboring the pro-aggregating P301L mutation into control hiPSC-derived neural progenitor cells followed by differentiation into cortical neurons. TAU aggregation and phosphorylation was quantified using AlphaLISA technology. Although no spontaneous aggregation was observed upon expressing TAU-P301L in neurons, seeding with preformed aggregates consisting of the TAU-microtubule binding repeat domain triggered robust TAU aggregation and hyperphosphorylation already after 2 weeks, without affecting general cell health. To validate our model, activity of two autophagy inducers was tested. Both rapamycin and trehalose significantly reduced TAU aggregation levels suggesting that iPSC-derived neurons allow for the generation of a biologically relevant human Tauopathy model, highly suitable to screen for compounds that modulate TAU aggregation. PMID:26720731

  10. Electrostatic origin of in vitro aggregation of human γ-crystallin

    PubMed Central

    Mohr, Benjamin G.; Dobson, Cassidy M.; Garman, Scott C.; Muthukumar, Murugappan

    2013-01-01

    The proteins α-, β-, and γ-crystallins are the major components of the lens in the human eye. Using dynamic light scattering method, we have performed in vitro investigations of protein-protein interactions in dilute solutions of human γ-crystallin and α-crystallin. We find that γ-crystallin spontaneously aggregates into finite-sized clusters in phosphate buffer solutions. There are two distinct populations of unaggregated and aggregated γ-crystallins in these solutions. On the other hand, α-crystallin molecules are not aggregated into large clusters in solutions of α-crystallin alone. When α-crystallin and γ-crystallin are mixed in phosphate buffer solutions, we demonstrate that the clusters of γ-crystallin are prevented. By further investigating the roles of temperature, protein concentration, pH, salt concentration, and a reducing agent, we show that the aggregation of γ-crystallin under our in vitro conditions arises from non-covalent electrostatic interactions. In addition, we show that aggregation of γ-crystallin occurs under the dilute in vitro conditions even in the absence of oxidizing agents that can induce disulfide cross-links, long considered to be responsible for human cataracts. Aggregation of γ-crystallin when maintained under reducing conditions suggests that oxidation does not contribute to the aggregation in dilute solutions. PMID:24089726

  11. RE-186 labeled 16.88 IgM and 88BV59 IgG human antibody studies to assess potential for radioimmunotherapy

    SciTech Connect

    Breitz, H.; Seiler, C.; Weiden, P. ||

    1994-05-01

    Two studies with Re-186-MAG{sub 2}GABA labeled human antibodies were carried out to assess feasibility for radioimmunotherapy. Antibodies 16.88 and 88BV59 react with different epitopes of CTA 16.88, a tumor associated antigen of colorectal carcinoma. In a phase I dose escalation study, 14 patients received 60 mg/m{sup 2} 16.88 IgM MoAb. The dose of Re-186 ranged from 25 mCi/m{sup 2} to 210 mCi/m{sup 2} divided into 3 weekly infusions. In a pilot study with 88BV59, a human IgG3k MoAb, 20 mg antibody was labeled with 25 mCi/m{sup 2} Re-186 and administered to 4 patients with colon carcinoma. Tumor targeting was seen in 12 of 14 patients with 16.88 and all 4 patients with 88BV59. Retention of antibody at the tumor was longer with 88BV59. One patient developed a rash. No other acute or delayed toxicities were observed. Human anti-human antibody did not develop in any patient. The slower metabolism of the 88BV59 IgG suggests that this form of immunoconjugate merits further investigation for use in radioimmunotherapy.

  12. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells

    PubMed Central

    Li, Tong; Paudel, Hemant K.

    2016-01-01

    Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer’s disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445–6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ. PMID:27548710

  13. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells.

    PubMed

    Li, Tong; Paudel, Hemant K

    2016-01-01

    Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer's disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445-6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ. PMID:27548710

  14. Spice active principles as the inhibitors of human platelet aggregation and thromboxane biosynthesis.

    PubMed

    Raghavendra, R H; Naidu, K Akhilender

    2009-07-01

    Spice active principles are reported to have anti-diabetic, anti-hypercholesterolemic, antilithogenic, anti-inflammatory, anti-microbial and anti-cancer properties. In our previous report we have shown that spices and their active principles inhibit 5-lipoxygenase and also formation of leukotriene C4. In this study, we report the modulatory effect of spice active principles viz., eugenol, capsaicin, piperine, quercetin, curcumin, cinnamaldehyde and allyl sulphide on in vitro human platelet aggregation. We have demonstrated that spice active principles inhibit platelet aggregation induced by different agonists, namely ADP (50microM), collagen (500microg/ml), arachidonic acid (AA) (1.0mM) and calcium ionophore A-23187 (20microM). Spice active principles showed preferential inhibition of arachidonic acid-induced platelet aggregation compared to other agonists. Among the spice active principles tested, eugenol and capsaicin are found to be most potent inhibitors of AA-induced platelet aggregation with IC50 values of 0.5 and 14.6microM, respectively. The order of potency of spice principles in inhibiting AA-induced platelet aggregation is eugenol>capsaicin>curcumin>cinnamaldehyde>piperine>allyl sulphide>quercetin. Eugenol is found to be 29-fold more potent than aspirin in inhibiting AA-induced human platelet aggregation. Eugenol and capsaicin inhibited thromboxane B2 (TXB2) formation in platelets in a dose-dependent manner challenged with AA apparently by the inhibition of the cyclooxygenase (COX-1). Eugenol-mediated inhibition of platelet aggregation is further confirmed by dose-dependent decrease in malondialdehyde (MDA) in platelets. Further, eugenol and capsaicin inhibited platelet aggregation induced by agonists-collagen, ADP and calcium ionophore but to a lesser degree compared to AA. These results clearly suggest that spice principles have beneficial effects in modulating human platelet aggregation. PMID:19501497

  15. A Complex Dance: The Importance of Glycosaminoglycans and Zinc in the Aggregation of Human Prolactin.

    PubMed

    Christensen, Line Friis Bakmann; Malmos, Kirsten Gade; Christiansen, Gunna; Otzen, Daniel Erik

    2016-07-01

    The zinc binding hormone pituitary human prolactin (hPRL) is stored in secretory granules of specialized cells in an aggregated form. Glycosaminoglycans (GAGs) are anionic polysaccharides commonly associated with secretory granules, indicating their involvement in granule formation. Here we, for the first time, study the impact of GAGs in combination with Zn(2+) on the reversible hPRL aggregation across the pH range of 7.4-5.5. Zn(2+) alone causes hPRL aggregation at pH 7.4, while aggregation between pH 7.4 and 5.5 requires both Zn(2+) and GAGs. GAGs alone cause hPRL aggregation below pH 5.5. Comprehensive thermal stability investigations show that hPRL is particularly destabilized toward thermal denaturation at pH 5.5 and that GAGs increasingly destabilize hPRL at decreasing pH values. We propose that Zn(2+) causes hPRL aggregation through low-affinity Zn(2+) binding sites on hPRL with GAGs facilitating Zn(2+) binding by neutralizing repulsive positive charges of hPRL in the acidic environments of the TGN and mature secretory granules. In a manner independent of the aggregation-causing agent(s), the different hPRL aggregates show very similar secondary structure and amorphous morphology. We speculate that this may be a recognizable sorting signal in the formation of hPRL granular vesicles. PMID:27305175

  16. Fate of Multimeric Oligomers, Submicron, and Micron Size Aggregates of Monoclonal Antibodies Upon Subcutaneous Injection in Mice.

    PubMed

    Kijanka, Grzegorz; Bee, Jared S; Bishop, Steven M; Que, Ivo; Löwik, Clemens; Jiskoot, Wim

    2016-05-01

    The aim of this study was to examine the fate of differently sized protein aggregates upon subcutaneous injection in mice. A murine and a human monoclonal immunoglobulin G 1 (IgG1) antibody were labeled with a fluorescent dye and subjected to stress conditions to create aggregates. Aggregates fractionated by centrifugation or gel permeation chromatography were administered subcutaneously into SKH1 mice. The biodistribution was measured by in vivo fluorescence imaging for up to 1 week post injection. At several time points, mice were sacrificed and selected organs and tissues were collected for ex vivo analysis. Part of injected aggregated IgGs persisted much longer at the injection site than unstressed controls. Aggregate fractions containing submicron (0.1-1 μm) or micron (1-100 μm) particles were retained to a similar extent. Highly fluorescent "hot-spots" were detected 24 h post injection in spleens of mice injected with submicron aggregates of murine IgG. Submicron aggregates of human IgG showed higher accumulation in draining lymph nodes 1 h post injection than unstressed controls or micron size aggregates. For both tested proteins, aggregated fractions seemed to be eliminated from circulation more rapidly than monomeric fractions. The biodistribution of monomers isolated from solutions subjected to stress conditions was similar to that of unstressed control. PMID:27044942

  17. Effects of the extract from Conyza canadensis on human blood platelet aggregation.

    PubMed

    Saluk-Juszczak, J; Olas, B; Pawlaczyk, I; Gancarz, R; Wachowicz, B

    2007-06-01

    The effects of different parts of extract from medicinal plant Conyza canadensis, used to control bleeding, on human blood platelet aggregation in vitro were investigated. Aqueous extract of Conyza c. from young or old plants, glycoconjugate part, polysaccharide part and aglycon part at the concentrations above 0.75 mg/ml strongly inhibited platelet aggregation induced by collagen (2 microg/ml) in dose-dependent manner. Polysaccharide part isolated from plant extract had the strongest inhibitory effect on aggregation stimulated by collagen and seems to be responsible for antiaggregatory properties. PMID:17660590

  18. Mixed Zwitterion-Based Self-Assembled Monolayer Interface for Impedimetric Glycomic Analyses of Human IgG Samples in an Array Format.

    PubMed

    Bertok, Tomas; Dosekova, Erika; Belicky, Stefan; Holazova, Alena; Lorencova, Lenka; Mislovicova, Danica; Paprckova, Darina; Vikartovska, Alica; Plicka, Robert; Krejci, Jan; Ilcikova, Marketa; Kasak, Peter; Tkac, Jan

    2016-07-19

    An impedimetric lectin biosensor for the detection of changes in the glycan structure of antibodies isolated from human serum is here correlated with the progression of rheumatoid arthritis (RA). The biosensor was built up from a mixed self-assembled monolayer (SAM) on gold consisting of two different thiolated zwitterionic derivatives, carboxybetaine and sulfobetaine, to resist nonspecific interactions. The carboxyl-terminated one was applied also for the covalent immobilization of lectin Ricinus communis agglutinin I (RCA-I). The process of building a bioreceptive layer was optimized and characterized using a diverse range of techniques. Impedimetric assays were integrated on a chip consisting of eight gold working electrodes, which is an important step toward the achievement of a moderate level of multiplexing for the analysis of human serum samples. At the end, the results obtained by the impedimetric analysis of immunoglobulins G (IgGs) isolated from serum samples were compared with those of two other standard bioanalytical methods employing lectins, that is, lectin microarrays (MAs) and enzyme-linked lectin binding assays (ELLBAs). The impedimetric results agreed very well with the DAS28 index (RA disease activity score 28), suggesting that impedimetric assays could be used for the development of a new diagnostic procedure sensitive to glycosylation changes in human IgGs and thus RA progression. PMID:27311591

  19. Loci associated with N-glycosylation of human IgG are not associated with rheumatoid arthritis: a Mendelian randomisation study

    PubMed Central

    Yarwood, Annie; Okada, Yukinori; Plenge, Robert; Yamamoto, Kazuhiko; Barton, Anne; Symmons, Deborah; Raychaudhuri, Soumya; Klareskog, Lars; Gregersen, Peter; Worthington, Jane; Eyre, Steve

    2016-01-01

    Objectives A recent study identified 16 genetic variants associated with N-glycosylation of human IgG. Several of the genomic regions where these single nucleotide polymorphisms (SNPs) reside have also been associated with autoimmune disease (AID) susceptibility, suggesting there may be pleiotropy (genetic sharing) between loci controlling both N-glycosylation and AIDs. We investigated this by testing variants associated with levels of IgG N-glycosylation for association with rheumatoid arthritis (RA) susceptibility using a Mendelian randomisation study, and testing a subset of these variants in a less well-powered study of treatment response and severity. Methods SNPs showing association with IgG N-glycosylation were analysed for association with RA susceptibility in 14 361 RA cases and 43 923 controls. Five SNPs were tested for association with response to anti-tumour necrosis factor (TNF) therapy in 1081 RA patient samples and for association with radiological disease severity in 342 patients. Results Only one SNP (rs9296009) associated with N-glycosylation showed an association (p=6.92×10–266) with RA susceptibility, although this was due to linkage disequilibrium with causal human leukocyte antigen (HLA) variants. Four regions of the genome harboured SNPs associated with both traits (shared loci); although statistical analysis indicated that the associations observed for the two traits are independent. No SNPs showed association with response to anti-TNF therapy. One SNP rs12342831 was modestly associated with Larsen score (p=0.05). Conclusions In a large, well-powered cohort of RA patients, we show SNPs driving levels of N-glycosylation have no association with RA susceptibility, indicating colocalisation of associated SNPs are not necessarily indicative of a shared genetic background or a role for glycosylation in disease susceptibility. PMID:26386125

  20. Cognitive defects are reversible in inducible mice expressing pro-aggregant full-length human Tau

    PubMed Central

    Sydow, Astrid; Hofmann, Anne; Wu, Dan; Messing, Lars; Balschun, Detlef; D'Hooge, Rudi; Mandelkow, Eva-Maria

    2016-01-01

    Neurofibrillary lesions of abnormal Tau are hallmarks of Alzheimer´s disease and frontotemporal dementias. Our regulatable (Tet-OFF) mouse models of tauopathy express variants of human full-length Tau in the forebrain (CaMKIIα promoter) either with mutation ΔK280 (pro-aggregant) or ΔK280/I277P/I308P (anti-aggregant). Co-expression of luciferase enables in vivo quantification of gene expression by bioluminescence imaging. Pro-aggregant mice develop synapse loss and Tau pathology including missorting, phosphorylation and early pretangle formation, whereas anti-aggregant mice do not. We correlated hippocampal Tau pathology with learning/memory performance and synaptic plasticity. Pro-aggregant mice at 16 months of gene expression exhibited severe cognitive deficits in Morris water-maze and in passive-avoidance paradigms, whereas anti-aggregant mice were comparable to controls. Cognitive impairment of pro-aggregant mice was accompanied by loss of hippocampal LTP in CA1 and CA3 areas and by a reduction of synaptic proteins and dendritic spines, although no neuronal loss was observed. Remarkably, memory and LTP recovered when pro-aggregant Tau was switched-OFF for ∼4 months, Tau phosphorylation and missorting were reversed, and synapses recovered. Moreover soluble and insoluble pro-aggregant hTau40 disappeared while insoluble mouse Tau was still present. This study links early Tau pathology without neurofibrillary tangles and neuronal death to cognitive decline and synaptic dysfunction. It demonstrates that Tau-induced impairments are reversible after switching-OFF pro-aggregant Tau. Therefore our mouse model may mimic an early phase of AD when the hippocampus does not yet suffer from irreversible cell death but cognitive deficits are already striking. It offers potential to evaluate drugs with regard to learning and memory performance. PMID:22532069

  1. Compaction, Fusion, and Functional Activation of Three-Dimensional Human Mesenchymal Stem Cell Aggregate

    PubMed Central

    Tsai, Ang-Chen; Liu, Yijun; Yuan, Xuegang

    2015-01-01

    Human mesenchymal stem cells (hMSCs) are primary candidates in cell therapy and tissue engineering and are being tested in clinical trials for a wide range of diseases. Originally isolated and expanded as plastic adherent cells, hMSCs have intriguing properties of in vitro self-assembly into three-dimensional (3D) aggregates that improve a range of biological properties, including multilineage potential, secretion of therapeutic factors, and resistance against ischemic condition. While cell–cell contacts and cell–extracellular matrix interactions mediate 3D cell aggregation, the adaptive changes of hMSC cytoskeleton during self-assembly and associated metabolic reconfiguration may also influence aggregate properties and functional activation. In this study, we investigated the role of actin in regulating 3D hMSC aggregate compaction, fusion, spreading and functional activation. Individual hMSC aggregates with controlled initial cell number were formed by seeding a known number of hMSCs (500, 2000, and 5000 cells/well) in multi-well plates of an ultra-low adherent surface to form multicellular aggregates in individual wells. To assess the influence of actin-mediated contractility on hMSC aggregation and properties, actin modulators, including cytochalasin D (cytoD), nocodazole, lysophosphatidic acid (LPA), and Y-27632, were added at different stages of aggregation and their impacts on hMSC aggregate compaction and apoptosis were monitored. The results suggest that actin-mediated contractility influences hMSC aggregation, compaction, fusion, and spreading on adherent surface. Formation of multi-cellular aggregates significantly upregulated caspase 3/7 expression, expression of C-X-C chemokine receptor type 4 (CXCR-4), cell migration, secretion of prostaglandin E2 (PGE-2) and interleukin 6 (IL-6), and resistance to in vitro ischemic stress. The functional enhancement, however, is dependent on caspase activation, because treatment with Q-VD-OPh, a pan

  2. Structural insights into the interaction of human IgG1 with FcγRI: no direct role of glycans in binding

    SciTech Connect

    Oganesyan, Vaheh Mazor, Yariv; Yang, Chunning; Cook, Kimberly E.; Woods, Robert M.; Ferguson, Andrew; Bowen, Michael A.; Martin, Tom; Zhu, Jie; Wu, Herren; Dall’Acqua, William F.

    2015-10-31

    In an effort to identify the critical structural features responsible for the high-affinity interaction of IgG1 Fc with FcγRI, the structure of the corresponding complex was solved at a resolution of 2.4 Å. The three-dimensional structure of a human IgG1 Fc fragment bound to wild-type human FcγRI is reported. The structure of the corresponding complex was solved at a resolution of 2.4 Å using molecular replacement; this is the highest resolution achieved for an unmutated FcγRI molecule. This study highlights the critical structural and functional role played by the second extracellular subdomain of FcγRI. It also explains the long-known major energetic contribution of the Fc ‘LLGG’ motif at positions 234–237, and particularly of Leu235, via a ‘lock-and-key’ mechanism. Finally, a previously held belief is corrected and a differing view is offered on the recently proposed direct role of Fc carbohydrates in the corresponding interaction. Structural evidence is provided that such glycan-related effects are strictly indirect.

  3. The release of nucleotides, 5-hydroxytryptamine and enzymes from human blood platelets during aggregation

    PubMed Central

    Mills, D. C. B.; Robb, I. A.; Roberts, G. C. K.

    1968-01-01

    1. Adenosine diphosphate (ADP) and adrenaline caused the aggregation of human platelets suspended in plasma containing citrate anticoagulant and stirred at 37° C. The aggregation occurred in two phases and the second phase was associated with the appearance in the plasma of up to 30% of the ATP and 55% of the ADP present in the platelets. The concentration of ADP appearing in the plasma was up to 7 times the concentration added. 2. Radioactivity was released by ADP and by adrenaline from platelets labelled with radioactive 5-hydroxytryptamine; this release was closely correlated with the second phase of aggregation and with the release of nucleotides. 3. Acid phosphatase, β-glucuronidase and adenylate kinase were released to a small extent during second phase aggregation by ADP or adrenaline; thrombin and collagen particles caused significantly greater release of β-glucuronidase than of either acid phosphatase or of adenylate kinase. 4. Morphological changes indicating degranulation of the platelets were observed during the second phase of aggregation produced by adrenaline and by ADP. 5. The second phase of aggregation, degranulation of platelets, and the release of nucleotides, of labelled 5-hydroxytryptamine and of enzymes, were all inhibited by concentrations of amitriptyline which did not inhibit aggregation. ImagesPlate 1Plate 2 PMID:5649642

  4. Sulfur mustard induces the formation of keratin aggregates in human epidermal keratinocytes.

    PubMed

    Dillman, James F; McGary, Kriston L; Schlager, John J

    2003-12-01

    The vesicant sulfur mustard is an alkylating agent that has the capacity to cross-link biological molecules. We are interested in identifying specific proteins that are altered upon sulfur mustard exposure. Keratins are particularly important for the structural integrity of skin, and several genetically inherited blistering diseases have been linked to mutations in keratin 5 and keratin 14. We examined whether sulfur mustard exposure alters keratin biochemistry in cultured human epidermal keratinocytes. Western blotting with specific monoclonal antibodies revealed the formation of stable high-molecular-weight "aggregates" containing keratin 14 and/or keratin 5. These aggregates begin to form within 15 min after sulfur mustard exposure. These aggregates display a complex gel electrophoresis pattern between approximately 100 and approximately 200 kDa. Purification and analysis of these aggregates by one- and two-dimensional gel electrophoresis and mass spectrometry confirmed the presence of keratin 14 and keratin 5 and indicate that at least some of the aggregates are composed of keratin 14-keratin 14, keratin 14-keratin 5, or keratin 5-keratin 5 dimers. These studies demonstrate that sulfur mustard induces keratin aggregation in keratinocytes and support further investigation into the role of keratin aggregation in sulfur mustard-induced vesication. PMID:14644625

  5. Sulfate anion delays the self-assembly of human insulin by modifying the aggregation pathway.

    PubMed

    Owczarz, Marta; Arosio, Paolo

    2014-07-01

    The understanding of the molecular mechanisms underlying protein self-assembly and of their dependence on solvent composition has implications in a large number of biological and biotechnological systems. In this work, we characterize the aggregation process of human insulin at acidic pH in the presence of sulfate ions using a combination of Thioflavin T fluorescence, dynamic light scattering, size exclusion chromatography, Fourier transform infrared spectroscopy, and transmission electron microscopy. It is found that the increase of sulfate concentration inhibits the conversion of insulin molecules into aggregates by modifying the aggregation pathway. At low sulfate concentrations (0-5 mM) insulin forms amyloid fibrils following the nucleated polymerization mechanism commonly observed under acidic conditions in the presence of monovalent anions. When the sulfate concentration is increased above 5 mM, the sulfate anion induces the salting-out of ∼18-20% of insulin molecules into reversible amorphous aggregates, which retain a large content of α-helix structures. During time these aggregates undergo structure rearrangements into β-sheet structures, which are able to recruit monomers and bind to the Thioflavin T dye. The alternative aggregation mechanism observed at large sulfate concentrations is characterized by a larger activation energy and leads to more polymorphic structures with respect to the self-assembly in the presence of chloride ions. The system shown in this work represents a case where amorphous aggregates on pathway to the formation of structures with amyloid features could be detected and analyzed. PMID:24988354

  6. Sulfate Anion Delays the Self-Assembly of Human Insulin by Modifying the Aggregation Pathway

    PubMed Central

    Owczarz, Marta; Arosio, Paolo

    2014-01-01

    The understanding of the molecular mechanisms underlying protein self-assembly and of their dependence on solvent composition has implications in a large number of biological and biotechnological systems. In this work, we characterize the aggregation process of human insulin at acidic pH in the presence of sulfate ions using a combination of Thioflavin T fluorescence, dynamic light scattering, size exclusion chromatography, Fourier transform infrared spectroscopy, and transmission electron microscopy. It is found that the increase of sulfate concentration inhibits the conversion of insulin molecules into aggregates by modifying the aggregation pathway. At low sulfate concentrations (0–5 mM) insulin forms amyloid fibrils following the nucleated polymerization mechanism commonly observed under acidic conditions in the presence of monovalent anions. When the sulfate concentration is increased above 5 mM, the sulfate anion induces the salting-out of ∼18–20% of insulin molecules into reversible amorphous aggregates, which retain a large content of α-helix structures. During time these aggregates undergo structure rearrangements into β-sheet structures, which are able to recruit monomers and bind to the Thioflavin T dye. The alternative aggregation mechanism observed at large sulfate concentrations is characterized by a larger activation energy and leads to more polymorphic structures with respect to the self-assembly in the presence of chloride ions. The system shown in this work represents a case where amorphous aggregates on pathway to the formation of structures with amyloid features could be detected and analyzed. PMID:24988354

  7. Structural insights into the interaction of human IgG1 with FcγRI: no direct role of glycans in binding

    PubMed Central

    Oganesyan, Vaheh; Mazor, Yariv; Yang, Chunning; Cook, Kimberly E.; Woods, Robert M.; Ferguson, Andrew; Bowen, Michael A.; Martin, Tom; Zhu, Jie; Wu, Herren; Dall’Acqua, William F.

    2015-01-01

    The three-dimensional structure of a human IgG1 Fc fragment bound to wild-type human FcγRI is reported. The structure of the corresponding complex was solved at a resolution of 2.4 Å using molecular replacement; this is the highest resolution achieved for an unmutated FcγRI molecule. This study highlights the critical structural and functional role played by the second extracellular subdomain of FcγRI. It also explains the long-known major energetic contribution of the Fc ‘LLGG’ motif at positions 234–237, and particularly of Leu235, via a ‘lock-and-key’ mechanism. Finally, a previously held belief is corrected and a differing view is offered on the recently proposed direct role of Fc carbohydrates in the corresponding interaction. Structural evidence is provided that such glycan-related effects are strictly indirect. PMID:26527150

  8. Effects of tetrandrine and fangchinoline on human platelet aggregation and thromboxane B2 formation.

    PubMed

    Kim, H S; Zhang, Y H; Fang, L H; Yun, Y P; Lee, H K

    1999-08-01

    Tetrandrine (TET) and fangchinoline (FAN) are two major components of the radix of Stephania tetrandra. The effects of TET and FAN on human platelet aggregation and formation of thromboxane (TX) B2, a stable metabolite of TXA2, were examined in the aspect of platelet aggregation. TET and FAN inhibited platelet-activating factor (PAF)-induced human platelet aggregation. IC50 values for TET and FAN were 28.6+/-3.24 microM and 21.7+/-2.61 microM, respectively. In the PAF-receptor binding assay, neither TET nor FAN showed any inhibitory effects on the specific bindings of PAF to its receptor. TET and FAN also inhibited PAF-, thrombin- and arachidonic acid-induced thromboxane B2 formation in human washed platelet. These results indicate that TET and FAN inhibit the platelet aggregation by interfering with the intracellular messengers system, but not by inhibiting the binding of PAF to PAF-receptor on the platelet membrane directly, and the suppression of TXA2 formation by TET and FAN may be responsible for their inhibitory activities on the platelet aggregation and further on the thrombosis. PMID:10433485

  9. Study on the production of IgG-, IgA- and IgM-antibodies to somatic antigens of Salmonella typhi in humans

    PubMed Central

    Chernokhvostova, Elena; Luxemburg, K. I.; Starshinova, Valentina; Andreeva, Natalia; German, Galina

    1969-01-01

    The immune response to O- and Vi-antigens of Salmonella typhi in humans was studied under a variety of conditions. In sera of persons immunized with various typhoid vaccines and with chemically purified Vi-antigen of S. typhi, anti-Vi-antibodies of three main immunoglobulin types (IgG, IgA and IgM) were found, but anti-O-antibodies were of IgM-type only. In sera of typhoid patients anti-O-antibodies of IgG-, IgA- and IgM-types were detected. Anti-Vi-antibodies appearing in the course of typhoid fever were heterogeneous to the same extent as anti-O-antibodies. The antibody response to Vi-antigen administered subcutaneously was quite similar in typhoid patients and in healthy individuals. Both anti-O- and anti-Vi-antibodies in sera of chronic typhoid carriers were usually of IgG-type only. Immunization of typhoid carriers with Vi-antigen was followed by the significant augmentation of IgG-antibody level, not preceded by IgM-antibody production. The possible reasons of IgM-deficiency in typhoid carrier state are discussed. ImagesFIG. 1 PMID:4182404

  10. The effects of selected drugs, including chlorpromazine and non-steroidal anti-inflammatory agents, on polyclonal IgG synthesis and interleukin 1 production by human peripheral blood mononuclear cells in vitro.

    PubMed

    Martinez, F; Coleman, J W

    1989-05-01

    We tested a range of drugs for their effects on in vitro polyclonal IgG synthesis by human peripheral blood mononuclear cells (PBMC) stimulated with the lectin pokeweed mitogen (PWM). The test drugs were selected on the basis of reported disruptive effects on immune function in vivo. IgG production between day 4 and days 7 or 8 of culture was measured by biotin-streptavidin sandwich ELISA. The anti-psychotic agent chlorpromazine (0.55-1.7 microM) enhanced IgG synthesis to approximately double control levels. In contrast, the non-steroidal anti-inflammatory drugs (NSAIDs) indomethacin, piroxicam, ibuprofen and aspirin inhibited IgG synthesis by up to 50%, with a rank order of potency that reflects their activity as inhibitors of cyclo-oxygenase. Phenytoin, procainamide, propylthiouracil, methimazole, D-penicillamine and D-penicillamine-L-cysteine all failed to modulate IgG synthesis at non-toxic concentrations. The potentiation and inhibition of IgG synthesis by chlorpromazine and indomethacin, respectively, was observed only when the drug was present during the first 24 h of culture. Neither chlorpromazine nor indomethacin, at non-toxic concentrations, affected PHA- and PWM-stimulated proliferation of PBMC. In addition, chlorpromazine, indomethacin and piroxicam, at concentrations which produced maximal modulation of IgG synthesis, and D-penicillamine and D-penicillamine-L-cysteine at 10 microM failed to influence production of interleukin-1-like activity. We conclude that chlorpromazine and NSAIDs, although they exert opposite effects on IgG synthesis, act at an early stage of B cell differentiation that appears to be independent of interleukin 1 synthesis and early proliferative events. PMID:2788047

  11. Influence of Inorganic Ions on Aggregation and Adsorption Behaviors of Human Adenovirus

    EPA Science Inventory

    In this study, we investigated the influence of inorganic ions on the aggregation and deposition (adsorption) behavior of human adenovirus (HAdV). Experiments were conducted to determine the surface charge and size of HAdV and viral adsorption capacity of sand in different salt c...

  12. Influence of Inorganic Ions and Aggregation and Adsorption Behaviors of Human Adenovirus

    EPA Science Inventory

    In this study, influence of solution chemistries to the transport properties (aggregation and attachment behavior) of human adenovirus (HAdV) was investigated. Results showed isoelectric point (IEP) of HAdV in different salt conditions varied minimally, and it ranged from pH 3.5 ...

  13. Solution structure, aggregation behavior, and flexibility of human relaxin-2.

    PubMed

    Haugaard-Kedström, Linda M; Hossain, Mohammed Akhter; Daly, Norelle L; Bathgate, Ross A D; Rinderknecht, Ernst; Wade, John D; Craik, David J; Rosengren, K Johan

    2015-03-20

    Relaxin is a member of the relaxin/insulin peptide hormone superfamily and is characterized by a two-chain structure constrained by three disulfide bonds. Relaxin is a pleiotropic hormone and involved in a number of physiological and pathogenic processes, including collagen and cardiovascular regulation and tissue remodelling during pregnancy and cancer. Crystallographic and ultracentrifugation experiments have revealed that the human form of relaxin, H2 relaxin, self-associates into dimers, but the significance of this is poorly understood. Here, we present the NMR structure of a monomeric, amidated form of H2 relaxin and compare its features and behavior in solution to those of native H2 relaxin. The overall structure of H2 relaxin is retained in the monomeric form. H2 relaxin amide is fully active at the relaxin receptor RXFP1 and thus dimerization is not required for biological activity. Analysis of NMR chemical shifts and relaxation parameters identified internal motion in H2 relaxin at the pico-nanosecond and milli-microsecond time scales, which is commonly seen in other relaxin and insulin peptides and might be related to function. PMID:25547165

  14. IgG and complement deposition and neuronal loss in cats and humans with epilepsy and voltage-gated potassium channel complex antibodies.

    PubMed

    Klang, Andrea; Schmidt, Peter; Kneissl, Sibylle; Bagó, Zoltán; Vincent, Angela; Lang, Bethan; Moloney, Teresa; Bien, Christian G; Halász, Péter; Bauer, Jan; Pákozdy, Akos

    2014-05-01

    Voltage-gated potassium channel complex (VGKC-complex) antibody (Ab) encephalitis is a well-recognized form of limbic encephalitis in humans, usually occurring in the absence of an underlying tumor. The patients have a subacute onset of seizures, magnetic resonance imaging findings suggestive of hippocampal inflammation, and high serum titers of Abs against proteins of the VGKC-complex, particularly leucine-rich, glioma-inactivated 1 (LGI1). Most patients are diagnosed promptly and recover substantially with immunotherapies; consequently, neuropathological data are limited. We have recently shown that feline complex partial cluster seizures with orofacial involvement (FEPSO) in cats can also be associated with Abs against VGKC-complexes/LGI1. Here we examined the brains of cats with FEPSO and compared the neuropathological findings with those in a human with VGKC-complex-Ab limbic encephalitis. Similar to humans, cats with VGKC-complex-Ab and FEPSO have hippocampal lesions with only moderate T-cell infiltrates but with marked IgG infiltration and complement C9neo deposition on hippocampal neurons, associated with neuronal loss. These findings provide further evidence that FEPSO is a feline form of VGKC-complex-Ab limbic encephalitis and provide a model for increasing understanding of the human disease. PMID:24709680

  15. Characterization of the biological anti-staphylococcal functionality of hUK-66 IgG1, a humanized monoclonal antibody as substantial component for an immunotherapeutic approach

    PubMed Central

    Oesterreich, Babett; Lorenz, Birgit; Schmitter, Tim; Kontermann, Roland; Zenn, Michael; Zimmermann, Bastian; Haake, Markus; Lorenz, Udo; Ohlsen, Knut

    2014-01-01

    Multi-antigen immunotherapy approaches against Staphylococcus aureus are expected to have the best chance of clinical success when used in combinatorial therapy, potentially incorporating opsonic killing of bacteria and toxin neutralization. We recently reported the development of a murine monoclonal antibody specific for the immunodominant staphylococcal antigen A (IsaA), which showed highly efficient staphylococcal killing in experimental infection models of S. aureus. If IsaA-specific antibodies are to be used as a component of combination therapy in humans, the binding specificity and biological activity of the humanized variant must be preserved. Here, we describe the functional characterization of a humanized monoclonal IgG1 variant designated, hUK-66. The humanized antibody showed comparable binding kinetics to those of its murine parent, and recognized the target antigen IsaA on the surface of clinically relevant S. aureus lineages. Furthermore, hUK-66 enhances the killing of S. aureus in whole blood (a physiological environment) samples from healthy subjects and patients prone to staphylococcal infections such as diabetes and dialysis patients, and patients with generalized artery occlusive disease indicating no interference with already present natural antibodies. Taken together, these data indicate that hUK-66 mediates bacterial killing even in high risk patients and thus, could play a role for immunotherapy strategies to combat severe S. aureus infections. PMID:24495867

  16. Amorphous silica nanoparticles aggregate human platelets: potential implications for vascular homeostasis

    PubMed Central

    Corbalan, J Jose; Medina, Carlos; Jacoby, Adam; Malinski, Tadeusz; Radomski, Marek W

    2012-01-01

    Background Amorphous silica nanoparticles (SiNP) can be used in medical technologies and other industries leading to human exposure. However, an increased number of studies indicate that this exposure may result in cardiovascular inflammation and damage. A high ratio of nitric oxide to peroxynitrite concentrations ([NO]/[ONOO−]) is crucial for cardiovascular homeostasis and platelet hemostasis. Therefore, we studied the influence of SiNP on the platelet [NO]/[ONOO−] balance and platelet aggregation. Methods Nanoparticle–platelet interaction was examined using transmission electron microscopy. Electrochemical nanosensors were used to measure the levels of NO and ONOO− released by platelets upon nanoparticle stimulation. Platelet aggregation was studied using light aggregometry, flow cytometry, and phase contrast microscopy. Results Amorphous SiNP induced NO release from platelets followed by a massive stimulation of ONOO− leading to an unfavorably low [NO]/[ONOO−] ratio. In addition, SiNP induced an upregulation of selectin P expression and glycoprotein IIb/IIIa activation on the platelet surface membrane, and led to platelet aggregation via adenosine diphosphate and matrix metalloproteinase 2-dependent mechanisms. Importantly, all the effects on platelet aggregation were inversely proportional to nanoparticle size. Conclusions The exposure of platelets to amorphous SiNP induces a critically low [NO]/[ONOO−] ratio leading to platelet aggregation. These findings provide new insights into the pharmacological profile of SiNP in platelets. PMID:22334785

  17. Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins.

    PubMed

    Kötter, Sebastian; Unger, Andreas; Hamdani, Nazha; Lang, Patrick; Vorgerd, Matthias; Nagel-Steger, Luitgard; Linke, Wolfgang A

    2014-01-20

    In myocytes, small heat shock proteins (sHSPs) are preferentially translocated under stress to the sarcomeres. The functional implications of this translocation are poorly understood. We show here that HSP27 and αB-crystallin associated with immunoglobulin-like (Ig) domain-containing regions, but not the disordered PEVK domain (titin region rich in proline, glutamate, valine, and lysine), of the titin springs. In sarcomeres, sHSP binding to titin was actin filament independent and promoted by factors that increased titin Ig unfolding, including sarcomere stretch and the expression of stiff titin isoforms. Titin spring elements behaved predominantly as monomers in vitro. However, unfolded Ig segments aggregated, preferentially under acidic conditions, and αB-crystallin prevented this aggregation. Disordered regions did not aggregate. Promoting titin Ig unfolding in cardiomyocytes caused elevated stiffness under acidic stress, but HSP27 or αB-crystallin suppressed this stiffening. In diseased human muscle and heart, both sHSPs associated with the titin springs, in contrast to the cytosolic/Z-disk localization seen in healthy muscle/heart. We conclude that aggregation of unfolded titin Ig domains stiffens myocytes and that sHSPs translocate to these domains to prevent this aggregation. PMID:24421331

  18. Aggregate formation and suspension culture of human pluripotent stem cells and differentiated progeny.

    PubMed

    Hookway, Tracy A; Butts, Jessica C; Lee, Emily; Tang, Hengli; McDevitt, Todd C

    2016-05-15

    Culture of human pluripotent stem cells (hPSC) as in vitro multicellular aggregates has been increasingly used as a method to model early embryonic development. Three-dimensional assemblies of hPSCs facilitate interactions between cells and their microenvironment to promote morphogenesis, analogous to the multicellular organization that accompanies embryogenesis. In this paper, we describe a method for reproducibly generating and maintaining populations of homogeneous three-dimensional hPSC aggregates using forced aggregation and rotary orbital suspension culture. We propose solutions to several challenges associated with the consistent formation and extended culture of cell spheroids generated from hPSCs and their differentiated progeny. Further, we provide examples to demonstrate how aggregation can be used as a tool to select specific subpopulations of cells to create homotypic spheroids, or as a means to introduce multiple cell types to create heterotypic tissue constructs. Finally, we demonstrate that the aggregation and rotary suspension method can be used to support culture and maintenance of hPSC-derived cell populations representing each of the three germ layers, underscoring the utility of this platform for culturing many different cell types. PMID:26658353

  19. Radioimmunotherapy of human head and neck squamous cell carcinoma xenografts with 131I-labelled monoclonal antibody E48 IgG.

    PubMed Central

    Gerretsen, M.; Schrijvers, A. H.; van Walsum, M.; Braakhuis, B. J.; Quak, J. J.; Meijer, C. J.; Snow, G. B.; van Dongen, G. A.

    1992-01-01

    Monoclonal antibody (MAb) E48 reacts with a 22 kD antigen exclusively expressed in squamous and transitional epithelia and their neoplastic counterparts. Radiolabelled with 99mTc, MAb E48 is capable of targeting metastatic and recurrent disease in patients with head and neck cancer. In this study, the capacity of 131I-labelled MAb E48 to eradicate xenografts of human squamous cell carcinoma of the head and neck (HNSCC) in nude mice was examined. Experimental groups received a single i.v. bolus injection of 400 microCi MAb E48 IgG (number of mice (n = 6, number of tumours (t) = 9) or 800 microCi MAb E48 IgG (n) = 5,t = 7), whereas control groups received either diluent (n = 3,t = 5), unlabelled MAb E48 IgG (n = 4,t = 5) or 800 microCi 131I-labelled isotype-matched control MAb (n = 6,t = 9). A 4.1-fold increase in the median tumour volume doubling time and regression of two out of ten tumours (20%) was observed in mice treated with 400 microCi. In mice treated with 800 microCi. In mice treated with 800 microCi, two out of seven tumours (29%) showed complete remission without regrowth during follow-up (greater than 3 months). Median tumour volume doubling time in the remaining five tumours was increased 7.8-fold. No antitumour effects were observed in mice injected with diluent, unlabelled MAb E48 or 131I-labelled control MAb. In the same xenograft model, chemotherapy with doxorubicin, 5-fluorouracil, cisplatin, bleomycin, methotrexate or 2',2'-difluorodeoxycytidine yielded a less profound effect on tumour volume doubling time. Increases in tumour volume doubling time with these chemotherapeutic agents were 4, 2.2, 2.1, 1.7, 0, and 2.6 respectively. Moreover, no cures were observed with any of these chemotherapeutic agents. From the tissue distribution of 800 microCi MAb E48, the absorbed cumulative radiation doses of tumour and various organs were calculated using the trapezoid integration method for the area under the curve. To tumour xenografts, 12,170 cGy was

  20. Mechanism-Based Competitive Binding Model to Investigate the Effect of Neonatal Fc Receptor Binding Affinity on the Pharmacokinetic of Humanized Anti-VEGF Monoclonal IgG1 Antibody in Cynomolgus Monkey.

    PubMed

    Ng, Chee M; Fielder, Paul J; Jin, Jin; Deng, Rong

    2016-07-01

    The quantitative relationship between neonatal Fc receptor (FcRn) binding affinity at both acidic and physiological pH and the pharmacokinetics of protein engineered FcRn IgG1 variants has not yet been reported. Our objective was to develop a quantitatively mechanism-based competitive binding model to describe the effects of FcRn binding affinity at acidic and physiological pH on the pharmacokinetics of anti-VEGF IgG1 antibodies when both endogenous and exogenous antibodies are competing for the same FcRn. Pharmacokinetic (PK) and FcRn binding data from five Fc variants of humanized anti-VEGF IgG1 monoclonal antibodies with wide range of FcRn binding affinity were used for the analysis. Sixty-seven anti-VEGF IgG1 antibody-treated animals and 25 control animals with simulated endogenous IgG levels were used to develop the final model. A hybrid iterative two stages and Monte Carlo parametric expectation-maximization method was used to obtain the final model parameters estimates. The final model well described the observed PK data. Quantitative FcRn binding affinity-pharmacokinetics relationships was constructed to provide important biological insights in better understanding of the FcRn binding effect on pharmacokinetics of anti-VEGF IgG1 antibodies in cynomolgus monkeys and served as an important model-based drug discovery platform to guide the design and development of the future generation of anti-VEGF or other therapeutic IgG1 antibodies. PMID:27075465

  1. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    SciTech Connect

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan; Li, Lin; Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen; Jiang, Shibo

    2010-07-30

    Research highlights: {yields} One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. {yields} N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. {yields} These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

  2. Inhibitory effects of kiwifruit extract on human platelet aggregation and plasma angiotensin-converting enzyme activity.

    PubMed

    Dizdarevic, Lili L; Biswas, Dipankar; Uddin, M D Main; Jørgenesen, Aud; Falch, Eva; Bastani, Nasser E; Duttaroy, Asim K

    2014-01-01

    Previous human studies suggest that supplementation with kiwifruits lowers several cardiovascular risk factors such as platelet hyperactivity, blood pressure and plasma lipids. The cardiovascular health benefit of fruit and vegetables is usually attributed to the complex mixture of phytochemicals therein; however, kiwifruit's cardioprotective factors are not well studied. In this study, we investigated the effects of kiwifruit extract on human blood platelet aggregation and plasma angiotensin-converting enzyme (ACE) activity. A sugar-free, heat-stable aqueous extract with molecular mass less than 1000 Da was prepared from kiwifruits. Typically, 100 g kiwifruits produced 66.3 ± 5.8 mg (1.2 ± 0.1 mg CE) of sugar-free kiwifruit extract (KFE). KFE inhibited both human platelet aggregation and plasma ACE activity in a dose-dependent manner. KFE inhibited platelet aggregation in response to ADP, collagen and arachidonic acid, and inhibitory action was mediated in part by reducing TxA2 synthesis. The IC50 for ADP-induced platelet aggregation was 1.6 ± 0.2 mg/ml (29.0 ± 3.0 μg CE/ml), whereas IC50 for serum ACE was 0.6 ± 0.1 mg/ml (11.0 ± 1.2 μg CE/ml). Consuming 500 mg of KFE (9.0 mg CE) in 10 g margarine inhibited ex vivo platelet aggregation by 12.7%, 2 h after consumption by healthy volunteers (n = 9). All these data indicate that kiwifruit contains very potent antiplatelet and anti-ACE components. Consuming kiwifruits might be beneficial as both preventive and therapeutic regime in cardiovascular disease. PMID:24219176

  3. Inhibitory Effects of Yuzu and Its Components on Human Platelet Aggregation

    PubMed Central

    Kim, Tae-Ho; Kim, Hye-Min; Park, Se Won; Jung, Yi-Sook

    2015-01-01

    Our previous study demonstrated that yuzu has an anti-platelet effect in rat blood. In the present study, we examined whether the anti-platelet effect of yuzu can be extended to human blood by investigating its ability to inhibit aggregations induced by various agonists in human platelet rich plasma (PRP). This study also investigated the underlying mechanism of yuzu focusing on ADP granule secretion, TXB2 formations, and PLCγ/Akt signaling. The results from this study showed that ethanolic yuzu extract (YE), and its components, hesperidin and naringin, inhibited human platelet aggregation in a concentration-dependent manner. YE, hesperidin and naringin also inhibited TXB2 formation and ADP release. The phosphorylation of PLCγ and Akt was significantly inhibited by YE, heperidin and naringin. Furthermore, we demonstrated that YE, heperidin and naringin has anti-platelet effects in rat ex vivo studies, and lower side effects in mice tail bleeding time studies. The results from this study suggest that YE, hesperidin and naringin can inhibit human platelet aggregation, at least partly through the inhibition of PLCγ and Akt, leading to a decrease in TXB2 formation and granule secretion. PMID:25767683

  4. Conformational and Colloidal Stabilities of Isolated Constant Domains of Human Immunoglobulin G and Their Impact on Antibody Aggregation under Acidic Conditions.

    PubMed

    Yageta, Seiki; Lauer, Timothy M; Trout, Bernhardt L; Honda, Shinya

    2015-05-01

    Antibody therapeutics are now in widespread use and provide a new approach for treating serious diseases such as rheumatic diseases and cancer. Monoclonal antibodies used as therapeutic agents must be of high quality, and their safety must be guaranteed. Aggregated antibody is a degradation product that may be generated during the manufacturing process. To maintain the high quality and safety of antibody therapeutics, it is necessary to understand the mechanism of aggregation and to develop technologies to strictly control aggregate formation. Here, we extensively investigated the conformational and colloidal characteristics of isolated antibody constant domains, and provided insights into the molecular mechanism of antibody aggregation. Isolated domains (CH2, CH3, CL, and CH1-CL dimer) of human immunoglobulin G were synthesized, solubilized using 49 sets of solution conditions (pH 2-8 and 0-300 mM NaCl), and characterized using circular dichroism, intrinsic tryptophan fluorescence, and dynamic light scattering. Salt-induced conformational changes and oligomer formation were kinetically analyzed by NaCl-jump measurements (from 0 to 300 mM at pH 3). Phase diagrams revealed that the domains have different conformational and colloidal stabilities. The unfolded fractions of CH3 and CH2 at pH 3 were larger than that of CL and CH1-CL dimer. The secondary and tertiary structures and particle sizes of CH3 and CH2 showed that, in non-native states, these domains were sensitive to salt concentration. Kinetic analyses suggest that oligomer formation by CH3 and CH2 proceeds through partially refolded conformations. The colloidal stability of CH3 in non-native states is the lowest of the four domains under the conditions tested. We propose that the impact of IgG constant domains on aggregation follows the order CH3 > CH2 > CH1-CL dimer > CL; furthermore, we suggest that CH3 plays the most critical role in driving intact antibody aggregation under acidic conditions. PMID

  5. Anion binding and controlled aggregation of human interleukin-1 receptor antagonist.

    PubMed

    Raibekas, Andrei A; Bures, Edward J; Siska, Christine C; Kohno, Tadahiko; Latypov, Ramil F; Kerwin, Bruce A

    2005-07-26

    Highly concentrated human recombinant interleukin-1 receptor antagonist (IL-1ra) aggregates at elevated temperature without perturbation in its secondary structure. The protein aggregation can be suppressed depending on the buffer ionic strength and the type of anion present in the sample solution. Phosphate is an approximately 4-fold weaker suppressant than either citrate or pyrophosphate on the basis of the measured protein aggregation rates. This is in agreement with the strength of protein-anion interactions at the IL-1ra single anion-binding site as judged by the estimated dissociation constant values of 2.9 mM, 3.8 mM, and 13.7 mM for pyrophosphate, citrate, and phosphate, respectively. The strength of binding also correlates with the anion size and with the number of ionized groups available per molecule at a given pH. Affinity probing of IL-1ra with methyl acetyl phosphate (MAP) in combination with proteolytic digestion and mass spectral analysis show that an anion-binding site location on the IL-1ra surface is contributed by lysine-93 and lysine-96 of the loop 84-98 as well as by lysine-6 of the unstructured N-terminal region 1-7. The replacement of lysine-93 with alanine by site-directed mutagenesis results in dramatically suppressed IL-1ra aggregation. Furthermore, when the unstructured N-terminal region of IL-1ra is removed by limited proteolysis, a 2-fold increase in the time course of the aggregation lag phase is observed for the truncated protein. An anion-controlled mechanism of IL-1ra aggregation is proposed by which the anion competition for the protein cationic site prevents formation of intermolecular cation-pi interactions and, thus, interferes with the protein asymmetric self-association pathway. PMID:16026159

  6. Pathogenicity and Epitope Characteristics Do Not Differ in IgG Subclass-Switched Anti-Desmoglein 3 IgG1 and IgG4 Autoantibodies in Pemphigus Vulgaris

    PubMed Central

    Ellebrecht, Christoph T.; Yu, Xiaocong; Posner, Marshall R.; Payne, Aimee S.

    2016-01-01

    Pemphigus vulgaris (PV) is characterized by IgG1 and IgG4 autoantibodies to desmoglein (Dsg) 3, causing suprabasal blistering of skin and mucous membranes. IgG4 is the dominant autoantibody subclass in PV and correlates with disease activity, whereas IgG1 can be associated with remittent disease. It is unknown if switching the same variable region between IgG4 and IgG1 directly impacts pathogenicity. Here, we tested whether three pathogenic PV monoclonal antibodies (mAbs) from three different patients demonstrate differences in antigen affinity, epitope specificity, or pathogenicity when expressed as IgG1 or IgG4. F706 anti-Dsg3 IgG4 and F779 anti-Dsg3 IgG1, previously isolated as heterohybridomas, and Px43, a monovalent anti-Dsg3/Dsg1 IgG antibody isolated by phage display, were subcloned to obtain paired sets of IgG1 and IgG4 mAbs. Using ELISA and cell surface staining assays, F706 and F779 demonstrated similar antigen binding affinities of IgG1 and IgG4, whereas Px43 showed 3- to 8-fold higher affinity of IgG4 versus IgG1 by ELISA, but identical binding affinities to human skin, perhaps due to targeting of a quaternary epitope best displayed in tissues. All 3 mAb pairs targeted the same extracellular cadherin (EC) domain on Dsg3, caused Dsg3 internalization in primary human keratinocytes, and caused suprabasal blisters in human skin at comparable doses. We conclude that switching IgG1 and IgG4 subclasses of pathogenic PV mAbs does not directly affect their antigen binding or pathogenic properties. PMID:27304671

  7. Uptake and Degradation of Protease-Sensitive and -Resistant Forms of Abnormal Human Prion Protein Aggregates by Human Astrocytes

    PubMed Central

    Choi, Young Pyo; Head, Mark W.; Ironside, James W.; Priola, Suzette A.

    2015-01-01

    Sporadic Creutzfeldt-Jakob disease is the most common of the human prion diseases, a group of rare, transmissible, and fatal neurologic diseases associated with the accumulation of an abnormal form (PrPSc) of the host prion protein. In sporadic Creutzfeldt-Jakob disease, disease-associated PrPSc is present not only as an aggregated, protease-resistant form but also as an aggregated protease-sensitive form (sPrPSc). Although evidence suggests that sPrPSc may play a role in prion pathogenesis, little is known about how it interacts with cells during prion infection. Here, we show that protease-sensitive abnormal PrP aggregates derived from patients with sporadic Creutzfeldt-Jakob disease are taken up and degraded by immortalized human astrocytes similarly to abnormal PrP aggregates that are resistant to proteases. Our data suggest that relative proteinase K resistance does not significantly influence the astrocyte's ability to degrade PrPSc. Furthermore, the cell does not appear to distinguish between sPrPSc and protease-resistant PrPSc, suggesting that sPrPSc could contribute to prion infection. PMID:25280631

  8. Retinoic acid-induced IgG production in TLR-activated human primary B cells involves ULK1-mediated autophagy

    PubMed Central

    Eriksen, Agnete Bratsberg; Torgersen, Maria Lyngaas; Holm, Kristine Lillebø; Abrahamsen, Greger; Spurkland, Anne; Moskaug, Jan Øivind; Simonsen, Anne; Blomhoff, Heidi Kiil

    2015-01-01

    In the present study we have established a vital role of autophagy in retinoic acid (RA)-induced differentiation of toll-like receptor (TLR)-stimulated human B cells into Ig-secreting cells. Thus, RA enhanced autophagy in TLR9- and CD180-stimulated peripheral blood B cells, as revealed by increased levels of the autophagosomal marker LC3B-II, enhanced colocalization between LC3B and the lysosomal marker Lyso-ID, by a larger percentage of cells with more than 5 characteristic LC3B puncta, and by the concomitant reduction in the level of SQSTM1/p62. Furthermore, RA induced expression of the autophagy-inducing protein ULK1 at the transcriptional level, in a process that required the retinoic acid receptor RAR. By inhibiting autophagy with specific inhibitors or by knocking down ULK1 by siRNA, the RA-stimulated IgG production in TLR9- and CD180-mediated cells was markedly reduced. We propose that the identified prominent role of autophagy in RA-mediated IgG-production in normal human B cells provides a novel mechanism whereby vitamin A exerts its important functions in the immune system. PMID:25749095

  9. Fluorescence resonance energy transfer based immunosensing of human IgG by using quantum dot/GIgG-gold nanoparticles/IgG conjugation.

    PubMed

    Luo, Lin; Liu, Zhao; Li, Jianjun; Zhu, Jian

    2014-06-01

    A novel immunosensor of human immune globulin (IgG) was fabricated based on the fluorescence transfer between luminescent semiconductor quantum dots (QDs) and gold nanoparticles (AuNPs). AuNPs and CdSe/ZnS QDs were respectively labeled with immune reaction pair:IgG and goat anti-human immunoglobulin (GIgG), by optimizing the conditions including pH value and protein amount. In the assembled QD-GIgG-IgG-AuNP fluorescence resonance energy transfer (FRET) immunocomplex system, the presence of AuNP-IgG directly reduced the fluorescence intensity of the GIgG conjugated QDs. As a result, the concentration of AuNP-IgG had a linear relationship with the fluorescence decrease in a range of 0-1.57 microg/mL. Furthermore, the mechanism of the QDs' fluorescence decay has also been discussed and attributed to the light-induced photobleaching. This novel sensing method achieves quantitative detection of trace proteins, suggesting the potential of biomolecule-AuNPs conjugation based analytical methods in further application. PMID:24738348

  10. Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide.

    PubMed

    Mirecka, Ewa Agnieszka; Gremer, Lothar; Schiefer, Stephanie; Oesterhelt, Filipp; Stoldt, Matthias; Willbold, Dieter; Hoyer, Wolfgang

    2014-12-10

    Human islet amyloid polypeptide (IAPP) is the major component of pancreatic amyloid deposits in type 2 diabetes. The structural conversion of IAPP from a monomeric state into amyloid assemblies is the subject of intense research. Recombinant production of IAPP is, however, difficult due to its extreme aggregation propensity. Here we describe a novel strategy for expression of IAPP in Escherichia coli, based on an engineered protein tag, which sequesters IAPP monomers and prevents IAPP aggregation. The IAPP-binding protein HI18 was selected by phage display from a β-wrapin library. Fusion of HI18 to IAPP enabled the soluble expression of the construct. IAPP was cleaved from the fusion construct and purified to homogeneity with a yield of 3mg of isotopically labeled peptide per liter of culture. In the monomeric state, IAPP was largely disordered as evidenced by far-UV CD and liquid-state NMR spectroscopy but competent to form amyloid fibrils according to atomic force microscopy. These results demonstrate the ability of the engineered β-wrapin HI18 for shielding the hydrophobic sequence of IAPP during expression and purification. Fusion of aggregation-inhibiting β-wrapins is a suitable approach for the recombinant production of aggregation-prone proteins. PMID:24928165

  11. Terminally differentiated human intestinal B cells. J chain expression of IgA and IgG subclass-producing immunocytes in the distal ileum compared with mesenteric and peripheral lymph nodes.

    PubMed Central

    Bjerke, K; Brandtzaeg, P

    1990-01-01

    Two-colour immunofluorescence staining for intracellular J chain and IgA (or J chain and IgG) was performed on tissue sections of normal human ileal mucosa (eight adult kidney donors), mesenteric lymph nodes (MLN), peripheral lymph nodes, and palatine tonsils. The most prominent J chain positivity was seen for IgA (97.3%) and IgG (81.7%) immunocytes in the ileal lamina propria (LP). Moreover, the proportion of J chain-expressing extrafollicular immunocytes was significantly higher (P less than 0.05) in MLN than in peripheral lymph nodes for the IgA class (58.5% versus 25.6%); the same proportion for the IgG class was 45.9% versus 30.4%. In clinically normal palatine tonsils of adults, extrafollicular J chain expression was much lower than in peripheral lymph nodes; 14.2% for IgA cells and 5.5% for IgG cells. When related to subclass production, J chain expression was found to be higher for IgA2 than for IgA1 cells in all tissues examined (palatine tonsils excluded because of a small number of IgA2 cells), the difference being significant in MLN and ileal LP (P less than 0.05). The J chain positivity tended to be higher for all IgG subclasses in MLN than in peripheral lymph nodes; this difference was significant (P less than 0.05) for IgG2-producing immunocytes. Taking J chain expression as a marker of clonal immaturity, our results may reflect to some extent distribution of newly generated memory B cell clones from gut-associated lymphoid tissue to MLN, peripheral lymph nodes, and palatine tonsils in a strikingly decreasing order. PMID:2122937

  12. Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

    PubMed Central

    2015-01-01

    The human lens contains three major protein families: α-, β-, and γ-crystallin. Among the several variants of γ-crystallin in the human lens, γD-crystallin is a major form. γD-Crystallin is primarily present in the nuclear region of the lens and contains a single lysine residue at the second position (K2). In this study, we investigated the acetylation of K2 in γD-crystallin in aging and cataractous human lenses. Our results indicated that K2 is acetylated at an early age and that the amount of K2-acetylated γD-crystallin increased with age. Mass spectrometric analysis revealed that in addition to K2, glycine 1 (G1) was acetylated in γD-crystallin from human lenses and in γD-crystallin acetylated in vitro. The chaperone ability of α-crystallin for acetylated γD-crystallin was lower than that for the nonacetylated protein. The tertiary structure and the microenvironment of the cysteine residues were significantly altered by acetylation. The acetylated protein exhibited higher surface hydrophobicity, was unstable against thermal and chemical denaturation, and exhibited a higher propensity to aggregate at 80 °C in comparison to the nonacetylated protein. Acetylation enhanced the GdnHCl-induced unfolding and slowed the subsequent refolding of γD-crystallin. Theoretical analysis indicated that the acetylation of K2 and G1 reduced the structural stability of the protein and brought the distal cysteine residues (C18 and C78) into close proximity. Collectively, these results indicate that the acetylation of G1 and K2 residues in γD-crystallin likely induced a molten globule-like structure, predisposing it to aggregation, which may account for the high content of aggregated proteins in the nucleus of aged and cataractous human lenses. PMID:25393041

  13. Generation in Human Plasma of Misfolded, Aggregation-Prone Electronegative Low Density Lipoprotein

    PubMed Central

    Greco, Giulia; Balogh, Gabor; Brunelli, Roberto; Costa, Graziella; De Spirito, Marco; Lenzi, Laura; Mei, Giampiero; Ursini, Fulvio; Parasassi, Tiziana

    2009-01-01

    Abstract Human plasma contains small amounts of a low density lipoprotein in which apoprotein is misfolded. Originally identified and isolated by means of anion-exchange chromatography, this component was subsequently described as electronegative low density lipoprotein (LDL)(−), with increased concentrations associated with elevated cardiovascular disease risk. It has been recognized recently as the trigger of LDL amyloidogenesis, which produces aggregates similar to subendothelial droplets observed in vivo in early atherogenesis. Although LDL(−) has been produced in vitro through various manipulations, the mechanisms involved in its generation in vivo remain obscure. By using a more physiological model, we demonstrate spontaneous, sustained and noticeable production of LDL(−) during incubation of unprocessed human plasma at 37°C. In addition to a higher fraction of amyloidogenic LDL(−), LDL purified from incubated plasma contains an increased level of lysophospholipids and free fatty acids; analysis of LDL lipids packing shows their loosening. As a result, during plasma incubation, lipid destabilization and protein misfolding take place, and aggregation-prone particles are generated. All these phenomena can be prevented by inhibiting calcium-dependent secretory phospholipases A2. Our plasma incubation model, without removal of reaction products, effectively shows a lipid-protein interplay in LDL, where lipid destabilization after lipolysis threatens the apoprotein's structure, which misfolds and becomes aggregation-prone. PMID:19619478

  14. Time dependent variation of human blood conductivity as a method for an estimation of RBC aggregation.

    PubMed

    Antonova, Nadia; Riha, Pavel; Ivanov, Ivan

    2008-01-01

    Time variation of whole human blood conductivity and shear stresses were investigated at rectangular and trapezium-shaped Couette viscometric flow under electric field of 2 kHz. The kinetics of conductivity signals were recorded both under transient flow and after the complete stoppage of shearing at shear rates from 0.94 to 94.5 s(-1) and temperatures T=25 degrees C and 37 degrees C. Contraves Low Shear 30 rotational viscometer as a base unit and a concurrent measuring system, including a device, developed by the conductometric method with a software for measurement of conductivity of biological fluids (data acquisition system), previously described (IFMBE Proceedings Series, Vol. 11, 2005, pp. 4247-4252; Clin. Hemorheol. Microcirc. 35(1/2) (2006), 19-29), were used for the experiments. The obtained experimental relationships show that the human blood conductivity is time, shear rate, hematocrit and temperature dependent under transient flow. It is also dependent on the regime of the applied shear rates. Non-linear curve approximation of the growth and relaxation curves is done. The results show that valuable information could be received about the kinetics of "rouleaux formation" and the time dependences of the blood conductivity follow the structural transformations of RBC aggregates during the aggregation-disaggregation processes. The results suggest that this technique may be used to clarify the mechanism of dynamics of RBC aggregates. PMID:18503112

  15. Effects of tetrandrine and fangchinoline on experimental thrombosis in mice and human platelet aggregation.

    PubMed

    Kim, H S; Zhang, Y H; Yun, Y P

    1999-03-01

    Tetrandrine (TET) and fangchinoline (FAN) are two naturally occurring analogues with a bisbenzylisoquinoline structure. The present study was undertaken to investigate the effects of TET and FAN on the experimental thrombosis induced by collagen plus epinephrine (EP) in mice, and platelet aggregation and blood coagulation in vitro. In the in vivo study, the administration (50 mg/kg, i.p.) of TET and FAN in mice showed the inhibition of thrombosis by 55% and 35%, respectively, while acetylsalicylic acid (ASA, 50 mg/kg, i.p.), a positive control, showed only 30% inhibition. In the vitro human platelet aggregations induced by the agonists used in tests, TET and FAN showed the inhibitions dose dependently. In addition, neither TET nor FAN showed any anticoagulation activities in the measurement of the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using human-citrated plasma. These results suggest that antithrombosis of TET and FAN in mice may be mainly related to the antiplatelet aggregation activities. PMID:10193204

  16. Modeling human protein aggregation cardiomyopathy using murine induced pluripotent stem cells.

    PubMed

    Limphong, Pattraranee; Zhang, Huali; Christians, Elisabeth; Liu, Qiang; Riedel, Michael; Ivey, Kathryn; Cheng, Paul; Mitzelfelt, Katie; Taylor, Graydon; Winge, Dennis; Srivastava, Deepak; Benjamin, Ivor

    2013-03-01

    Several mutations in αB-crystallin (CryAB), a heat shock protein with chaperone-like activities, are causally linked to skeletal and cardiac myopathies in humans. To better understand the underlying pathogenic mechanisms, we had previously generated transgenic (TG) mice expressing R120GCryAB, which recapitulated distinguishing features of the myopathic disorder (e.g., protein aggregates, hypertrophic cardiomyopathy). To determine whether induced pluripotent stem cell (iPSC)-derived cardiomyocytes, a new experimental approach for human disease modeling, would be relevant to aggregation-prone disorders, we decided to exploit the existing transgenic mouse model to derive iPSCs from tail tip fibroblasts. Several iPSC lines were generated from TG and non-TG mice and validated for pluripotency. TG iPSC-derived cardiomyocytes contained perinuclear aggregates positive for CryAB staining, whereas CryAB protein accumulated in both detergent-soluble and insoluble fractions. iPSC-derived cardiomyocytes identified by cardiac troponin T staining were significantly larger when expressing R120GCryAB at a high level in comparison with TG low expressor or non-TG cells. Expression of fetal genes such as atrial natriuretic factor, B-type natriuretic peptide, and α-skeletal α-actin, assessed by quantitative reverse transcription-polymerase chain reaction, were increased in TG cardiomyocytes compared with non-TG, indicating the activation of the hypertrophic genetic program in vitro. Our study demonstrates for the first time that differentiation of R120G iPSCs into cardiomyocytes causes protein aggregation and cellular hypertrophy, recapitulating in vitro key pathognomonic hallmarks found in both animal models and patients. Our findings pave the way for further studies exploiting this cell model system for mechanistic and therapeutic investigations. PMID:23430692

  17. Relative stabilities of IgG1 and IgG4 Fab domains: Influence of the light–heavy interchain disulfide bond architecture

    PubMed Central

    Heads, James T; Adams, Ralph; D'Hooghe, Lena E; Page, Matt J T; Humphreys, David P; Popplewell, Andrew G; Lawson, Alastair D; Henry, Alistair J

    2012-01-01

    The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 CH1 and IgG4 CH1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L–H) bond, did not affect thermal stability. Introducing the IgG1 type L–H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L–H interchain DSB with the IgG4 type L–H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 CH1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format. PMID:22761163

  18. Inhibition of protein phosphorylation by synthetic peptides from the Fc region of human IgG during the mixed lymphocyte response

    SciTech Connect

    McClurg, M.R.; Hahn, G.S.; Plummer, J.M.

    1986-03-01

    Certain synthetic peptides derived from the Fc region of human IgG suppressed protein, RNA, and DNA synthesis during mixed lymphocyte reactions. Responder mononuclear cells were incubated with medium or agents that alter phosphorylation of cellular proteins before immunomodulatory Fc peptides and stimulator cells were added. Incubating cells with trifluoperazine which inhibits calcium binding to calmodulin and inhibits protein kinase C (PKC) increased inhibition of the MLR induced by Fc peptides. Conversely, incubating cells with dubutyryl cyclic AMP (DBcAMP), calmodulin, 1,2-diolein, or phorbol myristate acetate (PMA) abolished inhibition of the MLR induced by Fc peptides. Inhibition of the MLR by Fc ..gamma.. peptides was not affected when DBcAMP or PMA was added after peptide addition. The PKC activity of cell homogenates was decreased by 69% when Fc..gamma.. peptides were present during the MLR. The in vitro phosphorylation of histone Hl by partially purified PKC from lymphocytes was inhibited 74% in the presence of Fc..gamma.. peptides. These results indicate that suppression of the MLR induced by Fc..gamma.. peptides is dependent on inhibition of protein phosphorylation by kinases including protein kinase C. The inhibition of phosphorylation may be related to the ability of Fc..gamma.. peptides to reverse animal models of autoimmune disease.

  19. Developmental Toxicity and Fertility Assessment in Rabbits with Tabalumab: A Human IgG4 Monoclonal Antibody.

    PubMed

    Breslin, William J; Hilbish, Kim G; Martin, Jennifer A; Halstead, Carolyn A; Edwards, Tammy L

    2015-06-01

    Tabalumab is a human immunoglobulin G subclass 4 monoclonal antibody that has been under development for autoimmune disorders. Tabalumab has full neutralizing activity against both soluble and membrane B-cell activating factor, a B-cell survival factor. The objectives of these studies were to assess the effects of tabalumab on embryo-fetal development and on male (M) and female (F) fertility in rabbits, a pharmacologically relevant species. Doses were administered at 0 (vehicle control), 0.3 (embryo-fetal study only), 1.0, and 30 mg/kg. In the embryo-fetal study, pregnant rabbits does were given a single dose by intravenous injection on gestation day (GD) 7. In the fertility studies, tabalumab was administered by intravenous injection every 7 days starting 2 (F) or 4 (M) weeks before mating, during cohabitation, and until necropsy (M) or through GD 18 (F). Treated animals were mated with untreated partners. Parental clinical signs, body weight, food consumption, blood lymphocyte phenotyping, organ weights, morphologic pathology, ovarian and uterine observations, sperm parameters, and fertility indices were evaluated along with conceptus viability, weight, and morphology. Exposure assessments were made in all main study animals and satellite animals. No adverse parental, reproductive, or developmental effects were observed in any study at any dose. A pharmacodynamic response consisting of dose-dependent decreases in the percent and number of total B lymphocytes and increases in the percent and/or number of total T lymphocytes was observed in parental rabbits at 1.0 and 30 mg/kg. In conclusion, no adverse reproductive or developmental effects were observed in rabbits following exposure to tabalumab at doses as high as 30 mg/kg and exposures at least 14-fold greater than human exposure levels. PMID:26195315

  20. An integrated practical implementation of continuous aqueous two-phase systems for the recovery of human IgG: From the microdevice to a multistage bench-scale mixer-settler device.

    PubMed

    Espitia-Saloma, Edith; Vâzquez-Villegas, Patricia; Rito-Palomares, Marco; Aguilar, Oscar

    2016-05-01

    Aqueous two-phase systems (ATPS) are a liquid-liquid extraction technology with clear process benefits; however, its lack of industrial embracement is still a challenge to overcome. Antibodies are a potential product to be recovered by ATPS in a commercial context. The objective of this work is to present a more integral approach of the different isolated strategies that have arisen in order to enable a practical, generic implementation of ATPS, using human immunoglobulin G (IgG) as experimental model. A microfluidic device is used for ATPS parameters preselection for product recovery. ATPS were continuously operated in a mixer-settler device in one stage, multistage and multistage with recirculation configuration. Single-stage pure IgG extraction with a polyethylene glycol (PEG) 3350-phophates ATPS within continuous operation allowed a 65% recovery. Further implementation of a multistage platform promoted a higher particle partitioning reaching a 90% recovery. The processing of IgG from a cell supernatant culture harvest in a multistage system with top phase recirculation resulted in 78% IgG recovery in bottom phase. This work conjugates three not widely spread methodologies for ATPS: microfluidics, continuous and multistage operation. PMID:26848821

  1. A fully human IgG1 anti-PD-L1 MAb in an in vitro assay enhances antigen-specific T-cell responses

    PubMed Central

    Grenga, Italia; Donahue, Renee N; Lepone, Lauren M; Richards, Jacob; Schlom, Jeffrey

    2016-01-01

    Monoclonal antibodies (MAbs) that interfere with checkpoint molecules are being investigated for the treatment of infectious diseases and cancer, with the aim of enhancing the function of an impaired immune system. Avelumab (MSB0010718C) is a fully human IgG1 MAb targeting programmed death-ligand 1 (PD-L1), which differs from other checkpoint-blocking antibodies in its ability to mediate antibody-dependent cell-mediated cytotoxicity. These studies were conducted to define whether avelumab could enhance the detection of antigen-specific immune response in in vitro assays. Peripheral blood mononuclear cells from 17 healthy donors were stimulated in vitro, with and without avelumab, with peptide pools encoding for cytomegalovirus, Epstein–Barr virus, influenza and tetanus toxin or the negative peptide control encoding for human leukocyte antigen. These studies show for the first time that the addition of avelumab to an antigen-specific IVS assay (a) increased the frequency of activated antigen-specific CD8+ T lymphocytes, and did so to a greater extent than that seen with commercially available PD-L1-blocking antibodies, (b) reduced CD4+ T-cell proliferation and (c) induced a switch in the production of Th2 to Th1 cytokines. Moreover, there was an inverse correlation between the enhancement of CD8+ T-cell activation and reduction in CD4+ T-cell proliferation induced by avelumab. These findings provide the rationale for the use of avelumab anti-PD-L1 in in vitro assays to monitor patient immune responses to immunotherapies. PMID:27350882

  2. Accelerated Human Mutant Tau Aggregation by Knocking Out Murine Tau in a Transgenic Mouse Model

    PubMed Central

    Ando, Kunie; Leroy, Karelle; Héraud, Céline; Yilmaz, Zehra; Authelet, Michèle; Suain, Valèrie; De Decker, Robert; Brion, Jean-Pierre

    2011-01-01

    Many models of human tauopathies have been generated in mice by expression of a human mutant tau with maintained expression of mouse endogenous tau. Because murine tau might interfere with the toxic effects of human mutant tau, we generated a model in which a pathogenic human tau protein is expressed in the absence of wild-type tau protein, with the aim of facilitating the study of the pathogenic role of the mutant tau and to reproduce more faithfully a human tauopathy. The Tg30 line is a tau transgenic mouse model overexpressing human 1N4R double-mutant tau (P301S and G272V) that develops Alzheimer's disease-like neurofibrillary tangles in an age-dependent manner. By crossing Tg30 mice with mice invalidated for their endogenous tau gene, we obtained Tg30xtau−/− mice that express only exogenous human double-mutant 1N4R tau. Although Tg30xtau−/− mice express less tau protein compared with Tg30, they exhibit signs of decreased survival, increased proportion of sarkosyl-insoluble tau in the brain and in the spinal cord, increased number of Gallyas-positive neurofibrillary tangles in the hippocampus, increased number of inclusions in the spinal cord, and a more severe motor phenotype. Deletion of murine tau accelerated tau aggregation during aging of this mutant tau transgenic model, suggesting that murine tau could interfere with the development of tau pathology in transgenic models of human tauopathies. PMID:21281813

  3. Resveratrol interferes with the aggregation of membrane-bound human-IAPP: a molecular dynamics study.

    PubMed

    Lolicato, Fabio; Raudino, Antonio; Milardi, Danilo; La Rosa, Carmelo

    2015-03-01

    Amyloid aggregation of islet amyloid polypeptide (IAPP) in pancreatic tissues is a typical feature of type 2 diabetes mellitus. Resveratrol, a natural product extensively studied for its wide range of biological effects, has been shown to inhibit IAPP aggregation. However, the mechanism by which resveratrol inhibits IAPP aggregation is still far from complete elucidation. Now, an increasing knowledge of the mechanism of amyloid toxicity shifts the target of research towards the development of compounds which can prevent amyloid-mediated membrane damage rather than merely inhibit fiber formation. In this study we used all atom molecular dynamics to investigate the interaction of resveratrol with full-length human IAPP in a negatively charged membrane environment. Our results show that the presence of resveratrol induces the formation of secondary structures (sheets and helices) by inserting in a hydrophobic pocket between the interaction surface of two IAPP molecules in aqueous solution. On the other hand, resveratrol significantly perturbs the interaction of IAPP with negatively charged membranes by anchoring specific hydrophobic regions (23FGA25 and 32VGS34) of the peptide and forming a stable 1:2 IAPP:resveratrol complex at the water/membrane interphase. PMID:25638571

  4. Elimination of soluble 123I-labelled aggregates of human immunoglobulin G in humans; the effect of splenectomy.

    PubMed Central

    Halma, C; Daha, M R; van Furth, R; Camps, J A; Evers-Schouten, J H; Pauwels, E K; Lobatto, S; Van Es, L A

    1989-01-01

    To study the role of the spleen in the elimination of immune complexes we examined mononuclear phagocyte system function in eight healthy controls and eight splenectomized patients, with soluble 123I-labelled aggregates of human immunoglobulin G (AIgG). No differences were found between the two groups in elimination and degradation of AIgG. The loss of splenic function was compensated for by increased uptake of AIgG by the liver. With the dose of 123I-AIgG used in this study (10 micrograms/kg body weight), significant generation of C3a was observed. No correlation was found between erythrocyte CR1 number and the fraction of aggregates that bound to erythrocytes. PMID:2788541

  5. Modulation of Invasive Phenotype by Interstitial Pressure-Driven Convection in Aggregates of Human Breast Cancer Cells

    PubMed Central

    Tien, Joe; Truslow, James G.; Nelson, Celeste M.

    2012-01-01

    This paper reports the effect of elevated pressure on the invasive phenotype of patterned three-dimensional (3D) aggregates of MDA-MB-231 human breast cancer cells. We found that the directionality of the interstitial pressure profile altered the frequency of invasion by cells located at the surface of an aggregate. In particular, application of pressure at one end of an aggregate suppressed invasion at the opposite end. Experimental alteration of the configuration of cell aggregates and computational modeling of the resulting flow and solute concentration profiles revealed that elevated pressure inhibited invasion by altering the chemical composition of the interstitial fluid near the surface of the aggregate. Our data reveal a link between hydrostatic pressure, interstitial convection, and invasion. PMID:23028839

  6. Preparation and characterization of novel IgG affinity resin coupling anti-Fc camelid single-domain antibodies.

    PubMed

    Tu, Zhui; Xu, Yang; Fu, Jinheng; Huang, Zhibing; Wang, Yao; Liu, Bin; Tao, Yong

    2015-03-01

    This work aimed to evaluate novel affinity resin used to purify immunoglobulin G (IgG) with a variable domain of the heavy chain of the heavy-chain antibody (VHH) as an affinity ligand. The VHH, isolated from a naïve camelid single-domain phage display library, exhibits not only affinity to the fragment crystallizable (Fc) region of IgG but also high thermal stability. This anti-Fc VHH (AFV) was expressed as a soluble protein in Escherichia coli and purified using a simple heat treatment procedure. The effects of pH and NaCl concentrations on the capacity of AFV resin were also investigated. Results showed a robust property of the AFV resin. It could bind IgGs at various pH conditions (from 6.0 to 9.0) and NaCl concentrations. The static binding capacities of AFV resin ranged from 3.40±0.53mg/ml to 15.04±0.37mg/ml measured using rabbit, mouse, and human IgGs. The bound IgGs can be efficiently eluted at pH 5.0, which is conducive to acid-sensitive IgGs and prevents the aggregation of IgGs. After 10 purification cycles or a 7-day period of storage at 37°C, recovery did not decrease. These findings suggested that VHHs from non-immunized library could also be robust and functional reagent as an affinity purification ligand. PMID:25614967

  7. The action of NIR (808nm) laser radiation and gold nanorods labeled with IgA and IgG human antibodies on methicillin-resistant and methicillin sensitive strains of Staphylococcus aureus

    NASA Astrophysics Data System (ADS)

    Tuchina, Elena S.; Petrov, Pavel O.; Ratto, Fulvio; Centi, Sonia; Pini, Roberto; Tuchin, Valery V.

    2015-03-01

    The effect of NIR laser radiation (808 nm) on methicillin-sensitive and methicillin resistant strains of Staphylococcus aureus incubated with gold nanorods is studied. Nanorods having length of 44 (± 4) nm and diameter of 10 (± 3) nm with the absorption maximum in the NIR (800 nm), functionalized with human immunoglobulins IgA and IgG, were synthesized and used in the studies. The killing ability up to 97% of the microorganism populations by using this nanotechnology was shown.

  8. Recognition of native and/or thermally induced denatured forms of the major food allergen, ovomucoid, by human IgE and mouse monoclonal IgG antibodies.

    PubMed

    Hirose, Junko; Kitabatake, Naofumi; Kimura, Akihiro; Narita, Hiroshi

    2004-12-01

    Human sera obtained from children with egg allergy reacted well with both native and heated ovomucoid (OM). Ovalbumin is present in egg white in a 5 times greater quantity than OM; however, it easily aggregates and becomes difficult to extract by heating. For accurate food allergen labeling of processed food, therefore, OM should be evaluated with the determination of egg white protein in consideration of heat denaturation. Three kinds of monoclonal antibodies and sandwich ELISA tests were established which are able to recognize the native and/or heat-denatured forms of OM. The usefulness of these characteristic mAbs and ELISA tests are discussed in relation to allergen labeling, monitoring food processing, and movement or change of dietary protein in vivo. PMID:15618619

  9. Adaptive Optics-Assisted Identification of Preferential Erythrocyte Aggregate Pathways in the Human Retinal Microvasculature

    PubMed Central

    Arichika, Shigeta; Uji, Akihito; Ooto, Sotaro; Miyamoto, Kazuaki; Yoshimura, Nagahisa

    2014-01-01

    Purpose To characterize human parafoveal blood flow using adaptive optics scanning laser ophthalmoscopy (AO-SLO). Methods In 5 normal subjects, erythrocyte aggregate distributions were analyzed on 3 different days. Erythrocyte aggregates were described as a “dark tail” in AO-SLO. The characteristics of the pathways with dark tail flow in the parafovea were measured. Additionally, the tendency for dark tail flow before and after bifurcations was analyzed to study the blood flow in detail. Results Average velocity in parent vessels with dark tail flow was 1.30±0.27 mm/s. Average velocity in daughter vessels with dark tail flow was 1.12±0.25 mm/s, and the average velocity of plasma gaps in daughter vessels without dark tail flow was 0.64±0.11 mm/s. Downstream from the bifurcations, the velocity in vessels with dark tail flow was higher than that in those without it (p<0.001), and the branching angles of vessels with dark tail flow were smaller than those of vessels without it (p<0.001). Conclusions Images from the AO-SLO noninvasively revealed pathways with and without dark tail flow in the human parafovea. Pathways with dark tail flow in the daughter vessels generally had faster flow and smaller bifurcation angles than daughter vessels without dark tail flow. Thus, AO-SLO is an instructive tool for analyzing retinal microcirculatory hemodynamics. PMID:24586959

  10. Electrophoretic and aggregation behavior of bovine, horse and human red blood cells in plasma and in polymer solutions.

    PubMed

    Bäumler, H; Neu, B; Mitlöhner, R; Georgieva, R; Meiselman, H J; Kiesewetter, H

    2001-01-01

    The electrophoretic mobility of native and glutaraldehyde-fixed bovine, human, and horse red blood cells (RBC) was investigated as a function of ionic strength (5-150 mM) and concentration of 464 kDa dextran (2 and 3 g/dl); RBC aggregation in autologous plasma and in dextran solutions was also measured. In agreement with previous observations, human and horse RBC form stable rouleaux whereas bovine RBC do not aggregate in either plasma or in dextran 464 kDa solutions. Electrophoretic measurements showed a species-dependent adsorption and depletion of dextran that can be theoretically evaluated. Adsorption of polymer is not a prerequisite for RBC aggregation (bovine RBC show the highest amount of adsorbed dextran yet do not aggregate). Aggregate formation thus occurs as long as the Gibbs free energy difference, given by the osmotic pressure difference between the bulk phase and the polymer-depleted region between two RBC, is larger than the steric and electrostatic repulsive energy contributed by the macromolecules present on the RBC surface. With increasing bulk-phase polymer concentration the depletion layer thickness decreases and the amount of adsorbed macromolecules increases, thereby resulting in an increase of the repulsive component of the interaction energy and decreased aggregation. We thus view electrophoretic measurements of RBC in various media as an important tool for understanding polymer behavior near the red cell surface and hence the mechanisms involved in RBC aggregation. PMID:11381164

  11. The different effector function capabilities of the seven equine IgG subclasses have implications for vaccine strategies

    PubMed Central

    Lewis, Melanie J.; Wagner, Bettina; Woof, Jenny M.

    2008-01-01

    Recombinant versions of the seven equine IgG subclasses were expressed in CHO cells. All assembled into intact immunoglobulins stabilised by disulphide bridges, although, reminiscent of human IgG4, a small proportion of equine IgG4 and IgG7 were held together by non-covalent bonds alone. All seven IgGs were N-glycosylated. In addition IgG3 appeared to be O-glycosylated and could bind the lectin jacalin. Staphylococcal protein A displayed weak binding for the equine IgGs in the order: IgG1 > IgG3 > IgG4 > IgG7 > IgG2 = IgG5 > IgG6. Streptococcal protein G bound strongly to IgG1, IgG4 and IgG7, moderately to IgG3, weakly to IgG2 and IgG6, and not at all to IgG5. Analysis of antibody effector functions revealed that IgG1, IgG3, IgG4, IgG5 and IgG7, but not IgG2 and IgG6, were able to elicit a strong respiratory burst from equine peripheral blood leukocytes, predicting that the former five IgG subclasses are able to interact with Fc receptors on effector cells. IgG1, IgG3, IgG4 and IgG7, but not IgG2, IgG5 and IgG6, were able to bind complement C1q and activate complement via the classical pathway. The differential effector function capabilities of the subclasses suggest that, for maximum efficacy, equine vaccine strategies should seek to elicit antibody responses of the IgG1, IgG3, IgG4, and IgG7 subclasses. PMID:17669496

  12. Reactivation of protein aggregates by mortalin and Tid1--the human mitochondrial Hsp70 chaperone system.

    PubMed

    Iosefson, Ohad; Sharon, Shelly; Goloubinoff, Pierre; Azem, Abdussalam

    2012-01-01

    The mitochondrial 70-kDa heat shock protein (mtHsp70), also known in humans as mortalin, is a central component of the mitochondrial protein import motor and plays a key role in the folding of matrix-localized mitochondrial proteins. MtHsp70 is assisted by a member of the 40-kDa heat shock protein co-chaperone family named Tid1 and a nucleotide exchange factor. Whereas, yeast mtHsp70 has been extensively studied in the context of protein import in the mitochondria, and the bacterial 70-kDa heat shock protein was recently shown to act as an ATP-fuelled unfolding enzyme capable of detoxifying stably misfolded polypeptides into harmless natively refolded proteins, little is known about the molecular functions of the human mortalin in protein homeostasis. Here, we developed novel and efficient purification protocols for mortalin and the two spliced versions of Tid1, Tid1-S, and Tid1-L and showed that mortalin can mediate the in vitro ATP-dependent reactivation of stable-preformed heat-denatured model aggregates, with the assistance of Mge1 and either Tid1-L or Tid1-S co-chaperones or yeast Mdj1. Thus, in addition of being a central component of the protein import machinery, human mortalin together with Tid1, may serve as a protein disaggregating machine which, for lack of Hsp100/ClpB disaggregating co-chaperones, may carry alone the scavenging of toxic protein aggregates in stressed, diseased, or aging human mitochondria. PMID:21811887

  13. Inverting microwell array chip for the cultivation of human induced pluripotent stem cells with controlled aggregate size and geometrical arrangement

    PubMed Central

    Satoh, Taku; Sugiura, Shinji; Sumaru, Kimio; Ozaki, Shigenori; Gomi, Shinichi; Kurakazu, Tomoaki; Oshima, Yasuhiro; Kanamori, Toshiyuki

    2014-01-01

    We present a novel cell culture chip, namely, “inverting microwell array chip,” for cultivation of human induced pluripotent stem cells. The chip comprises a lower hydrogel microwell array and an upper polystyrene culture surface. We demonstrate the formation of uniform cellular aggregates in the microwell array, and after inversion, a culture with controlled aggregate size and geometrical arrangement on the polystyrene surface. Here, we report effects of cell concentrations on a cultivation sequence in the chip. PMID:24803961

  14. New insights into side effect of solvents on the aggregation of human islet amyloid polypeptide 11-20.

    PubMed

    Mao, Yexuan; Yu, Lanlan; Yang, Ran; Ma, Chuanguo; Qu, Ling-bo; Harrington, Peter de B

    2016-02-01

    The formation of highly ordered fibrils for the human islet amyloid polypeptide (hIAPP) is considered as one of the precipitating factors of type 2 diabetes mellitus. In this study, an emerging new approach microscale thermophoresis and conventional ThT fluorescence assay were utilized to investigate the aggregation behavior of hIAPP(11-20), giving a new insight of the solvent effect on the aggregation of hIAPP(11-20). hIAPP(11-20) displayed different aggregation behaviors in various buffers, revealing that hIAPP(11-20) not only self-aggregates but also binds to solvent components. hIAPP(11-20) had a higher binding affinity for Tris than other selected buffers because multiple hydrogen bonds form, resulting in weaker self-aggregation of hIAPP(11-20) at the early stage of aggregation and prolonging the fibril formation process. hIAPP(11-20) displayed similar self-aggregation in both HEPES and pure water. Negatively charged phosphate ions in the PBS solution 'neutralize' the charges carried by hIAPP(11-20) itself to some extent, causing rapid aggregation of hIAPP(11-20), and leading to a shorter fibrillation process of hIAPP(11-20). These results revealed that solvents contribute to the aggregation of hIAPP(11-20) and demonstrated the affect of solvents on the activity of biomolecules. Additionally, as a new technique, microscale thermophoresis offers a powerful and promising approach to study the early stages of aggregation of peptides or proteins. PMID:26653463

  15. IgG4-related disease of the rectum

    PubMed Central

    Choi, Sung-Bong; Lim, Chul-Hyun; Cha, Myung-Guen

    2016-01-01

    IgG4-related disease is a relatively new disease entity characterized by elevated serum IgG4 levels and marked infiltration of IgG4-positive plasma cells in lesions. Organ enlargement or nodular lesions consisting of abundant infiltration of lymphocytes and IgG4-positive plasma cells and fibrosis are seen in various organs throughout. We encountered a patient with an inflammatory pseudotumor of the rectum, which was histopathologically confirmed to be an IgG4-related disease. The patient was a 28-year-old woman who had constipation for 3 months. The endoluminal ultrasonography showed a lesion that was heterogeneous and low echogenic in lower rectum. The result of colonoscopic biopsy findings was of chronic proctitis with lymphoid aggregates. For a confirmative diagnosis, excision was performed. Histopathological examination represented plasma cell infiltration and fibrosis. Immunohistochemistry revealed prominence of IgG4-positive plasma cells and confirmed the diagnosis of IgG4-related disease. The patient is currently under observation on low-dose oral prednisolone without relapse. PMID:27186575

  16. Potential Mechanisms for IgG4 Inhibition of Immediate Hypersensitivity Reactions.

    PubMed

    James, Louisa K; Till, Stephen J

    2016-03-01

    IgG4 is the least abundant IgG subclass in human serum, representing less than 5 % of all IgG. Increases in IgG4 occur following chronic exposure to antigen and are generally associated with states of immune tolerance. In line with this, IgG4 is regarded as an anti-inflammatory antibody with a limited ability to elicit effective immune responses. Furthermore, IgG4 attenuates allergic responses by inhibiting the activity of IgE. The mechanism by which IgG4 inhibits IgE-mediated hypersensitivity has been investigated using a variety of model systems leading to two proposed mechanisms. First by sequestering antigen, IgG4 can function as a blocking antibody, preventing cross-linking of receptor bound IgE. Second IgG4 has been proposed to co-stimulate the inhibitory IgG receptor FcγRIIb, which can negatively regulate FcεRI signaling and in turn inhibit effector cell activation. Recent advances in our understanding of the structural features of human IgG4 have shed light on the unique functional and immunologic properties of IgG4. The aim of this review is to evaluate our current understanding of IgG4 biology and reassess the mechanisms by which IgG4 functions to inhibit IgE-mediated allergic responses. PMID:26892721

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

    PubMed Central

    Luo, Shen; Zhang, Baolin

    2015-01-01

    Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5–6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5–6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development. PMID:26338058

  18. Molecular recognition of antibody (IgG) by cellular Fc receptor (FcRI).

    PubMed

    Burton, D R; Jefferis, R; Partridge, L J; Woof, J M

    1988-11-01

    Earlier studies from this and other laboratories have provided indirect evidence for the involvement of the C gamma 2 domain of human IgG in the binding of IgG to the high affinity monocyte Fc receptor (FcRI). Two approaches have been used to extend these studies and to further localize the site of interaction on human IgG. Firstly, monoclonal antibodies (MAbs) directed against different epitopes on IgG were assayed for their capacity to inhibit the binding of radiolabelled IgG to human monocytes or U937 cells. The capacity of the MAbs to interact with their respective epitopes on FcR-bound IgG was also studied using indirect radiobinding and immunofluorescence assays. Secondly, a number of IgGs from several different species and fragments of human IgGs were assayed for their ability to inhibit the binding of radiolabelled IgG to human monocytes. The amino acid sequences of those IgGs exhibiting relatively tight, intermediate or weak binding to monocyte FcRs were compared. On the basis of these studies a possible monocyte FcR-binding site on human IgG is postulated, involving the lower hinge region of IgG (residues Leu 234-Ser 239) with possible involvement of the nearby N-proximal bend and two beta-strands (Gly 316-Lys 338). PMID:2975762

  19. A comparison of the ability of the human IgG1 allotypes G1m3 and G1m1,17 to stimulate T-cell responses from allotype matched and mismatched donors

    PubMed Central

    Webster, Carl I.; Bryson, Christine J.; Cloake, Edward A.; Jones, Tim D.; Austin, Mark J.; Karle, Anette C.; Spindeldreher, Sebastian; Lowe, David C.; Baker, Matthew P.

    2016-01-01

    ABSTRACT The immunogenicity of clinically administered antibodies has clinical implications for the patients receiving them, ranging from mild consequences, such as increased clearance of the drug from the circulation, to life-threatening effects. The emergence of methods to engineer variable regions resulting in the generation of humanised and fully human antibodies as therapeutics has reduced the potential for adverse immunogenicity. However, due to differences in sequence referred to as allotypic variation, antibody constant regions are not homogeneous within the human population, even within sub-classes of the same immunoglobulin isotype. For therapeutically administered antibodies, the potential exists for an immune response from the patient to the antibody if the allotype of patient and antibody do not match. Allotypic distribution in the human population varies within and across ethnic groups making the choice of allotype for a therapeutic antibody difficult. This study investigated the potential of human IgG1 allotypes to stimulate responses in human CD4+ T cells from donors matched for homologous and heterologous IgG1 allotypes. Allotypic variants of the therapeutic monoclonal antibody trastuzumab were administered to genetically defined allotypic matched and mismatched donor T cells. No significant responses were observed in the mismatched T cells. To investigate the lack of T-cell responses in relation to mismatched allotypes, HLA-DR agretopes were identified via MHC associated peptide proteomics (MAPPs). As expected, many HLA-DR restricted peptides were presented. However, there were no peptides presented from the sequence regions containing the allotypic variations. Taken together, the results from the T-cell assay and MAPPs assay indicate that the allotypic differences in human IgG1 do not represent a significant risk for induction of immunogenicity. PMID:26821574

  20. Controlled aggregation of primary human pancreatic islet cells leads to glucose-responsive pseudoislets comparable to native islets

    PubMed Central

    Hilderink, Janneke; Spijker, Siebe; Carlotti, Françoise; Lange, Lydia; Engelse, Marten; van Blitterswijk, Clemens; de Koning, Eelco; Karperien, Marcel; van Apeldoorn, Aart

    2015-01-01

    Clinical islet transplantation is a promising treatment for patients with type 1 diabetes. However, pancreatic islets vary in size and shape affecting their survival and function after transplantation because of mass transport limitations. To reduce diffusion restrictions and improve islet cell survival, the generation of islets with optimal dimensions by dispersion followed by reassembly of islet cells, can help limit the length of diffusion pathways. This study describes a microwell platform that supports the controlled and reproducible production of three-dimensional pancreatic cell clusters of human donor islets. We observed that primary human islet cell aggregates with a diameter of 100–150 μm consisting of about 1000 cells best resembled intact pancreatic islets as they showed low apoptotic cell death (<2%), comparable glucose-responsiveness and increasing PDX1, MAFA and INSULIN gene expression with increasing aggregate size. The re-associated human islet cells showed an a-typical core shell configuration with beta cells predominantly on the outside unlike human islets, which became more randomized after implantation similar to native human islets. After transplantation of these islet cell aggregates under the kidney capsule of immunodeficient mice, human C-peptide was detected in the serum indicating that beta cells retained their endocrine function similar to human islets. The agarose microwell platform was shown to be an easy and very reproducible method to aggregate pancreatic islet cells with high accuracy providing a reliable tool to study cell–cell interactions between insuloma and/or primary islet cells. PMID:25782016

  1. Controlled aggregation of primary human pancreatic islet cells leads to glucose-responsive pseudoislets comparable to native islets.

    PubMed

    Hilderink, Janneke; Spijker, Siebe; Carlotti, Françoise; Lange, Lydia; Engelse, Marten; van Blitterswijk, Clemens; de Koning, Eelco; Karperien, Marcel; van Apeldoorn, Aart

    2015-08-01

    Clinical islet transplantation is a promising treatment for patients with type 1 diabetes. However, pancreatic islets vary in size and shape affecting their survival and function after transplantation because of mass transport limitations. To reduce diffusion restrictions and improve islet cell survival, the generation of islets with optimal dimensions by dispersion followed by reassembly of islet cells, can help limit the length of diffusion pathways. This study describes a microwell platform that supports the controlled and reproducible production of three-dimensional pancreatic cell clusters of human donor islets. We observed that primary human islet cell aggregates with a diameter of 100-150 μm consisting of about 1000 cells best resembled intact pancreatic islets as they showed low apoptotic cell death (<2%), comparable glucose-responsiveness and increasing PDX1, MAFA and INSULIN gene expression with increasing aggregate size. The re-associated human islet cells showed an a-typical core shell configuration with beta cells predominantly on the outside unlike human islets, which became more randomized after implantation similar to native human islets. After transplantation of these islet cell aggregates under the kidney capsule of immunodeficient mice, human C-peptide was detected in the serum indicating that beta cells retained their endocrine function similar to human islets. The agarose microwell platform was shown to be an easy and very reproducible method to aggregate pancreatic islet cells with high accuracy providing a reliable tool to study cell-cell interactions between insuloma and/or primary islet cells. PMID:25782016

  2. Inhibition of Unfolding and Aggregation of Lens Protein Human Gamma D Crystallin by Sodium Citrate

    PubMed Central

    Goulet, Daniel R.; Knee, Kelly M.; King, Jonathan A.

    2012-01-01

    Cataract affects 1 in 6 Americans over the age of 40, and is considered a global health problem. Cataract is caused by the aggregation of unfolded or damaged proteins in the lens, which accumulate as an individual ages. Currently, surgery is the only available treatment for cataract, however, small molecules have been suggested as potential preventative therapies. In this work, we study the effect of sodium citrate on the stability of Human γD Crystallin (HγD-Crys), a structural protein of the eye lens, and two cataract-related mutants, L5S HγD-Crys and I90F HγD-Crys. In equilibrium unfolding-refolding studies, the presence of 250 mM sodium citrate increased the transition midpoint of the N-td of WT HγD-Crys and L5S HγD-Crys by 0.3 M GuHCl, the C-td by 0.6M GuHCl, and the single transition of I90F HγD-Crys by 0.4M GuHCl. In kinetic unfolding reactions, sodium citrate demonstrates a measurable stabilization effect only for the mutant I90F HγD-Crys. In the presence of citrate, a kinetic unfolding intermediate of I90F HγD-Crys can be observed, which was not observed in the absence of citrate. Rate of aggregation was measured using solution turbidity, and sodium citrate demonstrates negligible effect on rate of aggregation of WT HγD-Crys, but considerably slows the rate of aggregation of both L5S HγD-Crys and I90F HγD-Crys. The presence of sodium citrate dramatically slows refolding of WT HγD-Crys and I90F HγD-Crys, but has a significantly smaller effect on the refolding of L5S HγD-Crys. The differential stabilizing effect of sodium citrate suggests that the ion is binding to a partially unfolded conformation of the C-td, but a solution-based Hofmeister effect cannot be eliminated as a possible explanation for the effects observed. These results suggest that sodium citrate may be a potential preventative agent for cataract. PMID:21600897

  3. Characterization of the subclasses and light chain types of IgG antibodies to rubella.

    PubMed Central

    Skvaril, F; Schilt, U

    1984-01-01

    IgG subclasses of antibodies to rubella were determined in indirect enzyme linked immunoassay (ELISA) with monoclonal antibodies specific for human IgG1, IgG2, IgG3 and IgG4. Eleven sera from women with long past history of rubella, two hyperimmune and five non-hyperimmune immunoglobulin preparations were tested. Light chain types of the antibodies were tested in ELISA with polyclonal specific antibodies to kappa and lambda chains. Antibodies to rubella in the sera as well as in the immunoglobulin preparations were found in the IgG1 subclass only. Both light chain types were present in the antibodies. PMID:6423327

  4. Designed Stem Cell Aggregates: Enhanced Biological Functions of Human Mesenchymal Stem-Cell Aggregates Incorporating E-Cadherin-Modified PLGA Microparticles (Adv. Healthcare Mater. 15/2016).

    PubMed

    Zhang, Yan; Mao, Hongli; Gao, Chao; Li, Suhua; Shuai, Qizhi; Xu, Jianbin; Xu, Ke; Cao, Lei; Lang, Ren; Gu, Zhongwei; Akaike, Toshihiro; Yang, Jun

    2016-08-01

    E-cadherin-modified poly(lactic-co-glycolic acid) (hE-cad-PLGA) microparticles were fabricated and then mediated the 3D cell aggregates of human mesenchymal stem cells (MSCs) on page 1949 by Jun Yang and co-workers. The hE-cad-Fc matrix and the PLGA microparticles synergistically regulate the proliferation and bioactive factors secretions of MSCs by activating EGFR, AKT and ERK1/2 signaling pathways. The hE-cad-PLGA microparticles offer a novel route to expand multipotent stem cell-based clinical applications. PMID:27511954

  5. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

    PubMed Central

    Gualda, Emilio J.; Simão, Daniel; Pinto, Catarina; Alves, Paula M.; Brito, Catarina

    2014-01-01

    The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment. PMID:25161607

  6. Human IgG response to a salivary peptide, gSG6-P1, as a new immuno-epidemiological tool for evaluating low-level exposure to Anopheles bites

    PubMed Central

    Poinsignon, Anne; Cornelie, Sylvie; Ba, Fatou; Boulanger, Denis; Sow, Cheikh; Rossignol, Marie; Sokhna, Cheikh; Cisse, Badara; Simondon, François; Remoue, Franck

    2009-01-01

    Background Human populations exposed to low malaria transmission present particular severe risks of malaria morbidity and mortality. In addition, in a context of low-level exposure to Anopheles vector, conventional entomological methods used for sampling Anopheles populations are insufficiently sensitive and probably under-estimate the real risk of malaria transmission. The evaluation of antibody (Ab) responses to arthropod salivary proteins constitutes a novel tool for estimating exposure level to insect bites. In the case of malaria, a recent study has shown that human IgG responses to the gSG6-P1 peptide represented a specific biomarker of exposure to Anopheles gambiae bites. The objective of this study was to investigate if this biomarker can be used to estimate low-level exposure of individuals to Anopheles vector. Methods The IgG Ab level to gSG6-P1 was evaluated at the peak and at the end of the An. gambiae exposure season in children living in Senegalese villages, where the Anopheles density was estimated to be very low by classical entomological trapping but where malaria transmission occurred during the studied season. Results Specific IgG responses to gSG6-P1 were observed in children exposed to very low-level of Anopheles bites. In addition, a significant increase in the specific IgG Ab level was observed during the Anopheles exposure season whereas classical entomological data have reported very few or no Anopheles during the studied period. Furthermore, this biomarker may also be applicable to evaluate the heterogeneity of individual exposure. Conclusion The results strengthen the hypothesis that the evaluation of IgG responses to gSG6-P1 during the season of exposure could reflect the real human contact with anthropophilic Anopheles and suggest that this biomarker of low exposure could be used at the individual level. This promising immuno-epidemiological marker could represent a useful tool to assess the risk to very low exposure to malaria vectors

  7. Proteosomes, emulsomes, and cholera toxin B improve nasal immunogenicity of human immunodeficiency virus gp160 in mice: induction of serum, intestinal, vaginal, and lung IgA and IgG.

    PubMed

    Lowell, G H; Kaminski, R W; VanCott, T C; Slike, B; Kersey, K; Zawoznik, E; Loomis-Price, L; Smith, G; Redfield, R R; Amselem, S; Birx, D L

    1997-02-01

    Intranasal immunization of mice with human immunodeficiency virus (HIV) rgp160 complexed to proteosomes improved anti-gp160 serum IgA and IgG titers, increased the number of gp160 peptides recognized, and stimulated anti-gp160 intestinal IgA compared with immunization with uncomplexed rgp160 in saline. These enhanced responses were especially evident when either a bioadhesive nanoemulsion (emulsomes) or cholera toxin B subunit (CTB) was added to the proteosome-rgp160 vaccine. Furthermore, anti-gp160 IgG and IgA in vaginal secretions and fecal extracts were induced after intranasal immunization with proteosome-rgp160 delivered either in saline or with emulsomes. Formulation of uncomplexed rgp160 with emulsomes or CTB also enhanced serum and selected mucosal IgA responses. Induction of serum, vaginal, bronchial, intestinal, and fecal IgA and IgG by intranasal proteosome-rgp160 vaccines delivered in saline or with emulsomes or CTB is encouraging for mucosal vaccine development to help control the spread of HIV transmission and AIDS. PMID:9203649

  8. Chrono-lume and magnesium potentiate aggregation of canine but not human platelets in citrated platelet-rich plasma.

    PubMed

    Callan, M B; Shofer, F S; Wojenski, C; Giger, U

    1998-07-01

    The effects of Chrono-lume (CL) and magnesium sulfate (Mg2+), a component of this luciferin-luciferase reagent, on platelet aggregation were studied in platelet-rich plasma (PRP) obtained from blood anticoagulated with sodium citrate from humans, dogs, cats, horses, and cows. The final added Mg2+ concentration of both solutions ranged from 0.75-3.7 mM. CL and Mg2+ had no effect on maximum aggregation of platelets from humans induced by sub-threshold concentrations of collagen and ADP. In contrast, addition of CL or Mg2+ to canine PRP resulted in a dose-dependent and equal potentiation of platelet aggregation in response to sub-threshold concentrations of collagen, ADP, and thrombin in normal and thrombopathic dogs. The effect of CL on platelet aggregation induced by sub-threshold concentrations of agonists was less pronounced and varied in other species according to the agonist. The reason for the marked difference in sensitivity of human and canine platelets to CL or Mg2+ is not clear, although a difference in releasable cation pools of the platelets from these two species has been recognized. Platelet aggregation studies of animals with suspected thrombopathias should be performed without CL to prevent masking of a platelet function defect. PMID:9684806

  9. Suppression of allo-human leucocyte antigen (HLA) antibodies secreted by B memory cells in vitro: intravenous immunoglobulin (IVIg) versus a monoclonal anti-HLA-E IgG that mimics HLA-I reactivities of IVIg

    PubMed Central

    Zhu, D; Ravindranath, M H; Terasaki, P I; Miyazaki, T; Pham, T; Jucaud, V

    2014-01-01

    B memory cells remain in circulation and secrete alloantibodies without antigen exposure > 20 years after alloimmunization postpartum or by transplantation. These long-lived B cells are resistant to cytostatic drugs. Therapeutically, intravenous immunoglobulin (IVIg) is administered to reduce allo-human leucocyte antigen (HLA) antibodies pre- and post-transplantation, but the mechanism of reduction remains unclear. Recently, we reported that IVIg reacts with several HLA-I alleles and the HLA reactivity of IVIg is lost after its HLA-E reactivity is adsorbed out. Therefore, we have generated an anti-HLA-E monoclonal antibody that mimics the HLA-reactivity of IVIg to investigate whether this antibody suppresses IgG secretion, as does IVIg. B cells were purified from the blood of a woman in whose blood the B memory cells remained without antigen exposure > 20 years after postpartum alloimmunization. The B cells were stimulated with cytokines using a well-defined culture system. The anti-HLA-E monoclonal antibody (mAb) significantly suppressed the allo-HLA class-II IgG produced by the B cells, and that this suppression was far superior to that by IVIg. These findings were confirmed with HLA-I antibody secreted by the immortalized B cell line, developed from the blood of another alloimmunized woman. The binding affinity of the anti-HLA-E mAb for peptide sequences shared (i.e. shared epitopes) between HLA-E and other β2-microglobulin-free HLA heavy chains (open conformers) on the cell surface of B cells may act as a ligand and signal suppression of IgG production of activated B memory cells. We propose that anti-HLA-E monoclonal antibody may also be useful to suppress allo-HLA IgG production in vivo. PMID:24611451

  10. Dose-dependent platelet stimulation and inhibition induced by anti-PIA1 IgG

    SciTech Connect

    Ryu, T.; Davis, J.M.; Schwartz, K.A. )

    1990-07-01

    The PIA1 antibody produces several clinically distinct and severe thrombocytopenias. Investigations have demonstrated divergent effects on platelet function; prior reports demonstrated inhibition, while a conflicting publication showed platelet activation. We have resolved this conflict using anti-PIA1 IgG produced by a patient with posttransfusion purpura. Relatively low concentrations stimulated platelet aggregation and release of adenosine triphosphate (ATP) whereas high concentrations inhibited platelet function, producing a thrombasthenia-like state. The number of molecules of platelet-associated IgG necessary to initiate aggregation and ATP release (2,086 +/- 556) or produce maximum aggregation (23,420 +/- 3,706) or complete inhibition (63,582 +/- 2654) were measured with a quantitative radiometric assay for bound anti-PIA1. Preincubation of platelets with high concentrations of PIA1 antibody inhibited platelet aggregation with 10 mumol/L adenosine diphosphate and blocked 125I-labeled fibrinogen platelet binding. Platelet activation with nonfibrinogen dependent agonist, 1 U/ml thrombin, was not inhibited by this high concentration of PIA1 IgG. In conclusion, anti-PIAI IgG produces (1) stimulation of platelet aggregation and ATP release that is initiated with 2000 molecules IgG per platelet and is associated with an increase of 125I-fibrinogen binding; (2) conversely, inhibition of platelet aggregation is observed with maximum antibody binding, 63,000 molecules IgG per platelet, and is mediated via a blockade of fibrinogen binding.

  11. In vivo metabolism of a monoclonal IgG cryoglobulin

    PubMed Central

    Abraham, G. N.; Waterhouse, Christine; Condemi, J. J.

    1979-01-01

    The metabolism of a monoclonal IgG cryoglobulin was studied in a patient with primary idiopathic cryoglobulinaemia under two different clinical conditions. Early in the illness, cryoprotein levels were diminished to 770 mg% by plasmapheresis and plasma disappearance assayed while serum levels of cryoprotein increased to 1100 mg%, i.e. the study was performed during a period of unstable cryoprotein synthesis. A second study was performed 1 year later when serum levels of cryoglobulin were stable at 1500 mg% which allowed a synthetic rate of 64 mg/kg of body weight per day to be computed. This was the upper limit of normal IgG synthesis. The final slopes of the plasma disappearance curves were nearly identical for both studies, indicating that the same fractional percentage of the cryoglobulin serum pool was degraded regardless of its serum level. T1/2 of 15·5 and 17·0 days were obtained when plasma levels were unstable and stable, respectively. For comparative purposes, the plasma disappearance of pooled normal IgG, IgG3 myeloma proteins and a highly aggregated IgG3 myeloma were also studied when cryoprotein levels were 1500 mg%. Normal IgG synthesis was suppressed and only 8·5 mg/kg body weight per day, but its plasma disappearance curve showed a final slope nearly identical to those for the cryoprotein, indicating that the ability to catabolize normal IgG was unimpaired. IgG3 myelomas were cleared at their normal accelerated rate, while aggregated IgG3 was totally cleared from the circulation within 2 days, indicating that the patient was able to catabolize circulating immune complexes. As controls, the catabolism of cryoprotein was shown to be identical to that of pooled normal IgG in two volunteers. The data support the concept that the build up of circulating IgG cryoprotein in the patient studied was due to an increase in cryoprotein synthesis and not to a lack of ability to catabolize it. PMID:428148

  12. Leveraging Human-environment Systems in Residential Buildings for Aggregate Energy Efficiency and Sustainability

    NASA Astrophysics Data System (ADS)

    Xu, Xiaoqi

    Reducing the energy consumed in the built environment is a key objective in many sustainability initiatives. Existing energy saving methods have consisted of physical interventions to buildings and/or behavioral modifications of occupants. However, such methods may not only suffer from their own disadvantages, e.g. high cost and transient effect, but also lose aggregate energy saving potential due to the oftentimes-associated single-building-focused view and an isolated examination of occupant behaviors. This dissertation attempts to overcome the limitations of traditional energy saving research and practical approaches, and enhance residential building energy efficiency and sustainability by proposing innovative energy strategies from a holistic perspective of the aggregate human-environment systems. This holistic perspective features: (1) viewing buildings as mutual influences in the built environment, (2) leveraging both the individual and contextualized social aspects of occupant behaviors, and (3) incorporating interactions between the built environment and human behaviors. First, I integrate three interlinked components: buildings, residents, and the surrounding neighborhood, and quantify the potential energy savings to be gained from renovating buildings at the inter-building level and leveraging neighborhood-contextualized occupant social networks. Following the confirmation of both the inter-building effect among buildings and occupants' interpersonal influence on energy conservation, I extend the research further by examining the synergy that may exist at the intersection between these "engineered" building networks and "social" peer networks, focusing specifically on the additional energy saving potential that could result from interactions between the two components. Finally, I seek to reach an alignment of the human and building environment subsystems by matching the thermostat preferences of each household with the thermal conditions within their

  13. The effects of 7.5% NaCl/6% dextran 70 on coagulation and platelet aggregation in humans

    NASA Technical Reports Server (NTRS)

    Hess, J. R.; Dubick, M. A.; Summary, J. J.; Bangal, N. R.; Wade, C. E.

    1992-01-01

    The combination solution of 7.5% NaCl/6% dextran 70 (HSD) administered IV gives hemodynamic improvement in the treatment of hemorrhagic hypotension. Since earlier dextran solutions were reported to interfere with blood coagulation, the effects of HSD on the prothrombin time (PT), the activated partial thromboplastin time (APTT), platelet aggregation, and platelet concentration were studied. The HSD mixed with human plasma (1:5 and 1:10) slightly prolonged PT, but had no effect on the APTT, compared with saline controls. The HSD also decreased human platelet aggregation at the 1:5 dilution. In separate mixing studies, the hypertonic saline component of HSD was associated with the prolongation of PT and decreased platelet aggregation. The data from these studies indicate that at its proposed therapeutic dose, HSD is expected to have minimal effect on blood coagulation.

  14. The B cell repertoire of patients with rheumatoid arthritis. Frequencies and specificities of peripheral blood B cells reacting with human IgG, human collagens, a mycobacterial heat shock protein and other antigens.

    PubMed Central

    Rudolphi, U; Hohlbaum, A; Lang, B; Peter, H H; Melchers, I

    1993-01-01

    Using a potent in vitro limiting dilution culture system, we have activated human peripheral blood B cells to proliferate and to differentiate into antibody-secreting cells (ASC). Under these conditions 25-100% of B cells are clonally expanded and produce IgM, IgG or IgA. Culture supernatants were tested for antibodies binding to human IgG-Fc fragments (RF), the 65-kD heat shock protein of Mycobacterium bovis (hsp60), human collagens type I, II, IV, V, transferrin, lactoferrin, albumins, and gelatine. All blood samples contained precursors of ASC (p-ASC) able to produce IgM binding to these antigens in frequencies above 0.03% of B cells. Most interestingly, a significant difference exists between rheumatoid arthritis (RA) patients and controls, concerning the relative frequencies of p-ASC able to produce monospecific or multireactive RF. Whereas most p-ASC(RF) in RA patients are monospecific (mean ratio 3.7), most p-ASC(RF) in healthy control persons are cross-reactive with at least one of five other antigens tested (mean ratio 0.2). The data suggest a disease-specific expansion of p-ASC committed to the production of monospecific rheumatoid factors. PMID:8099856

  15. Epitope location for two monoclonal antibodies against human cystatin C, representing opposite aggregation inhibitory properties.

    PubMed

    Behrendt, Izabela; Prądzińska, Martyna; Spodzieja, Marta; Kołodziejczyk, Aleksandra S; Rodziewicz-Motowidło, Sylwia; Szymańska, Aneta; Czaplewska, Paulina

    2016-07-01

    Human cystatin C (hCC), like many other amyloidogenic proteins, dimerizes and possibly makes aggregates by subdomain swapping. Inhibition of the process should suppress the fibrillogenesis leading to a specific amyloidosis (hereditary cystatin C amyloid angiopathy, HCCAA). It has been reported that exogenous agents like monoclonal antibodies against cystatin C are able to suppress formation of cystatin C dimers and presumably control the neurodegenerative disease. We have studied in detail two monoclonal antibodies (mAbs) representing very different aggregation inhibitory potency, Cyst10 and Cyst28, to find binding sites in hCC sequence responsible for the immunocomplex formation and pave the way for possible immunotherapy of HCCAA. We used the epitope extraction/excision mass spectrometry approach with the use of different enzymes complemented by affinity studies with synthetic hCC fragments as a basic technique for epitope identification. The results were analyzed in the context of hCC structure allowing us to discuss the binding sites for both antibodies. Epitopic sequences for clone Cyst28 which is a highly potent dimerization inhibitor were found in N-terminus, loop 1 and 2 (L1, L2) and fragments of β2 and β3 strands. The crucial difference between conformational epitope sequences found for both mAbs seems to be the lack of interactions with hCC via N-terminus and the loop 1 in the case of mAb Cyst10. Presumably the interactions of mAbs with hCC via L1 and β sheet fragments make the hCC structure rigid and unable to undergo the swapping process. PMID:27143169

  16. Upregulation of NRG-1 and VAMP-1 in human brain aggregates exposed to clozapine.

    PubMed

    Chana, Gursharan; Lucero, Ginger; Salaria, Shahid; Lozach, Jean; Du, Pinyi; Woelk, Christopher; Everall, Ian

    2009-09-01

    Growing genetic evidence has implicated a role for neuregulin-1 (NRG-1) in schizophrenia pathogenesis as well as alterations in SNAP receptor (SNARE) proteins at both gene and protein levels in post-mortem investigations. In relation to a potential therapeutic mechanism for atypical antipsychotic medications, clozapine has been shown to increase both NRG-1 levels and synaptic markers in rodents. As evidence continues to mount for a potential restoration in connectivity by antipsychotic medications being a mode of efficacy we chose to examine the effects of the atypical antipsychotic clozapine and the typical antipsychotic haloperidol on NRG-1 and SNARE protein transcripts in human brain aggregates exposed to plasma levels chronically for a period of three weeks. At the end of this exposure period we performed quantitative real-time PCR to investigate the mRNA levels of NRG-1, VAMP-1 and SNAP-25. Overall we found that clozapine had the ability to upregulate NRG-1 (+3.58 fold change) and VAMP-1 (+1.92) while SNAP-25 remained unchanged. Changes for haloperidol exposed aggregates were below our cut-off of +1.5. Overall the results of our investigation lend further support to atypical antipsychotic medications having the potential to increase levels of neurotrophic and synaptic markers such as NRG-1 and VAMP-1, the former being a strong candidate susceptibility gene for schizophrenia. In the absence of frank neuronal loss in schizophrenia, restoration of neuronal and synaptic functions by atypical antipsychotics in the brains of schizophrenics maybe a key mechanism of therapeutic efficacy by re-establishing normal connectivity and functioning. PMID:19502011

  17. Thermal stability of human plasma electronegative low-density lipoprotein: A paradoxical behavior of low-density lipoprotein aggregation.

    PubMed

    Rull, Anna; Jayaraman, Shobini; Gantz, Donald L; Rivas-Urbina, Andrea; Pérez-Cuellar, Montserrat; Ordóñez-Llanos, Jordi; Sánchez-Quesada, Jose Luis; Gursky, Olga

    2016-09-01

    Low-density lipoprotein (LDL) aggregation is central in triggering atherogenesis. A minor fraction of electronegative plasma LDL, termed LDL(-), plays a special role in atherogenesis. To better understand this role, we analyzed the kinetics of aggregation, fusion and disintegration of human LDL and its fractions, LDL(+) and LDL(-). Thermal denaturation of LDL was monitored by spectroscopy and electron microscopy. Initially, LDL(-) aggregated and fused faster than LDL(+), but later the order reversed. Most LDL(+) disintegrated and precipitated upon prolonged heating. In contrast, LDL(-) partially retained lipoprotein morphology and formed soluble aggregates. Biochemical analysis of all fractions showed no significant degradation of major lipids, mild phospholipid oxidation, and an increase in non-esterified fatty acid (NEFA) upon thermal denaturation. The main baseline difference between LDL subfractions was higher content of NEFA in LDL(-). Since NEFA promote lipoprotein fusion, increased NEFA content can explain rapid initial aggregation and fusion of LDL(-) but not its resistance to extensive disintegration. Partial hydrolysis of apoB upon heating was similar in LDL subfractions, suggesting that minor proteins importantly modulate LDL disintegration. Unlike LDL(+), LDL(-) contains small amounts of apoA-I and apoJ. Addition of exogenous apoA-I to LDL(+) hampered lipoprotein aggregation, fusion and precipitation, while depletion of endogenous apoJ had an opposite effect. Therefore, the initial rapid aggregation of LDL(-) is apparently counterbalanced by the stabilizing effects of minor proteins such as apoA-I and apoJ. These results help identify key determinants for LDL aggregation, fusion and coalescence into lipid droplets in vivo. PMID:27233433

  18. MotionFlow: Visual Abstraction and Aggregation of Sequential Patterns in Human Motion Tracking Data.

    PubMed

    Jang, Sujin; Elmqvist, Niklas; Ramani, Karthik

    2016-01-01

    Pattern analysis of human motions, which is useful in many research areas, requires understanding and comparison of different styles of motion patterns. However, working with human motion tracking data to support such analysis poses great challenges. In this paper, we propose MotionFlow, a visual analytics system that provides an effective overview of various motion patterns based on an interactive flow visualization. This visualization formulates a motion sequence as transitions between static poses, and aggregates these sequences into a tree diagram to construct a set of motion patterns. The system also allows the users to directly reflect the context of data and their perception of pose similarities in generating representative pose states. We provide local and global controls over the partition-based clustering process. To support the users in organizing unstructured motion data into pattern groups, we designed a set of interactions that enables searching for similar motion sequences from the data, detailed exploration of data subsets, and creating and modifying the group of motion patterns. To evaluate the usability of MotionFlow, we conducted a user study with six researchers with expertise in gesture-based interaction design. They used MotionFlow to explore and organize unstructured motion tracking data. Results show that the researchers were able to easily learn how to use MotionFlow, and the system effectively supported their pattern analysis activities, including leveraging their perception and domain knowledge. PMID:26529685

  19. Enhanced Biological Functions of Human Mesenchymal Stem-Cell Aggregates Incorporating E-Cadherin-Modified PLGA Microparticles.

    PubMed

    Zhang, Yan; Mao, Hongli; Gao, Chao; Li, Suhua; Shuai, Qizhi; Xu, Jianbin; Xu, Ke; Cao, Lei; Lang, Ren; Gu, Zhongwei; Akaike, Toshihiro; Yang, Jun

    2016-08-01

    Mesenchymal stem cells (MSCs) have emerged as a promising source of multipotent cells for various cell-based therapies due to their unique properties, and formation of 3D MSC aggregates has been explored as a potential strategy to enhance therapeutic efficacy. In this study, poly(lactic-co-glycolic acid) (PLGA) microparticles modified with human E-cadherin fusion protein (hE-cad-PLGA microparticles) have been fabricated and integrated with human MSCs to form 3D cell aggregates. The results show that, compared with the plain PLGA, the hE-cad-PLGA microparticles distribute within the aggregates more evenly and further result in a more significant improvement of cellular proliferation and secretion of a series of bioactive factors due to the synergistic effects from the bioactive E-cadherin fragments and the PLGA microparticles. Meanwhile, the hE-cad-PLGA microparticles incorporated in the aggregates upregulate the phosphorylation of epidermal growth factor receptors and activate the AKT and ERK1/2 signaling pathways in the MSCs. Additionally, the E-cadherin/β-catenin cellular membrane complex in the MSCs is markedly stimulated by the hE-cad-PLGA microparticles. Therefore, engineering 3D cell aggregates with hE-cad-PLGA microparticles can be a promising method for ex vivo multipotent stem-cell expansion with enhanced biological functions and may offer a novel route to expand multipotent stem-cell-based clinical applications. PMID:27245478

  20. IgG transport across mucosal barriers by neonatal Fc receptor for IgG and mucosal immunity.

    PubMed

    Yoshida, Masaru; Masuda, Atsuhiro; Kuo, Timothy T; Kobayashi, Kanna; Claypool, Steven M; Takagawa, Tetsuya; Kutsumi, Hiromu; Azuma, Takeshi; Lencer, Wayne I; Blumberg, Richard S

    2006-12-01

    Mucosal secretions of the human gastrointestinal, respiratory, and genital tracts contain significant quantities of IgG. The neonatal Fc receptor for IgG (FcRn) plays a major role in regulating host IgG levels and transporting IgG and associated antigens across polarized epithelial barriers. The FcRn can then recycle the IgG/antigen complex back across the intestinal barrier into the lamina propria for processing by dendritic cells and presentation to CD4(+) T cells in regional organized lymphoid structures. FcRn, through its ability to secrete and absorb IgG, thus integrates luminal antigen encounters with systemic immune compartments and, as such, provides essential host defense and immunoregulatory functions at the mucosal surfaces. PMID:17051393

  1. Effects of aggregation and solvent on the toxicity of amphotericin B to human erythrocytes.

    PubMed Central

    Legrand, P; Romero, E A; Cohen, B E; Bolard, J

    1992-01-01

    In aqueous suspensions of amphotericin B (AmB), a polyene antibiotic and antifungal agent, three forms of AmB coexist: monomers, water-soluble oligomers, and non-water-soluble aggregates. The toxicity of the water-soluble self-associated form of AmB compared with that of the non-water-soluble self-associated form was tested by measuring induction of K+ leakage from human erythrocytes, using different suspensions containing the antibiotic and phosphate-buffered saline. These suspensions were obtained from various stock solutions of the antibiotic in dimethyl formamide or dimethyl sulfoxide. Their circular dichroism spectra around 340 nm, indicative of the degree of AmB self-association, were strongly dependent on the concentration of organic solvent in the suspensions. The nonsoluble self-associated form was separated from the water-soluble form by centrifugation. The nonsoluble form was favored by a high concentration of AmB of the stock solution. The kinetics of AmB-induced K+ leakage from human erythrocytes also appeared to be strongly dependent on the AmB concentration of the stock solution being much weaker with concentrated stock solutions. It was concluded that the only form of AmB toxic to human erythrocytes is the water-soluble self-associated form (in contrast with fungal cells on which the monomeric form is also active). This result may be important in the design of new less toxic AmB derivatives and in the understanding of the mechanism of action of liposomal AmB. PMID:1489196

  2. Cysteine Racemization on IgG Heavy and Light Chains

    PubMed Central

    Zhang, Qingchun; Flynn, Gregory C.

    2013-01-01

    Under basic pH conditions, the heavy chain 220-light chain 214 (H220-L214) disulfide bond, found in the flexible hinge region of an IgG1, can convert to a thioether. Similar conditions also result in racemization of the H220 cysteine. Here, we report that racemization occurs on both H220 and L214 on an IgG1 with a λ light chain (IgG1λ) but almost entirely on H220 of an IgGl with a κ light chain (IgG1κ) under similar conditions. Likewise, racemization was detected at significant levels on H220 and L214 on endogenous human IgG1λ but only at the H220 position on IgG1κ. Low but measurable levels of d-cysteines were found on IgG2 cysteines in the hinge region, both with monoclonal antibodies incubated under basic pH conditions and on antibodies isolated from human serum. A simplified reaction mechanism involving reversible β-elimination on the cysteine is presented that accounts for both base-catalyzed racemization and thioether formation at the hinge disulfide. PMID:24142697

  3. Separate Fc-receptors for immunoglogulins IgG2a and IgG2b on an established cell line of mouse macrophages.

    PubMed

    Walker, W S

    1976-04-01

    The specificity of Fe-receptors on IC-21 cells, an established line of mouse peritoneal macrophages with antibody-dependent effector cell activity has been examined. Only IgG2a and IgG2b myeloma proteins bound readily to IC-21 Fc-receptors, the former in nonaggregated as well as aggregated form, the latter only as aggregated complexes. Thus, IgG2a bound in a manner characteristic of classically defined cytophilic antibody, whereas the binding of IgG2b appeared to be mediated by Fc-receptors for antigen-antibody complexes. Evidence is presented in support of the view that IC-21 macrophages possess separate and distinct Fc-receptor sites for these two immunoglobulins. PMID:1254971

  4. Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering.

    PubMed

    Geoghegan, James C; Fleming, Ryan; Damschroder, Melissa; Bishop, Steven M; Sathish, Hasige A; Esfandiary, Reza

    2016-07-01

    Undesired solution behaviors such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation can introduce substantial challenges during development of monoclonal antibody formulations. Although a global mechanistic understanding of RSA (i.e., native and reversible protein-protein interactions) is sufficient to develop robust formulation controls, its mitigation via protein engineering requires knowledge of the sites of protein-protein interactions. In the study reported here, we coupled our previous hydrogen-deuterium exchange mass spectrometry findings with structural modeling and in vitro screening to identify the residues responsible for RSA of a model IgG1 monoclonal antibody (mAb-C), and rationally engineered variants with improved solution properties (i.e., reduced RSA and viscosity). Our data show that mutation of either solvent-exposed aromatic residues within the heavy and light chain variable regions or buried residues within the heavy chain/light chain interface can significantly mitigate RSA and viscosity by reducing the IgG's surface hydrophobicity. The engineering strategy described here highlights the utility of integrating complementary experimental and in silico methods to identify mutations that can improve developability, in particular, high concentration solution properties, of candidate therapeutic antibodies. PMID:27050875

  5. Thermal unfolding and aggregation of human complement protein C9: a differential scanning calorimetry study.

    PubMed

    Lohner, K; Esser, A F

    1991-07-01

    The thermotropic behavior of purified human complement protein C9 was investigated by high-sensitivity differential scanning calorimetry. When dissolved in physiological buffers (pH 7.2, 150 mM NaCl), C9 underwent three endothermic transitions with transition temperatures (Tm) centered at about 32, 48, and 53 degrees C, respectively, and one exothermic transition above 64 degrees C that correlated with protein aggregation. The associated calorimetric enthalpies of the three endothermic transitions were about 45, 60, and 161 kcal/mol with cooperative ratios (delta Hcal/delta HvH) close to unity. The total calorimetric enthalphy for the unfolding process was in the range of 260-280 kcal/mol under all conditions. The exothermic aggregation temperature was strongly pH dependent, changing from 60 degrees C at pH 6.6 to 81.4 degrees C at pH 8.0, whereas none of the three endothermic transitions was significantly affected by pH changes. They were, however, sensitive to addition of calcium ions; most affected was Tm1 which shifted from 32 to 35.8 degrees C in the presence of 3 mM calcium, i.e., the normal blood concentration. Kosmotropic ions stabilized the protein by shifting the endothermic transitions to slightly higher temperatures whereas inclusion of chaotropic ions (such as choline), removal of bound calcium by addition of EDTA, or proteolysis with thrombin lowered the transition temperatures. Previous studies had indicated the formation of at least three different forms of C9 during membrane insertion or during heat polymerization, and it is suggested that the three endothermic transitions reflect the formation of such C9 conformers. Choline, which is present at high concentrations on the surface of biological membranes, and calcium ions have the ability to shift the transition temperatures of the first two transitions to be either close to or below body temperature. Thus, it is very likely that C9 is present in vivo in a partially unfolded state when bound to a

  6. Interactions between human serum proteins and oral streptococci reveal occurrence of receptors for aggregated beta 2-microglobulin.

    PubMed Central

    Ericson, D; Bratthall, D; Björck, L; Myhre, E; Kronvall, G

    1979-01-01

    A total of 31 strains of oral streptococci representing Streptococcus mutans, Streptococcus sanguis, Streptococcus mitior, Streptococcus salivarius, and Streptococcus milleri were tested for possible binding of human immunoglobulins G, G1, G2, G3, G4, A1, A2, M1, and M2 and haptoglobin, hemoglobin, fibrinogen, and aggregated beta 2-microglobulin. Radiolabeled beta 2-microglobulin in aggregated form showed affinity for 20 of the 31 strains tested. Binding activity for the protein was found in strains belonging to all five species. The bacterial receptor was resistant to trypsin. Monomeric, unlabeled beta 2-microglobulin did not interfere with the binding of the aggregated form. Of the other proteins tested, only the immunoglobulin A1 protein showed positive binding, and that was only with a single strain of S. milleri. beta 2-Microglobulin is present on all nucleated cell membranes in vivo. The reaction between aggregated beta 2-microglobulin and oral streptococci is a new type of human-bacterium interaction which should be considered in studies of bacterial adherence. PMID:90015

  7. Staphylococcal enterotoxin B initiates protein kinase C translocation and eicosanoid metabolism while inhibiting thrombin-induced aggregation in human platelets.

    PubMed

    Tran, Uyen; Boyle, Thomas; Shupp, Jeffrey W; Hammamieh, Rasha; Jett, Marti

    2006-08-01

    Staphylococcal enterotoxin (SE) B, a heat-stable toxin secreted by Staphylococcus aureus, has been implicated in the pathogenesis and exacerbation of several critical illnesses. It has been hypothesized that enterotoxins may interact with blood products such as platelets, in addition to T-lymphocytes and renal proximal tubule cells. The aim of this present study was to elucidate whether SEB directly alters human platelet function. Human platelet rich plasma (PRP) was pre-incubated with SEA, SEB, SEC or TSST-1, (at various concentrations and incubation times). After incubation, PRP was exposed to thrombin and aggregation was assessed. Incubation with all toxins tested resulted in decreased aggregation, specifically; exposure to 10mu g/ml of SEB for 30 min caused a 20% decrease and a 49% decrease at 90 min. A similar reduction in aggregation was seen in samples incubated with phorbol myristate acetate, a known stimulator of protein kinase C (PKC). Further, platelets exposed to SEB exhibited an increased plasma membrane PKC activity. Sphingosine, an inhibitor of PKC proved to block the SEB-induced reduction in aggregation. SEB effects on platelet metabolism were investigated using high performance liquid chromatography showing up to a 2-fold increase of active metabolites lipoxin A4 and 12-HETE, as compared to control. These data indicate that SEB is able to induce platelet dysfunction, and these effects may be mediated through activation of PKC. PMID:16550298

  8. Effect of lobe pumping on human albumin: investigating the underlying mechanisms of aggregate formation.

    PubMed

    Gomme, Peter T; Hunt, Ben M; Tatford, Owen C; Johnston, Anna; Bertolini, Joseph

    2006-02-01

    A common problem in the manufacture of liquid protein therapeutics is the tendency for aggregation and particle formation on extended storage. One aspect of processing that might contribute to particle formation is pumping. In the present study, we demonstrate that lobe pumps can promote aggregation in albumin preparations. This is accentuated where the clearance between the pump housing and lobes is increased. Under these conditions, the pump efficiency decreases, resulting in increased exposure of the protein to the pump environment. Depending on the inherent physicochemical stability of the protein, this can lead to aggregate formation, which can influence the long-term stability characteristics of the product. PMID:16246176

  9. Mechanism for benzyl alcohol-induced aggregation of recombinant human interleukin-1 receptor antagonist in aqueous solution.

    PubMed

    Zhang, Ye; Roy, Shouvik; Jones, Latoya S; Krishnan, Sampathkumar; Kerwin, Bruce A; Chang, Byeong S; Manning, Mark C; Randolph, Theodore W; Carpenter, John F

    2004-12-01

    Benzyl alcohol, an antimicrobial preservative, accelerates aggregation and precipitation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) in aqueous solution. The loss of native monomer during incubation at 37 degrees C was determined by analysis of sample aliquots with size exclusion high performance liquid chromatography (SE-HPLC). Benzyl alcohol caused minor perturbation of the tertiary structure of the protein without changing its secondary structure, documenting that the preservative caused a minor shift in the protein molecular population toward partially unfolded species. Consistent with this conclusion, in the presence of benzyl alcohol the rate of H-D exchange was accelerated and the fluorescence of 1-anilinonaphthalene-8-sulfonic acid in the presence of rhIL1ra was increased. Benzyl alcohol did not alter the free energy of unfolding based on unfolding experiments in urea or guanidine HCl. With differential scanning calorimetry it was determined that benzyl alcohol reduced the apparent Tm of rhIL-1ra, but this effect occurred because the preservative lowered the temperature at which the protein aggregated during heating. Isothermal calorimetry documented that the interaction of benzyl alcohol with rhIL-1ra is relatively weak and hydrophobically driven. Thus, benzyl alcohol accelerates protein aggregation by binding to the protein and favoring an increase in the level of partially unfolded, aggregation-competent species. Sucrose partially inhibited benzyl alcohol-induced aggregation and tertiary structural change. Sucrose is preferentially excluded from the surface of the protein, favoring most compact native state species over expanded aggregation-prone forms. PMID:15514986

  10. Evaluation of a novel human IgG1 anti-claudin3 antibody that specifically recognizes its aberrantly localized antigen in ovarian cancer cells and that is suitable for selective drug delivery

    PubMed Central

    Romani, Chiara; Cocco, Emiliano; Bignotti, Eliana; Moratto, Daniele; Bugatti, Antonella; Todeschini, Paola; Bandiera, Elisabetta; Tassi, Renata; Zanotti, Laura; Pecorelli, Sergio; Sartori, Enrico; Odicino, Franco E.; de Marco, Ario; Davide Santin, Alessandro; Ravaggi, Antonella; Mitola, Stefania

    2015-01-01

    Membrane protein claudin3 has been recently suggested as a marker for biologically aggressive tumors and a possible target for the therapeutic delivery of active anti-cancer compounds. Claudin3-binding molecules such as the Clostridium perfringens enterotoxin (CPE), CPE-related molecules, and murine and chimeric antibodies have shown promising antitumor efficacy in preclinical oncological settings. We first engineered a fully human anti-claudin3 IgG1 antibody (IgGH6) by fusing the human IgG1 Fc-domain to the anti-claudin3 scFvH6 previously isolated from a pre-immune phage display library. The construct was expressed in mammalian cells and specifically targeted claudin3 endogenously expressed on the surface of different human ovarian cancer cell lines. No detectable cross-reactivity with other homologous claudins was observed. The epitope recognized by IgGH6 is located within the minor extracellular domain of claudin3 and becomes accessible only in tumor cells characterized by incomplete junction formation. Confocal microscopy experiments demonstrated that IgGH6 was actively internalized in tumor cells after binding to native claudin3 and co-localized, likely within intracellular vesicles, with the C-CPE peptide. Preliminary results indicate that IgGH6 accumulated in vivo in free claudin3 ovarian carcinoma xenografts. For its selective uptake in tumor cells and its human nature, IgGH6 represents a valuable candidate for antibody-drug conjugate therapeutic applications in ovarian cancer patients. PMID:26416446

  11. N114S mutation causes loss of ATP-induced aggregation of human phosphoribosylpyrophosphate synthetase 1

    SciTech Connect

    Liu Honglin; Peng, Xiaohui; Zhao Fang; Zhang Guobin; Tao Ye; Luo Zhaofeng; Li Yang; Teng Maikun; Li Xu Wei Shiqiang

    2009-02-20

    This study examined recombinant wild-type human phosphoribosylpyrophosphate synthetase 1 (wt-PRS1, EC 2.7.6.1) and the point mutant Asn114Ser PRS1 (N114S-Mutant) in cells of a patient with primary gout. Dynamic light-scattering and sedimentation velocity experiments indicated that the monomeric wt-PRS1 in solution was assembled into hexamers after adding the substrate ATP. However, this ATP-induced aggregation effect was not observed with N114S-Mutant, which has a 50% higher enzymatic activity than that of wt-PRS1. Synchrotron radiation circular dichroism spectroscopy revealed that the point mutation causes an increase of {alpha}-helix content and a decrease of turn content. Examination of the crystal structure of wt-PRS1 indicated that 12 hydrogen bonds formed by 6 pairs of N114 and D139 have an important role in stabilizing the hexamer. We suggest that the substitution of S114 for N114 in N114S-Mutant leads to the rupture of 12 hydrogen bonds and breakage of the PO{sub 4}{sup 3-} allosteric site where PO{sub 4}{sup 3-} functions as a fixer of the ATP-binding loop. Therefore, we consider that formation of the hexamer as the structural basis of the ADP allosteric inhibition is greatly weakened by the N114S mutation, and that alteration of the ATP-binding loop conformation is the key factor in the increased activity of N114S-Mutant. These two factors could be responsible for the high level of activity of N114S-Mutant in this patient.

  12. [THE INFLUENCE OF HYDROGEN SULFIDE ON COLLAGEN-INDUCED AGGREGATION OF HUMAN PLATELETS].

    PubMed

    Petrova, I V; Trubacheva, O A; Mangataeva, O S; Suslova, T E; Kovalev, I V; Gusakova, S V

    2015-10-01

    Study the impact of hydrogen sulfide on collagen-induced platelet aggregation from healthy donors and patients with type 2 diabetes. In healthy individuals, in contrast to patients with type 2 diabetes, NaHS significantly inhibited platelet aggregation. Activators of cAMP signaling (forskolin and phosphodiesterase inhibitor) significantly reduced platelet aggregation in both groups of examinees. NO-synthase inhibitors increased platelet aggregation in healthy volunteers, but not in patients with type 2 diabetes. The presence of H2S donor did not alter the extent of platelet aggregation at high concentrations of cAMP or decreased production of nitric oxide. It is assumed that the antiplatelet effect of H2S is not associated with the effect on the signal system, mediated cAMP or nitric oxide. Change H2S-dependent regulation of platelet aggregation in patients with type 2 diabetes is caused by disorders have been reported with this disease: the increase of intracellular calcium ion concentration, oxidative damage to proteins, hyperhomocysteinemia, glycosylation of key proteins involved in this process. PMID:26827498

  13. New analogues of 13-hydroxyocatdecadienoic acid and 12-hydroxyeicosatetraenoic acid block human blood platelet aggregation and cyclooxygenase-1 activity

    PubMed Central

    2012-01-01

    Background Thromboxane A2 is derived from arachidonic acid through the action of cyclooxygenases and thromboxane synthase. It is mainly formed in blood platelets upon activation and plays an important role in aggregation. Aspirin is effective in reducing the incidence of complications following acute coronary syndrome and stroke. The anti-thrombotic effect of aspirin is obtained through the irreversible inhibition of cyclooxygenases. Analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid were shown previously to modulate platelet activation and to block thromboxane receptors. Results and discussion We synthesized 10 compounds based on the structures of analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid and evaluated their effect on platelet aggregation triggered by arachidonic acid. The structure activity relationship was evaluated. Five compounds showed a significant inhibition of platelet aggregation and highlighted the importance of the lipidic hydrophobic hydrocarbon chain and the phenol group. Their IC50 ranged from 7.5 ± 0.8 to 14.2 ± 5.7 μM (Mean ± S.E.M.). All five compounds decreased platelet aggregation and thromboxane synthesis in response to collagen whereas no modification of platelet aggregation in response to thromboxane receptor agonist, U46619, was observed. Using COS-7 cells overexpressing human cyclooxygenase-1, we showed that these compounds are specific inhibitors of cyclooxygenase-1 with IC50 ranging from 1.3 to 12 μM. Docking observation of human recombinant cyclooxygenase-1 supported a role of the phenol group in the fitting of cyclooxygenase-1, most likely related to hydrogen bonding with the Tyr 355 of cyclooxygenase-1. Conclusions In conclusion, the compounds we synthesized at first based on the structures of analogues of 12 lipoxygenase metabolites showed a role of the phenol group in the anti-platelet and anti-cyclooxygenase-1 activities. These compounds mediate their

  14. Transfer of IgG in the female genital tract by MHC class I-related neonatal Fc receptor (FcRn) confers protective immunity to vaginal infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    IgG is a major immunoglobulin subclass in mucosal secretions of human female genital tract, where it predominates over the IgA isotype. Despite the abundance of IgG, surprisingly little is known about whether and how IgG enters the lumen of the genital tract and the exact role of local IgG may play ...

  15. Human Islet Amyloid Polypeptide N-Terminus Fragment Self-Assembly: Effect of Conserved Disulfide Bond on Aggregation Propensity

    NASA Astrophysics Data System (ADS)

    Ilitchev, Alexandre I.; Giammona, Maxwell J.; Do, Thanh D.; Wong, Amy G.; Buratto, Steven K.; Shea, Joan-Emma; Raleigh, Daniel P.; Bowers, Michael T.

    2016-02-01

    Amyloid formation by human islet amyloid polypeptide (hIAPP) has long been implicated in the pathogeny of type 2 diabetes mellitus (T2DM) and failure of islet transplants, but the mechanism of IAPP self-assembly is still unclear. Numerous fragments of hIAPP are capable of self-association into oligomeric aggregates, both amyloid and non-amyloid in structure. The N-terminal region of IAPP contains a conserved disulfide bond between cysteines at position 2 and 7, which is important to hIAPP's in vivo function and may play a role in in vitro aggregation. The importance of the disulfide bond in this region was probed using a combination of ion mobility-based mass spectrometry experiments, molecular dynamics simulations, and high-resolution atomic force microscopy imaging on the wildtype 1-8 hIAPP fragment, a reduced fragment with no disulfide bond, and a fragment with both cysteines at positions 2 and 7 mutated to serine. The results indicate the wildtype fragment aggregates by a different pathway than either comparison peptide and that the intact disulfide bond may be protective against aggregation due to a reduction of inter-peptide hydrogen bonding.

  16. A Numerical Computation Model for Low-Density Lipoprotein (LDL) Aggregation and Deposition in the Human Artery

    NASA Astrophysics Data System (ADS)

    Zhao, Yongli; Cai, Shaobiao; Ratner, Albert

    2009-11-01

    Cholesterol caused cardiovascular events are commonly seen in human lives. These events are primarily believed to be caused by the built up of particles like low-density lipoprotein (LDL). When a large number of LDL circulates in the blood, it can gradually build up in the inner walls of the arteries. A thick, hard deposit plaque can be formed together with other substances. This type of plaque may clog those arteries and cause vascular problems. Clinical evidences suggest that LDL is related to cardiovascular events and the progression of coronary heart disease is due to its aggregation and deposition. This study presents an investigation of LDL aggregation and deposition based on particulate flow. A soft-sphere based particulate computational flow model is developed to represent LDL suspending in plasma. The transport, collision and adhesion phenomena of LDL particles are simulated to examine the physics involved in aggregation and deposition. A multiple-time step discrete-element approach is presented for efficiently simulating large number of LDL particles and their interactions. The roles the quality and quantity the LDL playing in the process of aggregation and deposition are determined. The study provides a new perspective for improving the understanding of the fundamentals as related to these particle-caused cardiovascular events.

  17. Aggregation of deamidated human βB2-crystallin and incomplete rescue by α-crystallin chaperone

    PubMed Central

    Michiel, Magalie; Duprat, Elodie; Skouri-Panet, Fériel; Finet, Stéphanie; Tardieu, Annette; Lampi, Kirsten J.

    2010-01-01

    Aging of the lens is accompanied by extensive deamidation of the lens specific proteins, the crystallins. Deamidated crystallins are increased in the insoluble proteins and may contribute to cataracts. Deamidation has been shown in vitro to alter the structure and decrease the stability of human lens βB1, βB2 and βA3-crystallin. Of particular interest, βB2 mutants were constructed to mimic the effect of in vivo deamidations at the interacting interface between domains, at Q70 in the N terminal domain and at Q162, its C terminal homologue. The double mutant was also constructed. We previously reported that deamidation at the critical interface sites decreased stability, while preserving the dimeric 3D structure. In the present study, dynamic light scattering, differential scanning calorimetry and small angle X-ray scattering were used to investigate the effect of deamidation on stability, thermal unfolding and aggregation. The bovine βLb fraction was used for comparative analysis. The chaperone requirements of the various samples were determined using bovine α-crystallins as the chaperone. Deamidation at both interface Gln residues or at Q70, but not Q162, significantly lowered the temperature for unfolding and aggregation, which was rapidly followed by precipitation. This deamidation-induced aggregation and precipitation was not completely prevented by α-crystallin chaperone. A potential mechanism for cataract formation in vivo involving accumulation of deamidated β-crystallin aggregates is discussed. PMID:20188088

  18. Human Islet Amyloid Polypeptide N-Terminus Fragment Self-Assembly: Effect of Conserved Disulfide Bond on Aggregation Propensity.

    PubMed

    Ilitchev, Alexandre I; Giammona, Maxwell J; Do, Thanh D; Wong, Amy G; Buratto, Steven K; Shea, Joan-Emma; Raleigh, Daniel P; Bowers, Michael T

    2016-06-01

    Amyloid formation by human islet amyloid polypeptide (hIAPP) has long been implicated in the pathogeny of type 2 diabetes mellitus (T2DM) and failure of islet transplants, but the mechanism of IAPP self-assembly is still unclear. Numerous fragments of hIAPP are capable of self-association into oligomeric aggregates, both amyloid and non-amyloid in structure. The N-terminal region of IAPP contains a conserved disulfide bond between cysteines at position 2 and 7, which is important to hIAPP's in vivo function and may play a role in in vitro aggregation. The importance of the disulfide bond in this region was probed using a combination of ion mobility-based mass spectrometry experiments, molecular dynamics simulations, and high-resolution atomic force microscopy imaging on the wildtype 1-8 hIAPP fragment, a reduced fragment with no disulfide bond, and a fragment with both cysteines at positions 2 and 7 mutated to serine. The results indicate the wildtype fragment aggregates by a different pathway than either comparison peptide and that the intact disulfide bond may be protective against aggregation due to a reduction of inter-peptide hydrogen bonding. Graphical Abstract ᅟ. PMID:26894887

  19. Human Islet Amyloid Polypeptide N-Terminus Fragment Self-Assembly: Effect of Conserved Disulfide Bond on Aggregation Propensity

    NASA Astrophysics Data System (ADS)

    Ilitchev, Alexandre I.; Giammona, Maxwell J.; Do, Thanh D.; Wong, Amy G.; Buratto, Steven K.; Shea, Joan-Emma; Raleigh, Daniel P.; Bowers, Michael T.

    2016-06-01

    Amyloid formation by human islet amyloid polypeptide (hIAPP) has long been implicated in the pathogeny of type 2 diabetes mellitus (T2DM) and failure of islet transplants, but the mechanism of IAPP self-assembly is still unclear. Numerous fragments of hIAPP are capable of self-association into oligomeric aggregates, both amyloid and non-amyloid in structure. The N-terminal region of IAPP contains a conserved disulfide bond between cysteines at position 2 and 7, which is important to hIAPP's in vivo function and may play a role in in vitro aggregation. The importance of the disulfide bond in this region was probed using a combination of ion mobility-based mass spectrometry experiments, molecular dynamics simulations, and high-resolution atomic force microscopy imaging on the wildtype 1-8 hIAPP fragment, a reduced fragment with no disulfide bond, and a fragment with both cysteines at positions 2 and 7 mutated to serine. The results indicate the wildtype fragment aggregates by a different pathway than either comparison peptide and that the intact disulfide bond may be protective against aggregation due to a reduction of inter-peptide hydrogen bonding.

  20. Construction aggregates

    USGS Publications Warehouse

    Tepordei, V.V.

    1996-01-01

    Part of the Annual Commodities Review 1995. Production of construction aggregates such as crushed stone and construction sand and gravel showed a marginal increase in 1995. Most of the 1995 increases were due to funding for highway construction work. The major areas of concern to the industry included issues relating to wetlands classification and the classification of crystalline silica as a probable human carcinogen. Despite this, an increase in demand is anticipated for 1996.

  1. Classification and Characterization of Therapeutic Antibody Aggregates

    PubMed Central

    Joubert, Marisa K.; Luo, Quanzhou; Nashed-Samuel, Yasser; Wypych, Jette; Narhi, Linda O.

    2011-01-01

    A host of diverse stress techniques was applied to a monoclonal antibody (IgG2) to yield protein particles with varying attributes and morphologies. Aggregated solutions were evaluated for percent aggregation, particle counts, size distribution, morphology, changes in secondary and tertiary structure, surface hydrophobicity, metal content, and reversibility. Chemical modifications were also identified in a separate report (Luo, Q., Joubert, M. K., Stevenson, R., Narhi, L. O., and Wypych, J. (2011) J. Biol. Chem. 286, 25134–25144). Aggregates were categorized into seven discrete classes, based on the traits described. Several additional molecules (from the IgG1 and IgG2 subtypes as well as intravenous IgG) were stressed and found to be defined with the same classification system. The mechanism of protein aggregation and the type of aggregate formed depends on the nature of the stress applied. Different IgG molecules appear to aggregate by a similar mechanism under the same applied stress. Aggregates created by harsh mechanical stress showed the largest number of subvisible particles, and the class generated by thermal stress displayed the largest number of visible particles. Most classes showed a disruption of the higher order structure, with the degree of disorder depending on the stress process. Particles in all classes (except thermal stress) were at least partially reversible upon dilution in pH 5 buffer. High copper content was detected in isolated metal-catalyzed aggregates, a stress previously shown to produce immunogenic aggregates. In conclusion, protein aggregates can be a very heterogeneous population, whose qualities are the result of the type of stress that was experienced. PMID:21454532

  2. IgG4 subclass antibodies impair antitumor immunity in melanoma

    PubMed Central

    Karagiannis, Panagiotis; Gilbert, Amy E.; Josephs, Debra H.; Ali, Niwa; Dodev, Tihomir; Saul, Louise; Correa, Isabel; Roberts, Luke; Beddowes, Emma; Koers, Alexander; Hobbs, Carl; Ferreira, Silvia; Geh, Jenny L.C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Blower, Philip J.; Mitchell, Tracey; Fear, David J.; Spicer, James F.; Lacy, Katie E.; Nestle, Frank O.; Karagiannis, Sophia N.

    2013-01-01

    Host-induced antibodies and their contributions to cancer inflammation are largely unexplored. IgG4 subclass antibodies are present in IL-10–driven Th2 immune responses in some inflammatory conditions. Since Th2-biased inflammation is a hallmark of tumor microenvironments, we investigated the presence and functional implications of IgG4 in malignant melanoma. Consistent with Th2 inflammation, CD22+ B cells and IgG4+-infiltrating cells accumulated in tumors, and IL-10, IL-4, and tumor-reactive IgG4 were expressed in situ. When compared with B cells from patient lymph nodes and blood, tumor-associated B cells were polarized to produce IgG4. Secreted B cells increased VEGF and IgG4, and tumor cells enhanced IL-10 secretion in cocultures. Unlike IgG1, an engineered tumor antigen-specific IgG4 was ineffective in triggering effector cell–mediated tumor killing in vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions. IgG4 blockade was mediated through reduction of FcγRI activation. Additionally, IgG4 significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. Furthermore, serum IgG4 was inversely correlated with patient survival. These findings suggest that IgG4 promoted by tumor-induced Th2-biased inflammation may restrict effector cell functions against tumors, providing a previously unexplored aspect of tumor-induced immune escape and a basis for biomarker development and patient-specific therapeutic approaches. PMID:23454746

  3. Mathematical modeling of electro-rotation spectra of small particles in liquid solutions: application to human erythrocyte aggregates.

    PubMed

    Zehe, A; Ramírez, A; Starostenko, O

    2004-02-01

    Electro-rotation can be used to determine the dielectric properties of cells, as well as to observe dynamic changes in both dielectric and morphological properties. Suspended biological cells and particles respond to alternating-field polarization by moving, deforming or rotating. While in linearly polarized alternating fields the particles are oriented along their axis of highest polarizability, in circularly polarized fields the axis of lowest polarizability aligns perpendicular to the plane of field rotation. Ellipsoidal models for cells are frequently applied, which include, beside sphere-shaped cells, also the limiting cases of rods and disks. Human erythrocyte cells, due to their particular shape, hardly resemble an ellipsoid. The additional effect of rouleaux formation with different numbers of aggregations suggests a model of circular cylinders of variable length. In the present study, the induced dipole moment of short cylinders was calculated and applied to rouleaux of human erythrocytes, which move freely in a suspending conductive medium under the effect of a rotating external field. Electro-rotation torque spectra are calculated for such aggregations of different length. Both the maximum rotation speeds and the peak frequencies of the torque are found to depend clearly on the size of the rouleaux. While the rotation speed grows with rouleaux length, the field frequency nu(p) is lowest for the largest cell aggregations where the torque shows a maximum. PMID:14762571

  4. Identification of immunodominant VP1 linear epitope of enterovirus 71 (EV71) using synthetic peptides for detecting human anti-EV71 IgG antibodies in Western blots.

    PubMed

    Foo, D G W; Ang, R X; Alonso, S; Chow, V T K; Quak, S H; Poh, C L

    2008-03-01

    A major IgG-specific immunodominant VP1 linear epitope of enterovirus 71 (EV71) strain 41 (5865/SIN/00009), defined by the core sequence LEGTTNPNG, was identified by Pepscan analysis. Oligonucleotides corresponding to the amino-acid sequence of synthetic peptide SP32 were cloned and over-expressed in Escherichia coli as a recombinant glutathione-S-transferase (GST)-SP32 fusion protein. In ELISAs, this protein did not react with human anti-EV71 IgG antibodies, but there was significant immunoreactivity according to western blot analysis. The amino-acid sequence of SP32 was highly specific for detecting EV71 strains in western blot analysis, and showed no immunoreactivity with monoclonal antibodies raised against other enteroviruses, e.g., CA9 and Echo 6. PMID:18076666

  5. Somatic mutations in the variable regions of a human IgG anti-double-stranded DNA autoantibody suggest a role for antigen in the induction of systemic lupus erythematosus.

    PubMed

    van Es, J H; Gmelig Meyling, F H; van de Akker, W R; Aanstoot, H; Derksen, R H; Logtenberg, T

    1991-02-01

    The processes that govern the generation of pathogenic anti-DNA autoantibodies in human systemic lupus erythematosus (SLE) are largely unknown. Autoantibodies may arise as a consequence of polyclonal B cell activation and/or antigen-driven B cell activation and selection. The role of these processes in humoral autoimmunity may be studied by molecular genetic analysis of immunoglobulin (Ig) variable (V) regions of antibodies that are characteristic of SLE. We have analyzed the gene elements that encode a high affinity, IgG anti-double-stranded DNA autoantibody secreted by a monoclonal Epstein-Barr virus (EBV)-transformed cell line derived from a patient with active SLE. In addition, we have identified, cloned, and sequenced the germline counterparts of the VH and VL genes expressed in this autoantibody. The comparison of both sets of gene elements shows that the autoantibody VH and VL regions harbor numerous somatic mutations characteristic of an antigen-driven immune response. The light chain expressed in this autoantibody is a somatically mutated variant of the kv325 germline gene that is frequently associated with paraproteins having autoantibody activity and with Ig molecules produced by malignant B cells that express the CD5 antigen. Furthermore, the utilized DH segment has been repeatedly found in multireactive, low affinity IgM anti-DNA autoantibodies from SLE patients and healthy individuals. These results suggest that pathogenic IgG anti-DNA autoantibodies in human SLE may arise through antigen-driven selection of somatic mutations in the gene elements that frequently encode multireactive IgM autoantibodies. PMID:1899104

  6. The role of copper(II) in the aggregation of human amylin.

    PubMed

    Sinopoli, Alessandro; Magrì, Antonio; Milardi, Danilo; Pappalardo, Matteo; Pucci, Pietro; Flagiello, Angela; Titman, Jeremy J; Nicoletti, Vincenzo Giuseppe; Caruso, Giuseppe; Pappalardo, Giuseppe; Grasso, Giuseppe

    2014-10-01

    Amylin is a 37-residue peptide hormone produced by the islet β-cells of pancreas and the formation of amylin aggregates is strongly associated with β-cell degeneration in type 2 diabetes, as demonstrated by more than 95% of patients exhibiting amylin amyloid upon autopsy. It is widely recognized that metal ions such as copper(II) have been implicated in the aggregation process of amyloidogenic peptides such as Aβ and α-synuclein and there is evidence that amylin self-assembly is also largely affected by copper(II). For this reason, in this work, the role of copper(II) in the aggregation of amylin has been investigated by several different experimental approaches. Mass spectrometric investigations show that copper(II) induces significant changes in the amylin structure, which decrease the protein fibrillogenesis as observed by ThT measurements. Accordingly, solid-state NMR experiments together with computational analysis carried out on a model amylin fragment confirmed the non-fibrillogenic nature of the copper(II) induced aggregated structure. Finally, the presence of copper(II) is also shown to have a major influence on amylin proneness to be degraded by proteases and cytotoxicity studies on different cell cultures are reported. PMID:25080969

  7. Decrease in erythrocyte glycophorin sialic acid content is associated with increased erythrocyte aggregation in human diabetes.

    PubMed

    Rogers, M E; Williams, D T; Niththyananthan, R; Rampling, M W; Heslop, K E; Johnston, D G

    1992-03-01

    1. Sialic acid moieties of erythrocyte membrane glycoproteins are the principal determinants of the negative charge on the cell surface. The resultant electrostatic repulsion between the cells reduces erythrocyte aggregation and hence the low shear rate viscosity and yield stress of blood. 2. Using g.c.-m.s., a decrease in sialic acid content has been observed in the major erythrocyte membrane glycoprotein, glycophorin A, obtained from nine diabetic patients compared with that from seven normal control subjects [median (range): 3.30 (0.01-11.90) versus 18.60 (3.20-32.60) micrograms/100 micrograms of protein, P less than 0.02]. 3. Erythrocyte aggregation, measured by viscometry as the ratio of suspension viscosity to supernatant viscosity (LS/S) in fibrinogen solution, was increased in ten diabetic patients compared with ten normal control subjects (mean +/- SEM, 37.6 +/- 1.3 versus 33.8 +/- 0.6, P less than 0.02). 4. In the patients in whom both viscometry and carbohydrate analysis were performed, the decrease in erythrocyte glycophorin sialylation and the increase in erythrocyte aggregation in fibrinogen solution were related statistically (LS/S correlated negatively with glycophorin sialic acid content, r = 0.73, P less than 0.05). 5. Decreased glycophorin sialylation provides an explanation at the molecular level for increased erythrocyte aggregation and it may be important in the pathogenesis of vascular disease in diabetes. PMID:1312416

  8. Enzymatically degradable poly(ethylene glycol) hydrogels for the 3D culture and release of human embryonic stem cell derived pancreatic precursor cell aggregates

    PubMed Central

    Amer, Luke D.; Holtzinger, Audrey; Keller, Gordon; Mahoney, Melissa J.; Bryant, Stephanie J.

    2015-01-01

    This study aimed to develop a three dimensional culture platform for aggregates of human embryonic stem cell (hESC)-derived pancreatic progenitors that enables long-term culture, maintains aggregate size and morphology, does not adversely affect differentiation and provides a means for aggregate recovery. A platform was developed with poly(ethylene glycol) hydrogels containing collagen type I, for cell-matrix interactions, and peptide crosslinkers, for facile recovery of aggregates. The platform was first demonstrated with RIN-m5F cells, showing encapsulation and subsequent release of single cells and aggregates without adversely affecting viability. Aggregates of hESC-derived pancreatic progenitors with an effective diameter of 82 (15) μm were either encapsulated in hydrogels or cultured in suspension for 28 days. At day 14, aggregate viability was maintained in the hydrogels, but significantly reduced (88%) in suspension culture. However by day 28, viability was reduced in both culture conditions. Aggregate size was maintained in the hydrogels, but in suspension was significantly higher (~2-fold) by day 28. The ability to release aggregates followed by a second enzyme treatment to achieve single cells enabled assessment by flow cytometry. Prior to encapsulation, there were 39% Pdx1+/Nkx6.1+ cells, key endocrine markers required for β-cell maturation. The fraction of doubly positive cells was not affected in hydrogels but was slightly and significantly lower in suspension culture by 28 days. In conclusion, we demonstrate that a MMP-sensitive PEG hydrogel containing collagen type I is a promising platform for hESC-derived pancreatic progenitors that maintains viable aggregates, aggregate size, and progenitor state and offers facile recovery of aggregates. PMID:25913222

  9. [Molecular heterogeneity of proteoglycan aggregates of human hyalin cartilage in normal conditions and in systemic bone dysplasia].

    PubMed

    Feshchenko, S P; Krasnopol'skaia, K D; Rebrin, I A; Rudakov, S S

    1989-01-01

    Components of proteoglycan aggregates of human hyalin cartilage were studied under conditions of normal state and in some forms of osteochondrodysplasia. Extraction of uronic acids and protein from the tissue, amount of fractions and electrophoretic mobility of proteoglycan monomers, rations protein/glycosaminoglycans, keratan sulfate/chondroitin sulfate, a level and type of sulfatation as well as molecular mass of chondroitin sulfate, amino acid composition of rod protein, heterogeneity of binding proteins (concerning their isoelectric points and molecular masses) and immunoreactivity of protein moiety in proteoglycan aggregates were studied in rib cartilage, knee joint and ala ossis ilii. Structural parameters of proteoglycan aggregates proved to be dissimilar and depended on cartilage localization and age of the donors. Impairments in the rate of chondroitin sulfate sulfatation were detected in achondrogenesis of the II type and in diastrophic dysplasia; an extraction ability and amount of proteoglycan fractions, relative content of glycosaminoglycans and binding proteins were altered in some other forms of osteochondrodysplasias. Numerous biochemical markers of extracellular matrix deterioration were detected, which are typical for various morphofunctional alterations in hyalin cartilage--hyperproliferative reactions, tissue prematuration, persistence of the embryonal type of metabolism. PMID:2472707

  10. Structure-activity relationships of alphaIIb 313-320 derived peptide inhibitors of human platelet aggregation.

    PubMed

    Stanica, Ruxandra Maria; Benaki, Dimitra; Rodis, Foteini I; Mikros, Emmanuel; Tsoukatos, Dimokritos; Tselepis, Alexandros; Tsikaris, Vasilios

    2008-11-01

    The alphaIIbbeta3 receptor, which is the most abundant receptor on the surface of platelets, can interact with a variety of adhesive proteins including fibrinogen, fibronectin and the von Willebrand factor. Fibrinogen binding on alphaIIbbeta3 is an event essential for platelet aggregation and thrombus formation. Mapping of the fibrinogen-binding domains on alphaIIb subunit suggested the sequence 313-332 as a possible binding site. This region was restricted to sequence alphaIIb 313-320 (Y313MESRADR320) using synthetic octapeptides overlapping by six residues. The YMESRADR octapeptide inhibits ADP-stimulated human platelets aggregation and binds to immobilized fibrinogen. In this study, we used the Ala scanning methodology within the sequence 313-320 aiming to evaluate the contribution of each amino acid in inhibiting platelet aggregation. It was found that the substitution of Y313, M314, E315 or S316 by A does not affect the activity of the parent octapeptide. The-RADR-motif seems to be the most essential for the biological activity of the alphaIIb 313-320 site. The conformational analysis of the YAESRADR, YMESAADR and YMESRAAR analogs by using NMR spectroscopy and distance geometry calculations revealed significant differences in their conformational states in DMSO-d6. PMID:18646252

  11. Water-soluble jack-knife prawn extract inhibits 5-hydroxytryptamine-induced vasoconstriction and platelet aggregation in humans.

    PubMed

    Gamoh, Shuji; Kanai, Tasuku; Tanaka-Totoribe, Naoko; Ohkura, Masamichi; Kuwabara, Masachika; Nakamura, Eisaku; Yokota, Atsuko; Yamasaki, Tetsuo; Watanabe, Akiko; Hayashi, Masahiro; Fujimoto, Shouichi; Yamamoto, Ryuichi

    2015-02-01

    Coronary artery spasm plays an important role in the pathogenesis of various ischemic heart diseases or serious arrhythmia. The aim of this study is to look for functional foods which have physiologically active substances preventing 5-hydroxytryptamine (5-HT)-related vasospastic diseases including peri- and postoperative ischemic complications of coronary artery bypass grafting (CABG) from ocean resources in Japanese coastal waters. First, we evaluated the effect of water-soluble ocean resource extracts on the response to 5-HT in HEK293 cells which have forcibly expressed cyan fluorescent protein-fused 5-HT2A receptors (5-HT2A-CFP). Among 5 different water-soluble extracts of ocean resources, the crude water-soluble jack-knife prawn extract (WJPE) significantly reduced maximal Ca(2+) influx induced by 0.1 μM 5-HT in a concentration-dependent manner. The Crude WJPE significantly inhibited, in a concentration-dependent manner, 5-HT-induced constriction of human saphenous vein. 5-HT released from activated platelets plays a crucial roles in the constriction of coronary artery. Next the WJPE was purified for applying the experiment of 5-HT-induced human platelet aggregation. The purified WJPE significantly inhibited 5-HT-induced human platelet aggregation also in a concentration-dependent manner. Based on our findings, jack-knife prawn could be one of a functional food with health-promoting benefits for most people with vasospastic diseases including patients who have gone CABG. PMID:25464143

  12. Oncocalyxone A inhibits human platelet aggregation by increasing cGMP and by binding to GP Ibα glycoprotein

    PubMed Central

    Ferreira, M A D; do Nascimento, N R F; de Sousa, C M; Pessoa, O D L; de Lemos, T L G; Ventura, J S; Schattner, M; Chudzinski-Tavassi, A M

    2008-01-01

    Background and purpose: Oncocalyxone A (OncoA) has a concentration-dependent anti-platelet activity. The present study aimed to further understand the mechanisms related to this effect. Experimental approach: Human platelet aggregation was measured by means of a turbidimetric method. OncoA (32–256 μM) was tested against several platelet-aggregating agents, such as adenosine diphosphate (ADP), collagen, arachidonic acid (AA), ristocetin and thrombin. Key results: OncoA completely inhibited platelet aggregation with a calculated mean inhibitory concentration (IC50-μM) of 122 for ADP, 161 for collagen, 159 for AA, 169 for ristocetin and 85 for thrombin. The anti-aggregatory activity of OncoA was not inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). OncoA, at a concentration that caused no significant anti-aggregatory activity, potentiated sodium nitroprusside (SNP) anti-aggregatory activity (18.8±2.9%-SNP vs 85.0±8.2%-SNP+OncoA). The levels of nitric oxide (NO) or cAMP were not altered by OncoA while cGMP levels were increased more than 10-fold by OncoA in resting or ADP-activated platelets. Flow cytometry revealed that OncoA does not interact with receptors for fibrinogen, collagen or P-selectin. Nevertheless, OncoA decreased the binding of antibodies to GP Ibα, a glycoprotein that is related both to von Willebrand factor and to thrombin-induced platelet aggregation. Conclusion and implications: OncoA showed anti-aggregatory activity in platelets that was associated with increased cGMP levels, not dependent on NO and with blocking GP Ibα glycoprotein. This new mechanism has the prospect of leading to new anti-thrombotic drugs. PMID:18516074

  13. Proteolytic components of serum IgG preparations

    PubMed Central

    Li, L; Kalaga, R; Paul, S

    2000-01-01

    Chemical catalysis, an effector mechanism utilized by fully assembled antibodies, can also be mediated by the isolated antibody subunits. Because trace amounts of free light chains (L chains) are present in IgG preparations, a detailed study was undertaken to identify the constituents responsible for the polyreactive proteolytic activity of IgG purified from human sera, determined as the extent of cleavage of the model peptide substrate Pro-Phe-Arg-methylcoumarinamide. Two proteolytic species with approximate mass of 50 kD and 150 kD were separated by repetitive gel filtration in a denaturing solvent (6 m guanidine hydrochloride). The activity of the renatured 50-kD fraction (in fluorescence units/μg protein) was more than 45-fold greater than of the 150-kD fraction. Both fractions lost the activity following immunoadsorption on immobilized anti-IgG antibody. Fab fragments prepared from the 150-kD IgG fraction retained the activity. Reducing and non-reducing SDS-electrophoresis suggested the 50-kD fraction isolated from the IgG preparations to be a mixture of heavy chain (H chain) monomers and disulphide bonded L chain dimers. Electrophoretically homogeneous monomers of 50-kD H chains and 25-kD L chains were prepared by gel filtration of reduced and alkylated IgG from seven human subjects. Each of the alkylated L chain preparations displayed the proteolytic activity. The activity in alkylated H chains was undetectable or only marginally greater than the background values. L chain dimers appear to be the major species responsible for the polyreactive proteolytic activity of serum IgG preparations, with a smaller contribution furnished by tetrameric IgG. PMID:10792374

  14. Biochemical characterization and three-dimensional structures of the Fab and Fc portions of a human IgG1 antibody

    NASA Astrophysics Data System (ADS)

    Bourne, Christina R.

    2003-07-01

    A monoclonal antibody was isolated from the serum of a patient (Nav) with multiple myeloma for investigation of mechanisms of antibody-mediated damage. It was believed that the over-expressed molecule possessed intrinsic properties contributing unfavorably to disease progression. The monoclonal component was present in large quantities prior to treatment, thus creating a window for uncontrolled and potentially damaging events. Amino acid sequences were determined for the Fab VH and V L domains. This information suggested probable positive selective pressure from antigen. Crystals were obtained in the P21 space group, and X-ray data were collected to 2.5A (99.4% complete). This structure was refined to an Rwork of 22.5% and an Rfree of 28.0%. Unusual structural properties were located in the antigen-binding site. The LCDR3 loop contained a double proline sequence that directed its extrusion into the bulk solvent and partitioned the binding site, which has a groove-type structure. Ligands conforming to this site were identified with a library of phage-displayed peptides. Crystal packing interactions of the Nav Fab were partially mediated by the antigen-binding site, with prevalent involvement of the LCDR3 loop. The crystallographic asymmetric unit was an atypical trimer. Two molecules interacted by edge beta-strand pairing; the third was accommodated through steric complementarity. Edge beta-strand pairing has become a dominant crystal packing motif in antibodies. The frequency of occurrence of this motif revealed sequence preferences for kappa-type light chains. These interactions may play a role in lethal aggregation and amyloid fibrillogenesis. Similar aggregation behavior of the Nav kappa-type Bence-Jones protein probably resulted in impaired renal function. Crystals of the Fc portion suitable for X-ray analysis could be obtained only in gelled media. The space group was P212 121. X-ray data collected at room temperature to 2.5A were 76.0% complete; the final

  15. Adenosine diphosphate-induced aggregation of human platelets in flow through tubes. I. Measurement of concentration and size of single platelets and aggregates.

    PubMed Central

    Bell, D N; Spain, S; Goldsmith, H L

    1989-01-01

    A double infusion flow system and particle sizing technique were developed to study the effect of time and shear rate on adenosine diphosphate-induced platelet aggregation in Poiseuille flow. Citrated platelet-rich plasma, PRP, and 2 microM ADP were simultaneously infused into a 40-microliters cylindrical mixing chamber at a fixed flow ratio, PRP/ADP = 9:1. After rapid mixing by a rotating magnetic stirbar, the platelet suspension flowed through 1.19 or 0.76 mm i.d. polyethylene tubing for mean transit times, t, from 0.1 to 86 s, over a range of mean tube shear rate, G, from 41.9 to 1,000 s-1. Known volumes of suspension were collected into 0.5% buffered glutaraldehyde, and all particles in the volume range 1-10(5) microns 3 were counted and sized using a model ZM particle counter (Coulter Electronics Inc., Hialeah, FL) and a logarithmic amplifier. The decrease in the single platelet concentration served as an overall index of aggregation. The decrease in the total particle concentration was used to calculate the collision capture efficiency during the early stages of aggregation, and aggregate growth was followed by changes in the volume fraction of particles of successively increasing size. Preliminary results demonstrate that both collision efficiency and particle volume fraction reveal important aspects of the aggregation process not indicated by changes in the single platelet concentration alone. PMID:2605298

  16. Dimeric FcγR Ectodomains as Probes of the Fc Receptor Function of Anti-Influenza Virus IgG.

    PubMed

    Wines, Bruce D; Vanderven, Hillary A; Esparon, Sandra E; Kristensen, Anne B; Kent, Stephen J; Hogarth, P Mark

    2016-08-15

    Ab-dependent cellular cytotoxicity, phagocytosis, and Ag presentation are key mechanisms of action of Abs arising in vaccine or naturally acquired immunity, as well of therapeutic mAbs. Cells expressing the low-affinity FcγRs (FcγRII or CD32 and FcγRIII or CD16) are activated for these functions when receptors are aggregated following the binding of IgG-opsonized targets. Despite the diversity of the Fc receptor proteins, IgG ligands, and potential responding cell types, the induction of all FcγR-mediated responses by opsonized targets requires the presentation of multiple Fc regions in close proximity to each other. We demonstrated that such "near-neighbor" Fc regions can be detected using defined recombinant soluble (rs) dimeric low-affinity ectodomains (rsFcγR) that have an absolute binding requirement for the simultaneous engagement of two IgG Fc regions. Like cell surface-expressed FcγRs, the binding of dimeric rsFcγR ectodomains to Ab immune complexes was affected by Ab subclass, presentation, opsonization density, Fc fucosylation, or mutation. The activation of an NK cell line and primary NK cells by human IgG-opsonized influenza A hemagglutinin correlated with dimeric rsFcγRIIIa binding activity but not with Ab titer. Furthermore, the dimeric rsFcγR binding assay sensitively detected greater Fc receptor activity to pandemic H1N1 hemagglutinin after the swine influenza pandemic of 2009 in pooled human polyclonal IgG. Thus these dimeric rsFcγR ectodomains are validated, defined probes that should prove valuable in measuring the immune-activating capacity of IgG Abs elicited by infection or vaccination or experimentally derived IgG and its variants. PMID:27385782

  17. Different Factors Affecting Human ANP Amyloid Aggregation and Their Implications in Congestive Heart Failure

    PubMed Central

    Millucci, Lia; Paccagnini, Eugenio; Ghezzi, Lorenzo; Bernardini, Giulia; Braconi, Daniela; Laschi, Marcella; Consumi, Marco; Spreafico, Adriano; Tanganelli, Piero; Lupetti, Pietro; Magnani, Agnese; Santucci, Annalisa

    2011-01-01

    Aims Atrial Natriuretic Peptide (ANP)-containing amyloid is frequently found in the elderly heart. No data exist regarding ANP aggregation process and its link to pathologies. Our aims were: i) to experimentally prove the presumptive association of Congestive Heart Failure (CHF) and Isolated Atrial Amyloidosis (IAA); ii) to characterize ANP aggregation, thereby elucidating IAA implication in the CHF pathogenesis. Methods and Results A significant prevalence (85%) of IAA was immunohistochemically proven ex vivo in biopsies from CHF patients. We investigated in vitro (using Congo Red, Thioflavin T, SDS-PAGE, transmission electron microscopy, infrared spectroscopy) ANP fibrillogenesis, starting from α-ANP as well as the ability of dimeric β-ANP to promote amyloid formation. Different conditions were adopted, including those reproducing β-ANP prevalence in CHF. Our results defined the uncommon rapidity of α-ANP self-assembly at acidic pH supporting the hypothesis that such aggregates constitute the onset of a fibrillization process subsequently proceeding at physiological pH. Interestingly, CHF-like conditions induced the production of the most stable and time-resistant ANP fibrils suggesting that CHF affected people may be prone to develop IAA. Conclusions We established a link between IAA and CHF by ex vivo examination and assessed that β-ANP is, in vitro, the seed of ANP fibrils. Our results indicate that β-ANP plays a crucial role in ANP amyloid deposition under physiopathological CHF conditions. Overall, our findings indicate that early IAA-related ANP deposition may occur in CHF and suggest that these latter patients should be monitored for the development of cardiac amyloidosis. PMID:21814559

  18. LON is the master protease that protects against protein aggregation in human mitochondria through direct degradation of misfolded proteins

    PubMed Central

    Bezawork-Geleta, Ayenachew; Brodie, Erica J.; Dougan, David A.; Truscott, Kaye N.

    2015-01-01

    Maintenance of mitochondrial protein homeostasis is critical for proper cellular function. Under normal conditions resident molecular chaperones and proteases maintain protein homeostasis within the organelle. Under conditions of stress however, misfolded proteins accumulate leading to the activation of the mitochondrial unfolded protein response (UPRmt). While molecular chaperone assisted refolding of proteins in mammalian mitochondria has been well documented, the contribution of AAA+ proteases to the maintenance of protein homeostasis in this organelle remains unclear. To address this gap in knowledge we examined the contribution of human mitochondrial matrix proteases, LONM and CLPXP, to the turnover of OTC-∆, a folding incompetent mutant of ornithine transcarbamylase, known to activate UPRmt. Contrary to a model whereby CLPXP is believed to degrade misfolded proteins, we found that LONM, and not CLPXP is responsible for the turnover of OTC-∆ in human mitochondria. To analyse the conformational state of proteins that are recognised by LONM, we examined the turnover of unfolded and aggregated forms of malate dehydrogenase (MDH) and OTC. This analysis revealed that LONM specifically recognises and degrades unfolded, but not aggregated proteins. Since LONM is not upregulated by UPRmt, this pathway may preferentially act to promote chaperone mediated refolding of proteins. PMID:26627475

  19. Investigation of cyclooxygenase and signaling pathways involved in human platelet aggregation mediated by synergistic interaction of various agonists

    PubMed Central

    Khan, Nadia; Farooq, Ahsana Dar; Sadek, Bassem

    2015-01-01

    In the present study, the mechanism(s) of synergistic interaction of various platelet mediators such as arachidonic acid (AA) when combined with 5-hydroxytryptamine (5-HT) or adenosine diphosphate (ADP) on human platelet aggregation were examined. The results demonstrated that 5-HT had no or negligible effect on aggregation but it did potentiate the aggregation response of AA. Similarly, the combination of subeffective concentrations of ADP and AA exhibited noticeable rise in platelet aggregation. Moreover, the observed synergistic effect of AA with 5-HT on platelets was inhibited by different cyclooxygenase (COX) inhibitors, namely ibuprofen and celecoxib, with half maximal inhibitory effect (IC50) values of 18.0±1.8 and 15.6±3.4 μmol/L, respectively. Interestingly, the synergistic effect observed for AA with 5-HT was, also, blocked by the 5-HT receptor blockers cyproheptadine (IC50=22.0±7 μmol/L), ketanserin (IC50=152±23 μmol/L), phospholipase C (PLC) inhibitor (U73122; IC50=6.1±0.8 μmol/L), and mitogen activated protein kinase (MAPK) inhibitor (PD98059; IC50=3.8±0.5 μmol/L). Likewise, the synergism of AA and ADP was, also, attenuated by COX inhibitors (ibuprofen; IC50=20±4 μmol/L and celecoxib; IC50=24±7 μmol/L), PLC inhibitor (U73122; IC50=3.7±0.3 μmol/L), and MAPK inhibitor (PD98059; IC50=2.8±1.1 μmol/L). Our observed data demonstrate that the combination of subthreshold concentrations of agonists amplifies platelet aggregation and that these synergistic effects largely depend on activation of COX/thromboxane A2, receptor-operated Ca2+ channels, Gq/PLC, and MAPK signaling pathways. Moreover, our data revealed that inhibition of COX pathways by using both selective and/or non-selective COX inhibitors blocks not only AA metabolism and thromboxane A2 formation, but also its binding to Gq receptors and activation of receptor-operated Ca2+ channels in platelets. Overall, our results show that PLC and MAPK inhibitors proved to inhibit the

  20. Investigation of cyclooxygenase and signaling pathways involved in human platelet aggregation mediated by synergistic interaction of various agonists.

    PubMed

    Khan, Nadia; Farooq, Ahsana Dar; Sadek, Bassem

    2015-01-01

    In the present study, the mechanism(s) of synergistic interaction of various platelet mediators such as arachidonic acid (AA) when combined with 5-hydroxytryptamine (5-HT) or adenosine diphosphate (ADP) on human platelet aggregation were examined. The results demonstrated that 5-HT had no or negligible effect on aggregation but it did potentiate the aggregation response of AA. Similarly, the combination of subeffective concentrations of ADP and AA exhibited noticeable rise in platelet aggregation. Moreover, the observed synergistic effect of AA with 5-HT on platelets was inhibited by different cyclooxygenase (COX) inhibitors, namely ibuprofen and celecoxib, with half maximal inhibitory effect (IC50) values of 18.0 ± 1.8 and 15.6 ± 3.4 μmol/L, respectively. Interestingly, the synergistic effect observed for AA with 5-HT was, also, blocked by the 5-HT receptor blockers cyproheptadine (IC50=22.0 ± 7 μmol/L), ketanserin (IC50=152 ± 23 μmol/L), phospholipase C (PLC) inhibitor (U73122; IC50=6.1 ± 0.8 μmol/L), and mitogen activated protein kinase (MAPK) inhibitor (PD98059; IC50=3.8 ± 0.5 μmol/L). Likewise, the synergism of AA and ADP was, also, attenuated by COX inhibitors (ibuprofen; IC50=20 ± 4 μmol/L and celecoxib; IC50=24 ± 7 μmol/L), PLC inhibitor (U73122; IC50=3.7 ± 0.3 μmol/L), and MAPK inhibitor (PD98059; IC50=2.8 ± 1.1 μmol/L). Our observed data demonstrate that the combination of subthreshold concentrations of agonists amplifies platelet aggregation and that these synergistic effects largely depend on activation of COX/thromboxane A2, receptor-operated Ca(2+) channels, Gq/PLC, and MAPK signaling pathways. Moreover, our data revealed that inhibition of COX pathways by using both selective and/or non-selective COX inhibitors blocks not only AA metabolism and thromboxane A2 formation, but also its binding to Gq receptors and activation of receptor-operated Ca(2+) channels in platelets. Overall, our results show that PLC and MAPK inhibitors proved

  1. Mild exposure of RIN-5F β-cells to human islet amyloid polypeptide aggregates upregulates antioxidant enzymes via NADPH oxidase-RAGE: An hormetic stimulus☆

    PubMed Central

    Borchi, Elisabetta; Bargelli, Valentina; Guidotti, Valentina; Berti, Andrea; Stefani, Massimo; Nediani, Chiara; Rigacci, Stefania

    2013-01-01

    The presence of amyloid aggregates of human islet amyloid polypeptide (hIAPP), a hallmark of type 2 diabetes, contributes to pancreatic β-cell impairment, where oxidative stress plays a key role. A contribution of NADPH oxidase to reactive oxygen species (ROS) generation after cell exposure to micromolar concentrations of hIAPP aggregates has been suggested. However, little is known about β-cells exposure to lower amounts of hIAPP aggregates, similar to those found in human pancreas. Thus, we aimed to investigate the events resulting from RIN-5F cells exposure to nanomolar concentrations of toxic hIAPP aggregates. We found an early and transient rise of NADPH oxidase activity resulting from increased Nox1 expression following the engagement of receptor for advanced glycation end-products (RAGE) by hIAPP aggregates. Unexpectedly, NADPH oxidase activation was not accompanied by a significant ROS increase and the lipoperoxidation level was significantly reduced. Indeed, cell exposure to hIAPP aggregates affected the antioxidant defences, inducing a significant increase of the expression and activity of catalase and glutathione peroxidase. We conclude that exposure of pancreatic β-cells to nanomolar concentrations of hIAPP aggregates for a short time induces an hormetic response via the RAGE-Nox1 axis; the latter stimulates the enzymatic antioxidant defences that preserve the cells against oxidative stress damage. PMID:24416718

  2. A RECOMBINANT IgG Fc THAT RECAPITULATES THE ANTI-INFLAMMATORY ACTIVITY OF IVIG

    PubMed Central

    Anthony, Robert M.; Nimmerjahn, Falk; Ashline, David J.; Reinhold, Vernon N.; Paulson, James C.; Ravetch, Jeffrey V.

    2008-01-01

    High doses of monomeric IgG purified from pooled human plasma confer anti-inflammatory activity for a wide variety of autoimmune diseases. The heterogeneity of IVIG, derived from its Fab specificity, IgG subclass distribution and variable glycosylation have confounded efforts to develop a recombinant substitute for this blood-derived product. Recent studies have demonstrated that this paradoxical anti-inflammatory activity of IgG is completely dependent on sialylation of the N-linked glycan of the IgG Fc fragment. Determining the precise glycan requirements for this anti-inflammatory activity allowed appropriate glycan engineering of an IgG1 Fc fragment, leading to the generation of a fully recombinant, sialylated IgG1 Fc with greatly enhanced potency. PMID:18420934

  3. An amphioxus gC1q protein binds human IgG and initiates the classical pathway: Implications for a C1q-mediated complement system in the basal chordate.

    PubMed

    Gao, Zhan; Li, Mengyang; Ma, Jie; Zhang, Shicui

    2014-12-01

    The origin of the classical complement pathway remains open during chordate evolution. A C1q-like member, BjC1q, was identified in the basal chordate amphioxus. It is predominantly expressed in the hepatic caecum, hindgut, and notochord, and is significantly upregulated following challenge with bacteria or lipoteichoic acid and LPS. Recombinant BjC1q and its globular head domain specifically interact with lipoteichoic acid and LPS, but BjC1q displays little lectin activity. Moreover, rBjC1q can assemble to form the high molecular weight oligomers necessary for binding to proteases C1r/C1s and for complement activation, and binds human C1r/C1s/mannan-binding lectin-associated serine protease-2 as well as amphioxus serine proteases involved in the cleavage of C4/C2, and C3 activation. Importantly, rBjC1q binds with human IgG as well as an amphioxus Ig domain containing protein, resulting in the activation of the classical complement pathway. This is the first report showing that a C1q-like protein in invertebrates is able to initiate classical pathway, raising the possibility that amphioxus possesses a C1q-mediated complement system. It also suggests a new scenario for the emergence of the classical complement pathway, in contrast to the proposal that the lectin pathway evolved into the classical pathway. PMID:25174509

  4. A Vaccine of L2 Epitope Repeats Fused with a Modified IgG1 Fc Induced Cross-Neutralizing Antibodies and Protective Immunity against Divergent Human Papillomavirus Types

    PubMed Central

    Zhang, Ting; Liu, Yanchun; Xie, Xixiu; Wang, Zhirong; Xu, Xuemei

    2014-01-01

    Current human papillomavirus (HPV) major capsid protein L1 virus-like particles (VLPs)-based vaccines in clinic induce strong HPV type-specific neutralizing antibody responses. To develop pan-HPV vaccines, here, we show that the fusion protein E3R4 consisting of three repeats of HPV16 L2 aa 17–36 epitope (E3) and a modified human IgG1 Fc scaffold (R4) induces cross-neutralizing antibodies and protective immunity against divergent HPV types. E3R4 was expressed as a secreted protein in baculovirus expression system and could be simply purified by one step Protein A affinity chromatography with the purity above 90%. Vaccination of E3R4 formulated with Freunds adjuvant not only induced cross-neutralizing antibodies against HPV pseudovirus types 16, 18, 45, 52, 58, 6, 11 and 5 in mice, but also protected mice against vaginal challenges with HPV pseudovirus types 16, 45, 52, 58, 11 and 5 for at least eleven months after the first immunization. Moreover, vaccination of E3R4 formulated with FDA approved adjuvant alum plus monophosphoryl lipid A also induced cross-neutralizing antibodies against HPV types 16, 18 and 6 in rabbits. Thus, our results demonstrate that delivery of L2 antigen as a modified Fc-fusion protein may facilitate pan-HPV vaccine development. PMID:24802101

  5. Expression, purification and characterization of a human single-chain Fv antibody fragment fused with the Fc of an IgG1 targeting a rabies antigen in Pichia pastoris.

    PubMed

    Wang, Ding-ding; Su, Man-man; Sun, Yan; Huang, Shu-lin; Wang, Ju; Yan, Wei-qun

    2012-11-01

    Because the demand for rabies post exposure prophylaxis (PEP) treatment has increased exponentially in recent years, the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide an adequate amount of the required passive immune component in PEP in countries where canine rabies is endemic. The replacement of HRIG and ERIG with a potentially cheaper and efficacious alternative biological for the treatment of rabies in humans, therefore, remains a high priority. In this study, we set out to assess a human single-chain Fv antibody fragment fused with the Fc of an IgG1 targeting a rabies antigen to develop a product that can be used as a component of the PEP cocktail. We cloned the ScFv fragment from a human ScFv library that was established previously and inserted this fragment into the expression vector pPICZαC/Fc. An active recombinant ScFv-Fc fusion protein was successfully expressed in Pichia pastoris. The production of ScFv-Fc was optimized and scaled up in an 80L fermentor with yields exceeding 60mg/L. The ScFv-Fc protein was purified to more than 95% purity using a two-step scheme: ammonium sulfate fractionation and Protein A Sepharose CL-4B. The ScFv-Fc fusion protein neutralized rabies virus in a standard in vivo neutralization assay in which the virus was incubated with the ScFv-Fc molecules before intracranial inoculation in mice. Our results suggest that functional antibodies can be produced in P. pastoris and that ScFv-Fc fusion proteins have the potential to serve as therapeutic candidates. PMID:22982755

  6. Protease inhibitors decrease IgG shedding from Staphylococcus aureus, increasing complement activation and phagocytosis efficiency.

    PubMed

    Fernandez Falcon, Maria F; Echague, Charlene G; Hair, Pamela S; Nyalwidhe, Julius O; Cunnion, Kenji M

    2011-10-01

    Staphylococcus aureus is a major pathogen for immunologically intact humans and its pathogenesis is a model system for evasion of host defences. Antibodies and complement are essential elements of the humoral immune system for prevention and control of S. aureus infections. The specific hypothesis for the proposed research is that S. aureus modifies humoral host defences by cleaving IgG that has bound to the bacterial surface, thereby inhibiting opsonophagocytosis. S. aureus was coated with pooled, purified human IgG and assayed for the shedding of cleaved IgG fragments using ELISA and Western blot analysis. Surface-bound IgG was shed efficiently from S. aureus in the absence of host blood proteins. Broad-spectrum protease inhibitors prevented cleavage of IgG from the S. aureus surface, suggesting that staphylococcal proteases are responsible for IgG cleavage. Serine protease inhibitors and cysteine protease inhibitors decreased the cleavage of surface-bound IgG; however, a metalloprotease inhibitor had no effect. Using protease inhibitors to prevent the cleavage of surface-bound IgG increased the binding of complement C3 fragments on the surface of S. aureus, increased the association with human neutrophils and increased phagocytosis by human neutrophils. PMID:21636671

  7. Inhibition of LFA-1-dependent human B-cell aggregation induced by CD40 antibodies and interleukin-4 leads to decreased IgE synthesis.

    PubMed Central

    Björck, P; Paulie, S

    1993-01-01

    Antibodies to CD40 have been shown to induce homotypic aggregation of human resting B cells and B-cell lines via an LFA-1-dependent mechanism. We show here that interleukin-4 (IL-4) is a strong potentiator of this process and stimulation of tonsillar B cells for 4 days with IL-4 and CD40 antibodies resulted in the formation of large, dense aggregates. Also in this case, aggregation appeared to be chiefly dependent on the activation of LFA-1, although the small clusters of cells remaining after blocking with LFA-1 antibodies suggest the involvement of another adhesion system(s). When testing the relationship between aggregation and IgE synthesis, a known consequences of IL-4/CD40 stimulation, IgE levels were found to be significantly decreased in the presence of LFA-1 antibodies. In contrast to these observations, proliferation occurring in response to the IL-4/CD40 stimulation was not inhibitable by LFA-1 antibodies. Rather, in most cases, this was slightly enhanced, suggesting that aggregation may have a limiting effect on cell growth. Isolated aggregates, each of which could comprise more than 10(5) cells, were also examined by electron microscopy. This revealed a tissue-like structure of the aggregates with large contact areas and with minimal intercellular space between the adjacent cells. As the apparent inhibitory effect of aggregation on proliferation may reflect a negative autocrine signalling, which is enhanced by the close cell contact, we also tested the effect of neutralizing antibodies to IL-6, one of the factors known to be produced in the system. Such treatment did not affect aggregation but in most experiments enhanced proliferation. The results suggest that a possible effect of aggregation may be to enhance differentiation of cells and that this may also be associated with the difficulties in growing B cells in vitro. Images Figure 1 Figure 3 PMID:7682536

  8. IgG Suppresses Antibody Responses in Mice Lacking C1q, C3, Complement Receptors 1 and 2, or IgG Fc-Receptors

    PubMed Central

    Bergström, Joakim J. E.; Heyman, Birgitta

    2015-01-01

    Antigen-specific IgG antibodies, passively administered to mice or humans together with large particulate antigens like erythrocytes, can completely suppress the antibody response against the antigen. This is used clinically in Rhesus prophylaxis, where administration of IgG anti-RhD prevents RhD-negative women from becoming immunized against RhD-positive fetal erythrocytes aquired transplacentally. The mechanisms by which IgG suppresses antibody responses are poorly understood. We have here addressed whether complement or Fc-receptors for IgG (FcγRs) are required for IgG-mediated suppression. IgG, specific for sheep red blood cells (SRBC), was administered to mice together with SRBC and the antibody responses analyzed. IgG was able to suppress early IgM- as well as longterm IgG-responses in wildtype mice equally well as in mice lacking FcγRIIB (FcγRIIB knockout mice) or FcγRI, III, and IV (FcRγ knockout mice). Moreover, IgG was able to suppress early IgM responses equally well in mice lacking C1q (C1qA knockout mice), C3 (C3 knockout mice), or complement receptors 1 and 2 (Cr2 knockout mice) as in wildtype mice. Owing to the previously described severely impaired IgG responses in the complement deficient mice, it was difficult to assess whether passively administered IgG further decreased their IgG response. In conclusion, Fc-receptor binding or complement-activation by IgG does not seem to be required for its ability to suppress antibody responses to xenogeneic erythrocytes. PMID:26619292

  9. Islet-Like Cell Aggregates Generated from Human Adipose Tissue Derived Stem Cells Ameliorate Experimental Diabetes in Mice

    PubMed Central

    Chandra, Vikash; G, Swetha; Muthyala, Sudhakar; Jaiswal, Amit K.; Bellare, Jayesh R.; Nair, Prabha D.; Bhonde, Ramesh R.

    2011-01-01

    Background Type 1 Diabetes Mellitus is caused by auto immune destruction of insulin producing beta cells in the pancreas. Currently available treatments include transplantation of isolated islets from donor pancreas to the patient. However, this method is limited by inadequate means of immuno-suppression to prevent islet rejection and importantly, limited supply of islets for transplantation. Autologous adult stem cells are now considered for cell replacement therapy in diabetes as it has the potential to generate neo-islets which are genetically part of the treated individual. Adopting methods of islet encapsulation in immuno-isolatory devices would eliminate the need for immuno-suppressants. Methodology/Principal Findings In the present study we explore the potential of human adipose tissue derived adult stem cells (h-ASCs) to differentiate into functional islet like cell aggregates (ICAs). Our stage specific differentiation protocol permit the conversion of mesodermic h-ASCs to definitive endoderm (Hnf3β, TCF2 and Sox17) and to PDX1, Ngn3, NeuroD, Pax4 positive pancreatic endoderm which further matures in vitro to secrete insulin. These ICAs are shown to produce human C-peptide in a glucose dependent manner exhibiting in-vitro functionality. Transplantation of mature ICAs, packed in immuno-isolatory biocompatible capsules to STZ induced diabetic mice restored near normoglycemia within 3–4 weeks. The detection of human C-peptide, 1155±165 pM in blood serum of experimental mice demonstrate the efficacy of our differentiation approach. Conclusions h-ASC is an ideal population of personal stem cells for cell replacement therapy, given that they are abundant, easily available and autologous in origin. Our findings present evidence that h-ASCs could be induced to differentiate into physiologically competent functional islet like cell aggregates, which may provide as a source of alternative islets for cell replacement therapy in type 1 diabetes. PMID:21687731

  10. α-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells.

    PubMed

    Hansen, Christian; Angot, Elodie; Bergström, Ann-Louise; Steiner, Jennifer A; Pieri, Laura; Paul, Gesine; Outeiro, Tiago F; Melki, Ronald; Kallunki, Pekka; Fog, Karina; Li, Jia-Yi; Brundin, Patrik

    2011-02-01

    Post-mortem analyses of brains from patients with Parkinson disease who received fetal mesencephalic transplants show that α-synuclein-containing (α-syn-containing) Lewy bodies gradually appear in grafted neurons. Here, we explored whether intercellular transfer of α-syn from host to graft, followed by seeding of α-syn aggregation in recipient neurons, can contribute to this phenomenon. We assessed α-syn cell-to-cell transfer using microscopy, flow cytometry, and high-content screening in several coculture model systems. Coculturing cells engineered to express either GFP- or DsRed-tagged α-syn resulted in a gradual increase in double-labeled cells. Importantly, α-syn-GFP derived from 1 neuroblastoma cell line localized to red fluorescent aggregates in other cells expressing DsRed-α-syn, suggesting a seeding effect of transmitted α-syn. Extracellular α-syn was taken up by cells through endocytosis and interacted with intracellular α-syn. Next, following intracortical injection of recombinant α-syn in rats, we found neuronal uptake was attenuated by coinjection of an endocytosis inhibitor. Finally, we demonstrated in vivo transfer of α-syn between host cells and grafted dopaminergic neurons in mice overexpressing human α-syn. In summary, intercellularly transferred α-syn interacts with cytoplasmic α-syn and can propagate α-syn pathology. These results suggest that α-syn propagation is a key element in the progression of Parkinson disease pathology. PMID:21245577

  11. The protonation state of histidine 111 regulates the aggregation of the evolutionary most conserved region of the human prion protein.

    PubMed

    Fonseca-Ornelas, Luis; Zweckstetter, Markus

    2016-08-01

    In a group of neurodegenerative diseases, collectively termed transmissible spongiform encephalopathies, the prion protein aggregates into β-sheet rich amyloid-like deposits. Because amyloid structure has been connected to different prion strains and cellular toxicity, it is important to obtain insight into the structural properties of prion fibrils. Using a combination of solution NMR spectroscopy, thioflavin-T fluorescence and electron microscopy we here show that within amyloid fibrils of a peptide containing residues 108-143 of the human prion protein [humPrP (108-143)]-the evolutionary most conserved part of the prion protein - residue H111 and S135 are in close spatial proximity and their interaction is critical for fibrillization. We further show that residues H111 and H140 share the same microenvironment in the unfolded, monomeric state of the peptide, but not in the fibrillar form. While protonation of H140 has little influence on fibrillization of humPrP (108-143), a positive charge at position 111 blocks the conformational change, which is necessary for amyloid formation of humPrP (108-143). Our study thus highlights the importance of protonation of histidine residues for protein aggregation and suggests point mutations to probe the structure of infectious prion particles. PMID:27184108

  12. The Effects of Lipid Membranes, Crowding and Osmolytes on the Aggregation, and Fibrillation Propensity of Human IAPP

    PubMed Central

    Gao, Mimi; Winter, Roland

    2015-01-01

    Type 2 diabetes mellitus (T2DM) is an age-related and metabolic disease. Its development is hallmarked, among others, by the dysfunction and degeneration of β-cells of the pancreatic islets of Langerhans. The major pathological characteristic thereby is the formation of extracellular amyloid deposits consisting of the islet amyloid polypeptide (IAPP). The process of human IAPP (hIAPP) self-association, and the intermediate structures formed as well as the interaction of hIAPP with membrane systems seem to be, at least to a major extent, responsible for the cytotoxicity. Here we present a summary and comparison of the amyloidogenic propensities of hIAPP in bulk solution and in the presence of various neutral and charged lipid bilayer systems as well as biological membranes. We also discuss the cellular effects of macromolecular crowding and osmolytes on the aggregation pathway of hIAPP. Understanding the influence of different cellular factors on hIAPP aggregation will provide more insight into the onset of T2DM and help to develop novel therapeutic strategies. PMID:26582333

  13. α-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells

    PubMed Central

    Hansen, Christian; Angot, Elodie; Bergström, Ann-Louise; Steiner, Jennifer A.; Pieri, Laura; Paul, Gesine; Outeiro, Tiago F.; Melki, Ronald; Kallunki, Pekka; Fog, Karina; Li, Jia-Yi; Brundin, Patrik

    2011-01-01

    Post-mortem analyses of brains from patients with Parkinson disease who received fetal mesencephalic transplants show that α-synuclein–containing (α-syn–containing) Lewy bodies gradually appear in grafted neurons. Here, we explored whether intercellular transfer of α-syn from host to graft, followed by seeding of α-syn aggregation in recipient neurons, can contribute to this phenomenon. We assessed α-syn cell-to-cell transfer using microscopy, flow cytometry, and high-content screening in several coculture model systems. Coculturing cells engineered to express either GFP– or DsRed-tagged α-syn resulted in a gradual increase in double-labeled cells. Importantly, α-syn–GFP derived from 1 neuroblastoma cell line localized to red fluorescent aggregates in other cells expressing DsRed–α-syn, suggesting a seeding effect of transmitted α-syn. Extracellular α-syn was taken up by cells through endocytosis and interacted with intracellular α-syn. Next, following intracortical injection of recombinant α-syn in rats, we found neuronal uptake was attenuated by coinjection of an endocytosis inhibitor. Finally, we demonstrated in vivo transfer of α-syn between host cells and grafted dopaminergic neurons in mice overexpressing human α-syn. In summary, intercellularly transferred α-syn interacts with cytoplasmic α-syn and can propagate α-syn pathology. These results suggest that α-syn propagation is a key element in the progression of Parkinson disease pathology. PMID:21245577

  14. Wild-type human γD-crystallin promotes aggregation of its oxidation-mimicking, misfolding-prone W42Q mutant.

    PubMed

    Serebryany, Eugene; King, Jonathan A

    2015-05-01

    Non-native protein conformers generated by mutation or chemical damage template aggregation of wild-type, undamaged polypeptides in diseases ranging from amyotrophic lateral sclerosis to cancer. We tested for such interactions in the natively monomeric human eye lens protein γd-crystallin, whose aggregation leads to cataract disease. The oxidation-mimicking W42Q mutant of γd-crystallin formed non-native polymers starting from a native-like state under physiological conditions. Aggregation occurred in the temperature range 35-45 °C, in which the mutant protein began to lose the native conformation of its N-terminal domain. Surprisingly, wild-type γd-crystallin promoted W42Q polymerization in a catalytic manner, even at mutant concentrations too low for homogeneous nucleation to occur. The presence of wild-type protein also downshifted the temperature range of W42Q aggregation. W42Q aggregation required formation of a non-native intramolecular disulfide bond but not intermolecular cross-linking. Transient WT/W42Q binding may catalyze this oxidative misfolding event in the mutant. That a more stable variant in a mixture can specifically promote aggregation of a less stable one rationalizes how extensive aggregation of rare damaged polypeptides can occur during the course of aging. PMID:25787081

  15. Aggregation of human platelets by endotoxic glycolipid-bearing Salmonella minnesota Re595 is prevented by synthetic peptide analogs of cell adhesion sites of fibrinogen and fibronectin

    SciTech Connect

    Timmons, S.; Grabarek, J.; Kloczewiak, M.; Hawiger, J.

    1986-03-01

    Thrombocytopenia often accompanies sepsis due to endotoxin producing gram-negative bacteria. The authors have observed that mutant Re595 of S. minnesota induced aggregation of human platelets separated from plasma fibrinogen (Theta) and other proteins. This aggregation is dependent on ADP secreted from storage granules in response to mutant Re595. Platelet aggregation induced by mutant Re595 was prevented by simultaneously added EDTA and EGTA (5mM), whereas secretion of /sup 14/C-serotonin was maintained. Preincubation of platelets with chelators (1 hr, 37/sup 0/C), known to dissociate irreversibly the platelet membrane glycoprotein IIb x IIIa complex, abolished aggregation while serotonin secretion was decreased by only one fourth. Since the GPIIb x IIIa complex constitutes the receptor for Theta, its role was examined using synthetic peptide analogs of sites on gamma and alpha chains of Theta. Gamma 400-411 (225 ..mu..M) inhibited platelet aggregation induced by mutant Re595 while serotonin secretion was unaffected. Alpha 572-575 (RGDS; 100 ..mu..M), analogous to cell adhesion site of fibronectin, also prevented aggregation induced by mutant Re595. Thus, mutant Re595 causes platelet aggregation which is divalent cation-dependent and proceeds via receptor pathway for secreted adhesive macromolecules.

  16. Characterization of the promoter of the human gene encoding the high-affinity IgG receptor: Transcriptional induction by. gamma. -interferon is mediated through common DNA response elements

    SciTech Connect

    Pearse, R.N.; Feinman, R.; Ravetch, J.V. )

    1991-12-15

    Expression of the high-affinity receptor for IgG (Fc{sub {gamma}}RI) is restricted to cells of myeloid lineage and is induced by {gamma}-interferon (IFN-{gamma}) but not by IFN-{alpha}/{beta}. The organization of the human Fc{sub {gamma}}RI gene has been determined and the DNA elements governing its cell type-restricted transcription and IFN-{gamma} induction are reported here. A 39-nucleotide sequence (IFN-{gamma} response region, or GRR) is defined that is both necessary and sufficient for IFN-{gamma} inducibility. Sequence analysis of the GRR reveals the presence of promoter elements initially defined for the major histocompatibility complex class 2 genes: i.e., X, H, and {gamma}-IRE sequences. Comparison of a number of genes whose expression is induced selectively by IFN-{gamma} indicated that the presence of these elements is a general feature of IFN-{gamma}-responsive genes. The studies suggest that the combination of X, H, and {gamma}-IRE elements is a common motif in the pathway of transcriptional induction by this lymphokine.

  17. Reactivity of human anti-alpha-galactosyl IgG antibody with alpha(1-->3)-linked galactosyl epitopes exposed on basement membranes and on glomerular epithelial cells: an in vitro and in vivo study in the mouse.

    PubMed Central

    Vecchi, M L; Davin, J C; Castronovo, V; Foidart, J M; Malaise, M; Foidart, J B; Dechene, C; Sangiorgi, G B; Mahieu, P

    1989-01-01

    Anti-alpha-galactosyl antibody (a-Gal Ab) is a human natural antibody belonging to the IgG class, found in high titres in all normal sera regardless of blood group, and specifically recognizing alpha (1-->3)-linked galactosyl residues. We have observed by radioimmunoassay, ELISA, passive haemagglutination and immunofluorescence blocking studies that affinity-purified a-Gal Ab reacted with mouse laminin, but not with the other mouse basement membrane proteins tested; it was able to fix complement in vitro. When injected intravenously into mice, the a-Gal Ab was found to mainly accumulate in kidneys, liver, spleen and lungs. No acute respiratory distress syndrome was observed shortly after the i.v. injection of 100 or 200 microg of antibodies. These doses of a-Gal Ab were also unable to induce acute glomerular injury. However, in primary cultures, the a-Gal Ab (100 or 200 microg per ml of medium) was shown to impair the attachment of mouse glomerular epithelial cells to mouse laminin and to elicit complement-dependent cell damage. The data indicate that the a-Gal Ab can interact in vitro and/or in vivo with alpha (1-->3)-linked galactosyl residues exposed on murine laminin or on murine cultured glomerular epithelial cells. Although this antibody fails to be pathogenic when administered at low doses in the intact animal, similar doses can alter some metabolic properties of these cells in vitro. PMID:12412761

  18. Comparison of the Fc fragment from a human IgG1 and its CH2, pFc', and tFc' subfragments. A study using reductive methylation and sup 13 C NMR

    SciTech Connect

    Jentoft, J.E.; Rayford, R. )

    1989-04-18

    The Fc fragment of a human monoclonal IgG1 was compared with subfragments containing (a) the intact CH2 domain (CH2 fragment) or (b) the intact CH3 domain (pFc' and tFc' fragments). All fragments were reductively {sup 13}C-methylated and their resulting dimethyllysyl resonances characterized in 0.1 M KCl as a function of pH by {sup 13}C NMR spectroscopy. Seven resonances were characterized for the 18 lysine residues of the Fc fragment, eight for the 12 lysines of the CH2 fragment, and five each for the 9 lysines of the pFc' and the 6 lysines of the tFc' fragments, respectively. The multiplicity of resonances indicates that the lysine residues in each fragment exist in a variety of microenvironments and that the fragments are all highly structured. The correspondence between 6 of the 12 or 13 perturbed lysine residues in the Fc fragment and the smaller subfragments indicates that the conformation of the CH2 and CH3 domains is largely unchanged in the smaller fragments. However, in addition to three lysines at the CH2-CH3 domain interface, whose environments were known to be disrupted in the smaller fragments, three or four lysine residues have somewhat different properties in the Fc fragment and in the subfragments, indicating that some local perturbations are included in the domain structure in the subfragments.

  19. Comparative functional characterization of canine IgG subclasses.

    PubMed

    Bergeron, Lisa M; McCandless, Erin E; Dunham, Steve; Dunkle, Bill; Zhu, Yaqi; Shelly, John; Lightle, Sandra; Gonzales, Andrea; Bainbridge, Graeme

    2014-01-15

    To date, very little is known about the functional characteristics of the four published canine IgG subclasses. It is not clear how each subclass engages the immune system via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC), or how long each antibody may last in serum. Such information is critical for understanding canine immunology and for the discovery of canine therapeutic monoclonal antibodies. Through both in vitro and ex vivo experiments to evaluate canine Fc's for effector function, complement binding, FcRn binding, and ADCC, we are now able to categorize canine subclasses by function. The subclasses share functional properties with the four human IgG subclasses and are reported herein with their function-based human analog. Canine Fc fusions, canine chimeras, and caninized antibodies were characterized. Canine subclasses A and D appear effector-function negative while subclasses B and C bind canine Fc gamma receptors and are positive for ADCC. All canine subclasses bind the neonatal Fc receptor except subclass C. By understanding canine IgGs in this way, we can apply what is known of human immunology toward translational and veterinary medicine. Thus, this body of work lays the foundation for evaluating canine IgG subclasses for therapeutic antibody development and builds upon the fundamental scholarship of canine immunology. PMID:24268690

  20. Comparing Gray Mineral Trioxide Aggregate and Diluted Formocresol in Pulpotomized Human Primary Molars

    PubMed Central

    Zealand, Cameron M.; Briskie, Daniel M.; Botero, Tatiana M.; Boynton, James R.; Hu, Jan C.C.

    2016-01-01

    Purpose The purpose of this multisite, multioperator, prospective, randomized, controlled clinical trial was to evaluate the 6-month outcomes of diluted formocresol (DFC) compared to gray mineral trioxide aggregate (GMTA) as pulpotomy medicament. Methods Determined by a power analysis, 252 molars of 152 children were recruited. The teeth were randomly assigned to receive GMTA or DFC. At the 6-month follow-up, 118 children with 203 treated teeth were evaluated. Results Four blinded and calibrated evaluators scored each radiograph for pathologies. Clinical success was similar for DFC (97%) and GMTA (100%), (P<.09). Radiographic success differed significantly (P<.04) for DFC (86%) and GMTA (95%). Pulp canal obliteration was radiographically observed in 25% of the DFC group and in 37% of the GMTA group (P=.07). Dentin bridging was observed in 22% of the GMTA group but was not found in the DFC group (P<.01). Conclusion Teeth treated with GMTA showed more favorable radiographic outcomes than DFC at 6 months post-treatment. PMID:21070705

  1. Purified IgG from Patients with Obstetric but not IgG from Non-obstetric Antiphospholipid Syndrome Inhibit Trophoblast Invasion

    PubMed Central

    Poulton, Katie; Ripoll, Vera M; Pericleous, Charis; Meroni, Pier Luigi; Gerosa, Maria; Ioannou, Yiannis; Rahman, Anisur; Giles, Ian P

    2015-01-01

    Problem Some patients with antiphospholipid syndrome (APS) suffer pregnancy morbidity (PM) but not vascular thrombosis (VT), whilst others suffer VT only. Therefore, we compared the effects of IgG from VT+/PM− and VT−/PM+ subjects on human first-trimester trophoblast (HTR8) cells. Method of study HTR-8 cells were incubated with APS VT+/PM−, APS VT−/PM+ or healthy control (HC) IgG. We measured trophoblast invasion by cell invasion assay; mRNA expression of TLR4 and adaptor proteins; phosphorylation of p38 MAPK, NFκB and ERK; and expression of interleukin (IL)-8 and IL-6. Results VT−/PM+ IgG, but not VT+/PM− IgG significantly reduced HTR-8 invasion. The effects on invasion were blocked by TLR-4 inhibition. Neither VT+/PM− nor VT−/PM+ IgG altered MyD88 mRNA expression, phosphorylation of signalling molecules or cytokine expression. Conclusions VT−/PM+ IgG exert functionally relevant effects on human trophoblast cells but VT+/PM− IgG do not. PMID:25469631

  2. Evaluation of a novel hexavalent humanized anti-IGF-1R antibody and its bivalent parental IgG in diverse cancer cell lines.

    PubMed

    Chang, Chien-Hsing; Wang, Yang; Trisal, Preeti; Li, Rongxiu; Rossi, Diane L; Nair, Anju; Gupta, Pankaj; Losman, Michele; Cardillo, Thomas M; Rossi, Edmund A; Goldenberg, David M

    2012-01-01

    A major mechanism of monoclonal antibodies that selectively target the insulin-like growth factor type 1 receptor (IGF-1R) to inhibit tumor growth is by downregulating the receptor, regardless whether they are capable (antagonistic) or incapable (agonistic) of blocking the binding of cognate ligands. We have developed and characterized a novel agonistic anti-IGF-1R humanized antibody, hR1, and used the Dock-and-Lock (DNL) method to construct Hex-hR1, the first multivalent antibody comprising 6 functional Fabs of hR1, with the aim of enhancing potency of hR1. Based on cross-blocking experiments, hR1 recognizes a region of cysteine-rich domain on the α-subunit, different from the epitopes mapped for existing anti-IGF-1R antibodies, yet hR1 is similar to other anti-IGF-1R antibodies in downregulating IGF-1R and inhibiting proliferation, colony formation, or invasion of selected cancer cell lines in vitro, as well as suppressing growth of the RH-30 rhabdomyosarcoma xenograft in nude mice when combined with the mTOR inhibitor, rapamycin. Hex-hR1 and hR1 are generally comparable in their bioactivities under the in-intro and in-vivo conditions investigated. Nevertheless, in selective experiments involving a direct comparison of potency, Hex-hR1 demonstrated a stronger effect on inhibiting cell proliferation stimulated by IGF-1 and could effectively downregulate IGF-1R at a concentration as low as 20 pM. PMID:22952934

  3. Evaluation of a Novel Hexavalent Humanized Anti-IGF-1R Antibody and Its Bivalent Parental IgG in Diverse Cancer Cell Lines

    PubMed Central

    Chang, Chien-Hsing; Wang, Yang; Trisal, Preeti; Li, Rongxiu; Rossi, Diane L.; Nair, Anju; Gupta, Pankaj; Losman, Michele; Cardillo, Thomas M.; Rossi, Edmund A.; Goldenberg, David M.

    2012-01-01

    A major mechanism of monoclonal antibodies that selectively target the insulin-like growth factor type 1 receptor (IGF-1R) to inhibit tumor growth is by downregulating the receptor, regardless whether they are capable (antagonistic) or incapable (agonistic) of blocking the binding of cognate ligands. We have developed and characterized a novel agonistic anti-IGF-1R humanized antibody, hR1, and used the Dock-and-Lock (DNL) method to construct Hex-hR1, the first multivalent antibody comprising 6 functional Fabs of hR1, with the aim of enhancing potency of hR1. Based on cross-blocking experiments, hR1 recognizes a region of cysteine-rich domain on the α-subunit, different from the epitopes mapped for existing anti-IGF-1R antibodies, yet hR1 is similar to other anti-IGF-1R antibodies in downregulating IGF-1R and inhibiting proliferation, colony formation, or invasion of selected cancer cell lines in vitro, as well as suppressing growth of the RH-30 rhabdomyosarcoma xenograft in nude mice when combined with the mTOR inhibitor, rapamycin. Hex-hR1 and hR1 are generally comparable in their bioactivities under the in-intro and in-vivo conditions investigated. Nevertheless, in selective experiments involving a direct comparison of potency, Hex-hR1 demonstrated a stronger effect on inhibiting cell proliferation stimulated by IGF-1 and could effectively downregulate IGF-1R at a concentration as low as 20 pM. PMID:22952934

  4. Bovine IgG subclasses and fertility of Echinococcus granulosus hydatid cysts.

    PubMed

    Riesle, Silke; García, María Pía; Hidalgo, Christian; Galanti, Norbel; Saenz, Leonardo; Paredes, Rodolfo

    2014-09-15

    Hydatidosis is an important zoonotic disease of worldwide distribution, causing important health problems to humans and major economical losses in infected livestock. Echinococcus granulosus, the etiological agent of hydatid disease, induces a humoral immune response in the intermediate host (human and herbivorous) against hydatid cyst antigens. Specifically, IgGs are found in the laminar and germinal layers and inside the lumen of fertile and infertile hydatid cysts. In the germinal layer of infertile cysts IgGs are found in an order of magnitude greater than in the germinal layer of fertile cysts; a fraction of those IgGs are associated with high affinity to germinal layer proteins, suggesting their binding to specific parasite antigens. We have previously shown that those immunoglobulins, bound with high affinity to the germinal layer of hydatid cysts, induce apoptosis leading to cyst infertility. In the present work the presence of IgG1 and IgG2 subclasses in the germinal layer of both fertile and infertile hydatid cysts is reported. IgG1 is the most relevant immunoglobulin subclass present in the germinal layer of infertile cysts and bound with high affinity to that parasite structure. Contrarily, though the IgG2 subclass was also found in the germinal and adventitial layers, those immunoglobulins show low affinity to parasite antigens. We propose that the binding of an IgG1 subclass to parasite antigens present in the germinal layer is involved in the mechanism of cyst infertility. PMID:24962125

  5. Enhanced HIV-1 neutralization by a CD4-VH3-IgG1 fusion protein

    SciTech Connect

    Meyuhas, Ronit; Noy, Hava; Fishman, Sigal; Margalit, Alon; Montefiori, David C.; Gross, Gideon

    2009-08-21

    HIV-1 gp120 is an alleged B cell superantigen, binding certain VH3+ human antibodies. We reasoned that a CD4-VH3 fusion protein could possess higher affinity for gp120 and improved HIV-1 inhibitory capacity. To test this we produced several human IgG1 immunoligands harboring VH3. Unlike VH3-IgG1 or VH3-CD4-IgG1, CD4-VH3-IgG1 bound gp120 considerably stronger than CD4-IgG1. CD4-VH3-IgG1 exhibited {approx}1.5-2.5-fold increase in neutralization of two T-cell laboratory-adapted strains when compared to CD4-IgG1. CD4-VH3-IgG1 improved neutralization of 7/10 clade B primary isolates or pseudoviruses, exceeding 20-fold for JR-FL and 13-fold for Ba-L. It enhanced neutralization of 4/8 clade C viruses, and had negligible effect on 1/4 clade A pseudoviruses. We attribute this improvement to possible pairing of VH3 with CD4 D1 and stabilization of an Ig Fv-like structure, rather than to superantigen interactions. These novel findings support the current notion that CD4 fusion proteins can act as better HIV-1 entry inhibitors with potential clinical implications.

  6. Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity.

    PubMed

    Quast, Isaak; Keller, Christian W; Maurer, Michael A; Giddens, John P; Tackenberg, Björn; Wang, Lai-Xi; Münz, Christian; Nimmerjahn, Falk; Dalakas, Marinos C; Lünemann, Jan D

    2015-11-01

    IgG molecules exert both pro- and antiinflammatory effector functions based on the composition of the fragment crystallizable (Fc) domain glycan. Sialylated IgG Fc domains have antiinflammatory properties that are attributed to their ability to increase the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we report that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-mediated cytotoxicity (CDC). Fc sialylation of a CD20-targeting antibody had no impact on antibody-dependent cellular cytotoxicity and did not change the affinity of the antibody for activating Fcγ receptors. In contrast, the presence of sialic acid abrogated the increased binding of C1q to Fc-galactosylated IgG1 and resulted in decreased levels of C3b deposition on the cell surface. Similar to monoclonal antibodies, sialic acid inhibited the increased C1q binding to galactosylated Fc fragments in human polyclonal IgG. In sera derived from patients with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system in which humoral immune responses mediate tissue damage, induction of IgG Fc sialylation was associated with clinical disease remission. Thus, impairment of CDC represents an FcγR-independent mechanism by which Fc-sialylated glycovariants might limit proinflammatory IgG effector functions. PMID:26436649

  7. Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity

    PubMed Central

    Quast, Isaak; Keller, Christian W.; Maurer, Michael A.; Giddens, John P.; Tackenberg, Björn; Wang, Lai-Xi; Münz, Christian; Nimmerjahn, Falk; Dalakas, Marinos C.; Lünemann, Jan D.

    2015-01-01

    IgG molecules exert both pro- and antiinflammatory effector functions based on the composition of the fragment crystallizable (Fc) domain glycan. Sialylated IgG Fc domains have antiinflammatory properties that are attributed to their ability to increase the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we report that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-mediated cytotoxicity (CDC). Fc sialylation of a CD20-targeting antibody had no impact on antibody-dependent cellular cytotoxicity and did not change the affinity of the antibody for activating Fcγ receptors. In contrast, the presence of sialic acid abrogated the increased binding of C1q to Fc-galactosylated IgG1 and resulted in decreased levels of C3b deposition on the cell surface. Similar to monoclonal antibodies, sialic acid inhibited the increased C1q binding to galactosylated Fc fragments in human polyclonal IgG. In sera derived from patients with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system in which humoral immune responses mediate tissue damage, induction of IgG Fc sialylation was associated with clinical disease remission. Thus, impairment of CDC represents an FcγR-independent mechanism by which Fc-sialylated glycovariants might limit proinflammatory IgG effector functions. PMID:26436649

  8. IgG4-related skin disease.

    PubMed

    Tokura, Y; Yagi, H; Yanaguchi, H; Majima, Y; Kasuya, A; Ito, T; Maekawa, M; Hashizume, H

    2014-11-01

    IgG4-related disease (IgG4-RD) is a recently established clinical entity characterized by high levels of circulating IgG4, and tissue infiltration of IgG4(+) plasma cells. IgG4-RD exhibits a distinctive fibroinflammatory change involving multiple organs, such as the pancreas and salivary and lacrimal glands. The skin lesions of IgG4-RD have been poorly characterized and may stem not only from direct infiltration of plasma cells but also from IgG4-mediated inflammation. Based on the documented cases together with ours, we categorized the skin lesions into seven subtypes: (1) cutaneous plasmacytosis (multiple papulonodules or indurations on the trunk and proximal part of the limbs), (2) pseudolymphoma and angiolymphoid hyperplasia with eosinophilia (plaques and papulonodules mainly on the periauricular, cheek and mandible regions), (3) Mikulicz disease (palpebral swelling, sicca syndrome and exophthalmos), (4) psoriasis-like eruption (strikingly mimicking psoriasis vulgaris), (5) unspecified maculopapular or erythematous eruptions, (6) hypergammaglobulinaemic purpura (bilateral asymmetrical palpable purpuric lesions on the lower extremities) and urticarial vasculitis (prolonged urticarial lesions occasionally with purpura) and (7) ischaemic digit (Raynaud phenomenon and digital gangrene). It is considered that subtypes 1-3 are induced by direct infiltration of IgG4(+) plasma cells, while the other types (4-7) are caused by secondary mechanisms. IgG4-related skin disease is defined as IgG4(+) plasma-cell-infiltrating skin lesions that form plaques, nodules or tumours (types 1-3), but may manifest secondary lesions caused by IgG4(+) plasma cells and/or IgG4 (types 4-7). PMID:25065694

  9. Misfolding, Aggregation, and Disordered Segments in c-Abl and p53 in Human Cancer

    PubMed Central

    de Oliveira, Guilherme A. P.; Rangel, Luciana P.; Costa, Danielly C.; Silva, Jerson L.

    2015-01-01

    The current understanding of the molecular mechanisms that lead to cancer is not sufficient to explain the loss or gain of function in proteins related to tumorigenic processes. Among them, more than 100 oncogenes, 20–30 tumor-suppressor genes, and hundreds of genes participating in DNA repair and replication have been found to play a role in the origins of cancer over the last 25 years. The phosphorylation of serine, threonine, or tyrosine residues is a critical step in cellular growth and development and is achieved through the tight regulation of protein kinases. Phosphorylation plays a major role in eukaryotic signaling as kinase domains are found in 2% of our genes. The deregulation of kinase control mechanisms has disastrous consequences, often leading to gains of function, cell transformation, and cancer. The c-Abl kinase protein is one of the most studied targets in the fight against cancer and is a hotspot for drug development because it participates in several solid tumors and is the hallmark of chronic myelogenous leukemia. Tumor suppressors have the opposite effects. Their fundamental role in the maintenance of genomic integrity has awarded them a role as the guardians of DNA. Among the tumor suppressors, p53 is the most studied. The p53 protein has been shown to be a transcription factor that recognizes and binds to specific DNA response elements and activates gene transcription. Stress triggered by ionizing radiation or other mutagenic events leads to p53 phosphorylation and cell-cycle arrest, senescence, or programed cell death. The p53 gene is the most frequently mutated gene in cancer. Mutations in the DNA-binding domain are classified as class I or class II depending on whether substitutions occur in the DNA contact sites or in the protein core, respectively. Tumor-associated p53 mutations often lead to the loss of protein function, but recent investigations have also indicated gain-of-function mutations. The prion-like aggregation of mutant p

  10. Intermediate conformation between native β-sheet and non-native α-helix is a precursor of trifluoroethanol-induced aggregation of Human Carbonic Anhydrase-II

    SciTech Connect

    Gupta, Preeti; Deep, Shashank

    2014-06-20

    Highlights: • HCAII forms amyloid-like aggregates at moderate concentration of trifluoroethanol. • Protein adopts a state between β-sheet and α-helix at moderate % of TFE. • Hydrophobic surface(s) of partially structured conformation forms amyloid. • High % of TFE induces stable α-helical state preventing aggregation. - Abstract: In the present work, we examined the correlation between 2,2,2-trifluoroethanol (TFE)-induced conformational transitions of human carbonic anhydrase II (HCAII) and its aggregation propensity. Circular dichroism data indicates that protein undergoes a transition from β-sheet to α-helix on addition of TFE. The protein was found to aggregate maximally at moderate concentration of TFE at which it exists somewhere between β-sheet and α-helix, probably in extended non-native β-sheet conformation. Thioflavin-T (ThT) and Congo-Red (CR) assays along with fluorescence microscopy and transmission electron microscopy (TEM) data suggest that the protein aggregates induced by TFE possess amyloid-like features. Anilino-8-naphthalene sulfonate (ANS) binding studies reveal that the exposure of hydrophobic surface(s) was maximum in intermediate conformation. Our study suggests that the exposed hydrophobic surface and/or the disruption of the structural features protecting a β-sheet protein might be the major reason(s) for the high aggregation propensity of non-native intermediate conformation of HCAII.

  11. Soluble Monomeric IgG1 Fc*

    PubMed Central

    Ying, Tianlei; Chen, Weizao; Gong, Rui; Feng, Yang; Dimitrov, Dimiter S.

    2012-01-01

    Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small size and other unique properties. However, compared with full-size antibodies, these antibody fragments lack the ability to bind the neonatal Fc receptor (FcRn) and have reduced half-lives. Fc engineered to bind antigens but preserve interactions with FcRn and Fc fused with monomeric proteins currently are being developed as candidate therapeutics with prolonged half-lives; in these and other cases, Fc is a dimer of two CH2-CH3 chains. To further reduce the size of Fc but preserve FcRn binding, we generated three human soluble monomeric IgG1 Fcs (mFcs) by using a combination of structure-based rational protein design combined with multiple screening strategies. These mFcs were highly soluble and retained binding to human FcRn comparable with that of Fc. These results provide direct experimental evidence that efficient binding to human FcRn does not require human Fc dimerization. The newly identified mFcs are promising for the development of mFc fusion proteins and for novel types of mFc-based therapeutic antibodies of small size and long half-lives. PMID:22518843

  12. MuSK induced experimental autoimmune myasthenia gravis does not require IgG1 antibody to MuSK.

    PubMed

    Küçükerden, Melike; Huda, Ruksana; Tüzün, Erdem; Yılmaz, Abdullah; Skriapa, Lamprini; Trakas, Nikos; Strait, Richard T; Finkelman, Fred D; Kabadayı, Sevil; Zisimopoulou, Paraskevi; Tzartos, Socrates; Christadoss, Premkumar

    2016-06-15

    Sera of myasthenia gravis (MG) patients with muscle-specific receptor kinase-antibody (MuSK-Ab) predominantly display the non-complement fixing IgG4 isotype. Similarly, mouse IgG1, which is the analog of human IgG4, is the predominant isotype in mice with experimental autoimmune myasthenia gravis (EAMG) induced by MuSK immunization. The present study was performed to determine whether IgG1 anti-MuSK antibody is required for immunized mice to develop EAMG. Results demonstrated a significant correlation between clinical severity of EAMG and levels of MuSK-binding IgG1+, IgG2+ and IgG3+ peripheral blood B cells in MuSK-immunized wild-type (WT) mice. Moreover, MuSK-immunized IgG1 knockout (KO) and WT mice showed similar EAMG severity, serum MuSK-Ab levels, muscle acetylcholine receptor concentrations, neuromuscular junction immunoglobulin and complement deposit ratios. IgG1 and IgG3 were the predominant anti-MuSK isotypes in WT and IgG1 KO mice, respectively. These observations demonstrate that non-IgG1 isotypes can mediate MuSK-EAMG pathogenesis. PMID:27235354

  13. X-ray small angle scattering of the human transferrin protein aggregates. A fractal study.

    PubMed Central

    Castellano, A C; Barteri, M; Bianconi, A; Borghi, E; Cassiano, L; Castagnola, M; Della Longa, S

    1993-01-01

    X-ray small angle scattering experiments, using a pin hole SAXS camera with Synchrotron radiation source, have been performed to study the conformational changes of lyophilized samples of Apo-, Mono-, and Diferric- human transferrin. We report the experimental evidence that the analysis of the scattered intensity through the fractal theory may give information on the particle size and its variation upon iron binding. PMID:8457675

  14. MRP4 knockdown enhances migration, suppresses apoptosis, and produces aggregated morphology in human retinal vascular endothelial cells

    SciTech Connect

    Tagami, Mizuki; Kusuhara, Sentaro; Imai, Hisanori; Uemura, Akiyoshi; Honda, Shigeru; Tsukahara, Yasutomo; Negi, Akira

    2010-10-01

    Research highlights: {yields} Exogenous VEGF decreases MRP4 expression in a dose-dependent manner. {yields} MRP4 knockdown leads to enhanced cell migration. {yields} MRP4 knockdown suppresses caspase-3-mediated cell apoptosis. {yields} MRP4 knockdown produces cell assembly and cell aggregation. -- Abstract: The multidrug resistance protein (MRP) MRP4/ABCC4 is an ATP-binding cassette transporter that actively effluxes endogenous and xenobiotic substrates out of cells. In the rodent retina, Mrp4 mRNA and protein are exclusively expressed in vascular endothelial cells, but the angiogenic properties of Mrp4 are poorly understood so far. This study aims to explore the angiogenic properties of MRP4 in human retinal microvascular endothelial cells (HRECs) utilizing the RNA interference (RNAi) technique. MRP4 expression was decreased at the mRNA and protein levels after stimulation with exogenous vascular endothelial growth factor in a dose-dependent manner. RNAi-mediated MRP4 knockdown in HRECs do not affect cell proliferation but enhances cell migration. Moreover, cell apoptosis induced by serum starvation was less prominent in MRP4 siRNA-treated HRECs as compared to control siRNA-treated HRECs. In a Matrigel-based tube-formation assay, although MRP4 knockdown did not lead to a significant change in the total tube length, MRP4 siRNA-treated HRECs assembled and aggregated into a massive tube-like structure, which was not observed in control siRNA-treated HRECs. These results suggest that MRP4 is uniquely involved in retinal angiogenesis.

  15. Age-related changes in the composition, the molecular stoichiometry and the stability of proteoglycan aggregates extracted from human articular cartilage.

    PubMed Central

    Wells, Terri; Davidson, Catherine; Mörgelin, Matthias; Bird, Joseph L E; Bayliss, Michael T; Dudhia, Jayesh

    2003-01-01

    The heterogeneity of the components of proteoglycan aggregates, their stoichiometry within the aggregate and the aggregates' stability was investigated in normal human articular cartilage specimens (age-range newborn to 63 years). Proteoglycans were extracted from tissue by sequentially extracting them with PBS alone, PBS containing oligosaccharides of hyaluronan, and PBS containing solutions of increasing guanidinium chloride concentration (1 M, 2 M, 3 M and 4 M). A high proportion of each of the components of the proteoglycan aggregate, i.e. uronic acid, sulphated glycosaminoglycan, hyaluronan binding domain of aggrecan (G1-domain), link protein (LP) and hyaluronan, was extracted from immature cartilage by PBS alone and PBS containing oligosaccharides of hyaluronan. This was in marked contrast to adult cartilage, which required high concentrations of guanidinium chloride for the efficient extraction of these components. The molar ratios of total G1-domain:LP and the G1-domain associated with aggrecan:LP also differed markedly between immature and mature cartilage and between each of the sequential extracts. The concentration of LP was less than that of the G1-domain in all extracts of cartilage from individuals over 13 years, but this was particularly noticeable in the 1 M guanidinium chloride extracts, and it was surmised that a deficiency in LP produces unstable aggregates in situ. The fragmentation of LP, which is known to occur with advancing age, did not influence the extractability of LP, and fragments were present in each of the sequential extracts. Therefore the generally accepted model of proteoglycan aggregation presented in the literature, which is mostly derived from analysis of immature animal cartilage, cannot be used to describe the structure and organization of aggregates in adult human articular cartilage, where a heterogeneous population of complexes exist that have varying degrees of stability. PMID:12431185

  16. von Willebrand factor fibers promote cancer-associated platelet aggregation in malignant melanoma of mice and humans.

    PubMed

    Bauer, Alexander T; Suckau, Jan; Frank, Kathrin; Desch, Anna; Goertz, Lukas; Wagner, Andreas H; Hecker, Markus; Goerge, Tobias; Umansky, Ludmila; Beckhove, Philipp; Utikal, Jochen; Gorzelanny, Christian; Diaz-Valdes, Nancy; Umansky, Viktor; Schneider, Stefan W

    2015-05-14

    Tumor-mediated procoagulatory activity leads to venous thromboembolism and supports metastasis in cancer patients. A prerequisite for metastasis formation is the interaction of cancer cells with endothelial cells (ECs) followed by their extravasation. Although it is known that activation of ECs and the release of the procoagulatory protein von Willebrand factor (VWF) is essential for malignancy, the underlying mechanisms remain poorly understood. We hypothesized that VWF fibers in tumor vessels promote tumor-associated thromboembolism and metastasis. Using in vitro settings, mouse models, and human tumor samples, we showed that melanoma cells activate ECs followed by the luminal release of VWF fibers and platelet aggregation in tumor microvessels. Analysis of human blood samples and tumor tissue revealed that a promoted VWF release combined with a local inhibition of proteolytic activity and protein expression of ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type I repeats 13) accounts for this procoagulatory milieu. Blocking endothelial cell activation by the low-molecular-weight heparin tinzaparin was accompanied by a lack of VWF networks and inhibited tumor progression in a transgenic mouse model. Our findings implicate a mechanism wherein tumor-derived vascular endothelial growth factor-A (VEGF-A) promotes tumor progression and angiogenesis. Thus, targeting EC activation envisions new therapeutic strategies attenuating tumor-related angiogenesis and coagulation. PMID:25977583

  17. In Vitro and In Vivo Comparison of Lymphocytes Transduced with a Human CD16 or with a Chimeric Antigen Receptor Reveals Potential Off-Target Interactions due to the IgG2 CH2-CH3 CAR-Spacer.

    PubMed

    Clémenceau, Béatrice; Valsesia-Wittmann, Sandrine; Jallas, Anne-Catherine; Vivien, Régine; Rousseau, Raphaël; Marabelle, Aurélien; Caux, Christophe; Vié, Henri

    2015-01-01

    The present work was designed to compare two mechanisms of cellular recognition based on Ab specificity: firstly, when the anti-HER2 mAb trastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (ADCC) and, secondly, when HER2 positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (CAR). To compare these two mechanisms, we used the same cellular effector (NK-92) and the same signaling domain (FcεRIγ). The NK-92 cytotoxic cell line was transfected with either a FcγRIIIa-FcεRIγ (NK-92(CD16)) or a trastuzumab-based scFv-FcεRIγ chimeric receptor (NK-92(CAR)). In vitro, the cytotoxic activity against HER2 positive target cells after indirect recognition by NK-92(CD16) was always inferior to that observed after direct recognition by NK-92(CAR). In contrast, and somehow unexpectedly, in vivo, adoptive transfer of NK-92(CD16) + trastuzumab but not of NK-92(CAR) induced tumor regression. Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells. This interaction, leading to blockage of the NK-92(CAR) in the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain. PMID:26665156

  18. In Vitro and In Vivo Comparison of Lymphocytes Transduced with a Human CD16 or with a Chimeric Antigen Receptor Reveals Potential Off-Target Interactions due to the IgG2 CH2-CH3 CAR-Spacer

    PubMed Central

    Clémenceau, Béatrice; Valsesia-Wittmann, Sandrine; Jallas, Anne-Catherine; Vivien, Régine; Rousseau, Raphaël; Marabelle, Aurélien; Caux, Christophe; Vié, Henri

    2015-01-01

    The present work was designed to compare two mechanisms of cellular recognition based on Ab specificity: firstly, when the anti-HER2 mAb trastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (ADCC) and, secondly, when HER2 positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (CAR). To compare these two mechanisms, we used the same cellular effector (NK-92) and the same signaling domain (FcεRIγ). The NK-92 cytotoxic cell line was transfected with either a FcγRIIIa-FcεRIγ (NK-92CD16) or a trastuzumab-based scFv-FcεRIγ chimeric receptor (NK-92CAR). In vitro, the cytotoxic activity against HER2 positive target cells after indirect recognition by NK-92CD16 was always inferior to that observed after direct recognition by NK-92CAR. In contrast, and somehow unexpectedly, in vivo, adoptive transfer of NK-92CD16 + trastuzumab but not of NK-92CAR induced tumor regression. Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells. This interaction, leading to blockage of the NK-92CAR in the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain. PMID:26665156

  19. Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis.

    PubMed

    Ohmi, Yuhsuke; Ise, Wataru; Harazono, Akira; Takakura, Daisuke; Fukuyama, Hidehiro; Baba, Yoshihiro; Narazaki, Masashi; Shoda, Hirofumi; Takahashi, Nobunori; Ohkawa, Yuki; Ji, Shuting; Sugiyama, Fumihiro; Fujio, Keishi; Kumanogoh, Atsushi; Yamamoto, Kazuhiko; Kawasaki, Nana; Kurosaki, Tomohiro; Takahashi, Yoshimasa; Furukawa, Koichi

    2016-01-01

    Rheumatoid arthritis (RA)-associated IgG antibodies such as anti-citrullinated protein antibodies (ACPAs) have diverse glycosylation variants; however, key sugar chains modulating the arthritogenic activity of IgG remain to be clarified. Here, we show that reduced sialylation is a common feature of RA-associated IgG in humans and in mouse models of arthritis. Genetically blocking sialylation in activated B cells results in exacerbation of joint inflammation in a collagen-induced arthritis (CIA) model. On the other hand, artificial sialylation of anti-type II collagen antibodies, including ACPAs, not only attenuates arthritogenic activity, but also suppresses the development of CIA in the antibody-infused mice, whereas sialylation of other IgG does not prevent CIA. Thus, our data demonstrate that sialylation levels control the arthritogenicity of RA-associated IgG, presenting a potential target for antigen-specific immunotherapy. PMID:27046227

  20. Alum Directly Modulates Murine B Lymphocytes to Produce IgG1 Isotype

    PubMed Central

    Jin, Bo-Ra; Kim, Sun-Jin; Lee, Jeong-Min; Kang, Seong-Ho; Han, Hye-Ju; Jang, Young-Saeng; Seo, Goo-young

    2013-01-01

    Aluminum hydroxide (alum) is the most widely used adjuvant in human vaccines. Nevertheless, it is virtually unknown whether alum acts on B cells. In the present study, we explored the direct effect of alum on Ig expression by murine B cells in vitro. LPS-activated mouse spleen B cells were cultured with alum, and the level of isotype-specific Ig secretion, IgG1 secreting cell numbers, and Ig germ-line transcripts (GLT) were measured using ELISA, ELISPOT, and RT-PCR, respectively. Alum consistently enhanced total IgG1 production, numbers of IgG1 secreting cells, and GLTγ1 expression. These results demonstrate that alum can directly cause IgG1 isotype switching leading to IgG1 production. PMID:23559895

  1. Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis

    PubMed Central

    Ohmi, Yuhsuke; Ise, Wataru; Harazono, Akira; Takakura, Daisuke; Fukuyama, Hidehiro; Baba, Yoshihiro; Narazaki, Masashi; Shoda, Hirofumi; Takahashi, Nobunori; Ohkawa, Yuki; Ji, Shuting; Sugiyama, Fumihiro; Fujio, Keishi; Kumanogoh, Atsushi; Yamamoto, Kazuhiko; Kawasaki, Nana; Kurosaki, Tomohiro; Takahashi, Yoshimasa; Furukawa, Koichi

    2016-01-01

    Rheumatoid arthritis (RA)-associated IgG antibodies such as anti-citrullinated protein antibodies (ACPAs) have diverse glycosylation variants; however, key sugar chains modulating the arthritogenic activity of IgG remain to be clarified. Here, we show that reduced sialylation is a common feature of RA-associated IgG in humans and in mouse models of arthritis. Genetically blocking sialylation in activated B cells results in exacerbation of joint inflammation in a collagen-induced arthritis (CIA) model. On the other hand, artificial sialylation of anti-type II collagen antibodies, including ACPAs, not only attenuates arthritogenic activity, but also suppresses the development of CIA in the antibody-infused mice, whereas sialylation of other IgG does not prevent CIA. Thus, our data demonstrate that sialylation levels control the arthritogenicity of RA-associated IgG, presenting a potential target for antigen-specific immunotherapy. PMID:27046227

  2. X-ray diffraction from intact tau aggregates in human brain tissue

    PubMed Central

    Landahl, Eric C.; Antipova, Olga; Bongaarts, Angela; Barrea, Raul; Berry, Robert; Binder, Lester I.; Irving, Thomas; Orgel, Joseph; Vana, Laurel

    2011-01-01

    We describe an instrument to record x-ray diffraction patterns from diseased regions of human brain tissue by combining an in-line visible light fluorescence microscope with an x-ray diffraction microprobe. We use thiazine red fluorescence to specifically label and detect the filamentous tau protein pathology associated with Pick’s disease, as several labs have done previously. We demonstrate that thiazine red-enhanced regions within the tissue show periodic structure in x-ray diffraction that is not observed in healthy tissue. One observed periodicity (4.2 Å) is characteristic of cross-beta sheet structure, consistent with previous results from powder diffraction studies performed on purified, dried tau protein. PMID:21876609

  3. X-ray diffraction from intact tau aggregates in human brain tissue.

    PubMed

    Landahl, Eric C; Antipova, Olga; Bongaarts, Angela; Barrea, Raul; Berry, Robert; Binder, Lester I; Irving, Thomas; Orgel, Joseph; Vana, Laurel; Rice, Sarah E

    2011-09-01

    We describe an instrument to record x-ray diffraction patterns from diseased regions of human brain tissue by combining an in-line visible light fluorescence microscope with an x-ray diffraction microprobe. We use thiazine red fluorescence to specifically label and detect the filamentous tau protein pathology associated with Pick's disease, as several labs have done previously. We demonstrate that thiazine red-enhanced regions within the tissue show periodic structure in x-ray diffraction that is not observed in healthy tissue. One observed periodicity (4.2 Å) is characteristic of cross-beta sheet structure, consistent with previous results from powder diffraction studies performed on purified, dried tau protein. PMID:21876609

  4. X-ray diffraction from intact tau aggregates in human brain tissue

    NASA Astrophysics Data System (ADS)

    Landahl, Eric C.; Antipova, Olga; Bongaarts, Angela; Barrea, Raul; Berry, Robert; Binder, Lester I.; Irving, Thomas; Orgel, Joseph; Vana, Laurel; Rice, Sarah E.

    2011-09-01

    We describe an instrument to record X-ray diffraction patterns from diseased regions of human brain tissue by combining an in-line visible light fluorescence microscope with an X-ray diffraction microprobe. We use thiazine red fluorescence to specifically label and detect the filamentous tau protein pathology associated with Pick's disease, as several laboratories have done previously. We demonstrate that thiazine red-enhanced regions within the tissue show periodic structure in X-ray diffraction, which is not observed in healthy tissue. One observed periodicity (4.2 Å) is characteristic of cross-beta sheet structure, consistent with previous results from powder diffraction studies performed on purified, dried tau protein.

  5. X-ray diffraction from intact tau aggregates in human brain tissue

    SciTech Connect

    Landahl, Eric C.; Antipova, Olga; Bongaarts, Angela; Barrea, Raul; Berry, Robert; Binder, Lester I.; Irving, Thomas; Orgel, Joseph; Vana, Laurel; Rice, Sarah E.

    2011-09-15

    We describe an instrument to record X-ray diffraction patterns from diseased regions of human brain tissue by combining an in-line visible light fluorescence microscope with an X-ray diffraction microprobe. We use thiazine red fluorescence to specifically label and detect the filamentous tau protein pathology associated with Pick's disease, as several laboratories have done previously. We demonstrate that thiazine red-enhanced regions within the tissue show periodic structure in X-ray diffraction, which is not observed in healthy tissue. One observed periodicity (4.2 {angstrom}) is characteristic of cross-beta sheet structure, consistent with previous results from powder diffraction studies performed on purified, dried tau protein.

  6. Construction aggregates

    USGS Publications Warehouse

    Tepordei, V.V.

    1995-01-01

    Part of the 1994 Industrial Minerals Review. The production, consumption, and applications of construction aggregates are reviewed. In 1994, the production of construction aggregates, which includes crushed stone and construction sand and gravel combined, increased 7.7 percent to 2.14 Gt compared with the previous year. These record production levels are mostly a result of funding for highway construction work provided by the Intermodal Surface Transportation Efficiency Act of 1991. Demand is expected to increase for construction aggregates in 1995.

  7. Temperature induced morphological transitions from native to unfolded aggregated States of human serum albumin.

    PubMed

    Das, Nirmal Kumar; Ghosh, Narayani; Kale, Ajit Prabhakar; Mondal, Ramakanta; Anand, Uttam; Ghosh, Subhadip; Tiwari, Virendra Kumar; Kapur, Manmohan; Mukherjee, Saptarshi

    2014-07-01

    The circulatory protein, human serum albumin (HSA), is known to have two melting point temperatures, 56 and 62 °C. In this present manuscript, we investigate the interaction of HSA with a synthesized bioactive molecule 3-pyrazolyl 2-pyrazoline (PZ). The sole tryptophan amino acid residue (Trp214) of HSA and PZ forms an excellent FRET pair and has been used to monitor the conformational dynamics in HSA as a function of temperature. Molecular docking studies reveal that the PZ binds to a site which is in the immediate vicinity of Trp214, and such data are also supported by time-resolved FRET studies. Steady-state and time-resolved anisotropy of PZ conclusively proved that the structural and morphological changes in HSA mainly occur beyond its first melting temperature. Although the protein undergoes thermal denaturation at elevated temperatures, the Trp214 gets buried inside the protein scaffolds; this fact has been substantiated by acrylamide quenching studies. Finally, we have used atomic force microscopy to establish that at around 70 °C, HSA undergoes self-assembly to form fibrillar structures. Such an observation may be attributed to the loss of α-helical content of the protein and a subsequent rise in β-sheet structure. PMID:24915234

  8. Aggregation of Trp > Glu point mutants of human gamma-D crystallin provides a model for hereditary or UV-induced cataract.

    PubMed

    Serebryany, Eugene; Takata, Takumi; Erickson, Erika; Schafheimer, Nathaniel; Wang, Yongting; King, Jonathan A

    2016-06-01

    Numerous mutations and covalent modifications of the highly abundant, long-lived crystallins of the eye lens cause their aggregation leading to progressive opacification of the lens, cataract. The nature and biochemical mechanisms of the aggregation process are poorly understood, as neither amyloid nor native-state polymers are commonly found in opaque lenses. The βγ-crystallin fold contains four highly conserved buried tryptophans, which can be oxidized to more hydrophilic products, such as kynurenine, upon UV-B irradiation. We mimicked this class of oxidative damage using Trp→Glu point mutants of human γD-crystallin. Such substitutions may represent a model of UV-induced photodamage-introduction of a charged group into the hydrophobic core generating "denaturation from within." The effects of Trp→Glu substitutions were highly position dependent. While each was destabilizing, only the two located in the bottom of the double Greek key fold-W42E and W130E-yielded robust aggregation of partially unfolded intermediates at 37°C and pH 7. The αB-crystallin chaperone suppressed aggregation of W130E, but not W42E, indicating distinct aggregation pathways from damage in the N-terminal vs C-terminal domain. The W130E aggregates had loosely fibrillar morphology, yet were nonamyloid, noncovalent, showed little surface hydrophobicity, and formed at least 20°C below the melting temperature of the native β-sheets. These features are most consistent with domain-swapped polymerization. Aggregation of partially destabilized crystallins under physiological conditions, as occurs in this class of point mutants, could provide a simple in vitro model system for drug discovery and optimization. PMID:26991007

  9. High-pressure studies of aggregation of recombinant human interleukin-1 receptor antagonist: Thermodynamics, kinetics, and application to accelerated formulation studies

    PubMed Central

    Seefeldt, Matthew B.; Kim, Yong-Sung; Tolley, Kevin P.; Seely, Jim; Carpenter, John F.; Randolph, Theodore W.

    2005-01-01

    Recombinant human interleukin-1 receptor antagonist (IL-1ra) in aqueous solutions unfolds and aggregates when subjected to hydrostatic pressures greater than about 180 MPa. This study examined the mechanism and thermodynamics of pressure-induced unfolding and aggregation of IL-1ra. The activation free energy for growth of aggregates (ΔG∓aggregation) was found to be 37 ± 3 kJ/mol, whereas the activation volume (ΔV∓aggregation) was −120 ± 20 mL/mol. These values compare closely with equilibrium values for denaturation: The free energy for denaturation, ΔGdenaturation, was 20 ± 5 kJ/mol, whereas the partial specific volume change for denaturation, ΔVdenaturation, was −110 ± 30 mL/mol. When IL-1ra begins to denature at pressures near 140 MPa, cysteines that are normally buried in the native state become exposed. Under oxidizing conditions, this results in the formation of covalently cross-linked aggregates containing nonnative, intermolecular disulfide bonds. The apparent activation free energy for nucleation of aggregates, ΔG∓nuc, was 42 ± 4 kJ/mol, and the activation volume for nucleation, ΔV∓nuc,was −175 ± 37 mL/mol, suggesting that a highly solvent-exposed conformation is needed for nucleation. We hypothesize that the large specific volume of IL-1ra, 0.752 ± 0.004 mL/g, coupled with its relatively low conformational stability, leads to its susceptibility to denaturation at relatively low pressures. The positive partial specific adiabatic compressibility of IL-1ra, 4.5 ± 0.7 ± 10−12 cm2/dyn, suggests that a significant component of the ΔVdenaturation is attributable to the elimination of solvent-free cavities. Lastly, we propose that hydrostatic pressure is a useful variable to conduct accelerated formulation studies of therapeutic proteins. PMID:16081653

  10. IgG4-related spinal pachymeningitis.

    PubMed

    Lu, Zhang; Tongxi, Liu; Jie, Luo; Yujuan, Jiao; Wei, Jiang; Xia, Liu; Yumin, Zheng; Xin, Lu

    2016-06-01

    The aim of this study is to study the clinical, laboratory, imaging pathology, and prognosis features of IgG4-related spinal pachymeningitis. We worked with a 55-year-old man suffering from IgG4-related spinal pachymeningitis who had the most widespread lesion in his dura mater. We also review previous related studies and discuss the clinical characteristics of this rare disease. In total, eight IgG4-related spinal pachymeningitis patients have been reported in the literature since 2009. They were mostly male patients, 51.7 ± 11.9 years old on average. Cervical and thoracic vertebrae were the most common sites for lesions. The most prominent symptom was varying numbness and weakness of the limbs and/or body associated with spinal cord compression. There was one patient (1/5) with elevated serum IgG4 levels and three patients (3/3) with increased cerebrospinal fluid (CSF) IgG4 index. Positive histopathologic findings are the strongest basis for a diagnosis. All the patients with IgG4-related spinal pachymeningitis responded well to glucocorticoid therapy. IgG4-related spinal pachymeningitis is an orphan disease that mainly occurs in cervical and thoracic vertebrae. Older males are the most susceptible group. Serum IgG4 levels were consistently normal in these cases, so analysis of CSF for IgG4 production (IgG4 index) could become a useful tool. Pathological findings remain the gold standard for diagnosis. Most patients responded favorably to glucocorticoid treatment. PMID:26567899

  11. The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation

    PubMed Central

    Lee, Mihwa; Sadowska, Agata; Bekere, Indra; Ho, Diwei; Gully, Benjamin S.; Lu, Yanling; Iyer, K. Swaminathan; Trewhella, Jill; Fox, Archa H.; Bond, Charles S.

    2015-01-01

    SFPQ, (a.k.a. PSF), is a human tumor suppressor protein that regulates many important functions in the cell nucleus including coordination of long non-coding RNA molecules into nuclear bodies. Here we describe the first crystal structures of Splicing Factor Proline and Glutamine Rich (SFPQ), revealing structural similarity to the related PSPC1/NONO heterodimer and a strikingly extended structure (over 265 Å long) formed by an unusual anti-parallel coiled-coil that results in an infinite linear polymer of SFPQ dimers within the crystals. Small-angle X-ray scattering and transmission electron microscopy experiments show that polymerization is reversible in solution and can be templated by DNA. We demonstrate that the ability to polymerize is essential for the cellular functions of SFPQ: disruptive mutation of the coiled-coil interaction motif results in SFPQ mislocalization, reduced formation of nuclear bodies, abrogated molecular interactions and deficient transcriptional regulation. The coiled-coil interaction motif thus provides a molecular explanation for the functional aggregation of SFPQ that directs its role in regulating many aspects of cellular nucleic acid metabolism. PMID:25765647

  12. A dimer model of human calcitonin13-32 forms an α-helical structure and robustly aggregates in 50% aqueous 2,2,2-trifluoroethanol solution.

    PubMed

    Kawashima, Hiroyuki; Katayama, Mei; Yoshida, Ryota; Akaji, Kenichi; Asano, Akiko; Doi, Mitsunobu

    2016-07-01

    Determining the cause of human calcitonin (hCT) aggregation could be of help in the effort to utilize hCT for treatment of hypercalcemia. Here we report that a dimer model of hCT13-32 aggregated to a greater degree than native hCT under aqueous 2,2,2-trifluoroethanol conditions. Analyses using circular dichroism spectroscopy, thioflavine-T binding assays and atomic force microscopy suggest that the α-helical portion of hCT is important for initiation of the aggregation process, which yields long fibrils. Dimerization, which stabilizes the β-sheet structure of hCT, enhances aggregation potency. Dimerization of hCT stabilizes the α-helix under aqueous TFE conditions, leading to the long fibril formation. Up to now, there have been no reports of using a dimer model to investigate the properties of hCT aggregation. Our findings could potentially serve as the basis for development of novel hCT derivatives that could be utilized for treatment of hypercalcemia, as well as for development of novel therapeutics for other ailments caused by amyloid peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. PMID:27238594

  13. Engraftment of human induced pluripotent stem cell-derived hepatocytes in immunocompetent mice via 3D co-aggregation and encapsulation

    PubMed Central

    Song, Wei; Lu, Yen-Chun; Frankel, Angela S.; An, Duo; Schwartz, Robert E.; Ma, Minglin

    2015-01-01

    Cellular therapies for liver diseases and in vitro models for drug testing both require functional human hepatocytes (Hum-H), which have unfortunately been limited due to the paucity of donor liver tissues. Human pluripotent stem cells (hPSCs) represent a promising and potentially unlimited cell source to derive Hum-H. However, the hepatic functions of these hPSC-derived cells to date are not fully comparable to adult Hum-H and are more similar to fetal ones. In addition, it has been challenging to obtain functional hepatic engraftment of these cells with prior studies having been done in immunocompromised animals. In this report, we demonstrated successful engraftment of human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iPS-H) in immunocompetent mice by pre-engineering 3D cell co-aggregates with stromal cells (SCs) followed by encapsulation in recently developed biocompatible hydrogel capsules. Notably, upon transplantation, human albumin and α1-antitrypsin (A1AT) in mouse sera secreted by encapsulated iPS-H/SCs aggregates reached a level comparable to the primary Hum-H/SCs control. Further immunohistochemistry of human albumin in retrieved cell aggregates confirmed the survival and function of iPS-H. This proof-of-concept study provides a simple yet robust approach to improve the engraftment of iPS-H, and may be applicable to many stem cell-based therapies. PMID:26592180

  14. Extraction and purification of active IgG from SSPE and MS brain.

    PubMed

    Owens, G P; Burgoon, M P; Devlin, M E; Gilden, D H

    1997-11-01

    Immunoglobulin (Ig) G was purified from soluble and membrane fractions of postmortem subacute sclerosing panencephalitis (SSPE) brain, multiple sclerosis (MS) brain plaque-periplaque white matter, and normal human brain (NHB) white matter. After homogenization in 0.32 M sucrose and removal of cell debris and nuclei by low-speed centrifugation, soluble and crude membrane fractions were separated by ultracentrifugation. After removal of sucrose by dialysis, IgG was isolated from the soluble fraction by protein A affinity chromatography. IgG was obtained from the membrane fraction by elution at low pH and purification from the eluate by protein A chromatography. Whereas very little IgG was in NHB white matter, significant levels of IgG were recovered from both SSPE and MS brain. Both immunocytochemical staining of measles virus-infected cells in tissue culture and protein immunoblotting of virus-infected cell lysates showed that the IgG from SSPE brain contained activity specific for measles virus protein. The abundance, purity and functional activity of IgG extracted from SSPE and MS brain indicate that IgG extracted from the brain of humans with an inflammatory disease of unknown etiology can be used to identify its corresponding antigen. PMID:9389401

  15. Weighted aggregation

    NASA Technical Reports Server (NTRS)

    Feiveson, A. H. (Principal Investigator)

    1979-01-01

    The use of a weighted aggregation technique to improve the precision of the overall LACIE estimate is considered. The manner in which a weighted aggregation technique is implemented given a set of weights is described. The problem of variance estimation is discussed and the question of how to obtain the weights in an operational environment is addressed.

  16. Passive immunization with allergen-specific IgG antibodies for treatment and prevention of allergy

    PubMed Central

    Flicker, Sabine; Linhart, Birgit; Wild, Carmen; Wiedermann, Ursula; Valenta, Rudolf

    2013-01-01

    IgE antibody-mediated allergies affect more than 25% of the population worldwide. To investigate therapeutic and preventive effects of passive immunization with allergen-specific IgG antibodies on allergy in mouse models we used clinically relevant pollen allergens. In a treatment model, mice were sensitized to the major birch pollen allergen Bet v 1 and to the major grass pollen allergens, Phl p 1 and Phl p 5 and then received passive immunization with rabbit IgG antibodies specific for the sensitizing or an unrelated allergen. In a prevention model, mice obtained passive immunization with allergen-specific rabbit IgG before sensitization. Kinetics of the levels of administered IgG antibodies, effects of administered allergen-specific IgG on allergen-specific IgE reactivity, the development of IgE and IgG responses and on immediate allergic reactions were studied by ELISA, rat basophil leukaemia degranulation assays and skin testing, respectively. Treated mice showed an approximately 80% reduction of allergen-specific IgE binding and basophil degranulation which was associated with the levels of administered allergen-specific IgG antibodies. Preventive administration of allergen-specific IgG antibodies suppressed the development of allergen-specific IgE and IgG1 antibody responses as well as allergen-induced basophil degranulation and skin reactivity. Our results show that passive immunization with allergen-specific IgG antibodies is effective for treatment and prevention of allergy to clinically important pollen allergens in a mouse model and thus may pave the road for the clinical application of allergen-specific antibodies in humans. PMID:23182706

  17. Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice

    PubMed Central

    Diepenbroek, Meike; Casadei, Nicolas; Esmer, Hakan; Saido, Takaomi C.; Takano, Jiro; Kahle, Philipp J.; Nixon, Ralph A; Rao, Mala V.; Melki, Ronald; Pieri, Laura; Helling, Stefan; Marcus, Katrin; Krueger, Rejko; Masliah, Eliezer; Riess, Olaf; Nuber, Silke

    2014-01-01

    Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (αSyn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of αSyn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here, we investigated the role of calpain cleavage of αSyn in vivo by generating two opposing mouse models. We crossed into human [A30P]αSyn transgenic (i) mice deficient for calpastatin, a calpain-specific inhibitor, thus enhancing calpain activity (SynCAST(−)) and (ii) mice overexpressing human calpastatin leading to reduced calpain activity (SynCAST(+)). As anticipated, a reduced calpain activity led to a decreased number of αSyn-positive aggregates, whereas loss of calpastatin led to increased truncation of αSyn in SynCAST(−). Furthermore, overexpression of calpastatin decreased astrogliosis and the calpain-dependent degradation of synaptic proteins, potentially ameliorating the observed neuropathology in [A30P]αSyn and SynCAST(+) mice. Overall, our data further support a crucial role of calpains, particularly of calpain 1, in the pathogenesis of PD and in disease-associated aggregation of αSyn, indicating a therapeutic potential of calpain inhibition in PD. PMID:24619358

  18. Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice.

    PubMed

    Diepenbroek, Meike; Casadei, Nicolas; Esmer, Hakan; Saido, Takaomi C; Takano, Jiro; Kahle, Philipp J; Nixon, Ralph A; Rao, Mala V; Melki, Ronald; Pieri, Laura; Helling, Stefan; Marcus, Katrin; Krueger, Rejko; Masliah, Eliezer; Riess, Olaf; Nuber, Silke

    2014-08-01

    Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (αSyn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of αSyn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here, we investigated the role of calpain cleavage of αSyn in vivo by generating two opposing mouse models. We crossed into human [A30P]αSyn transgenic (i) mice deficient for calpastatin, a calpain-specific inhibitor, thus enhancing calpain activity (SynCAST(-)) and (ii) mice overexpressing human calpastatin leading to reduced calpain activity (SynCAST(+)). As anticipated, a reduced calpain activity led to a decreased number of αSyn-positive aggregates, whereas loss of calpastatin led to increased truncation of αSyn in SynCAST(-). Furthermore, overexpression of calpastatin decreased astrogliosis and the calpain-dependent degradation of synaptic proteins, potentially ameliorating the observed neuropathology in [A30P]αSyn and SynCAST(+) mice. Overall, our data further support a crucial role of calpains, particularly of calpain 1, in the pathogenesis of PD and in disease-associated aggregation of αSyn, indicating a therapeutic potential of calpain inhibition in PD. PMID:24619358

  19. Adverse effects of IgG therapy.

    PubMed

    Berger, Melvin

    2013-01-01

    IgG is widely used for patients with immune deficiencies and in a broad range of autoimmune and inflammatory disorders. Up to 40% of intravenous infusions of IgG may be associated with adverse effects (AEs), which are mostly uncomfortable or unpleasant but often are not serious. The most common infusion-related AE is headache. More serious reactions, including true anaphylaxis and anaphylactoid reactions, occur less frequently. Most reactions are related to the rate of infusion and can be prevented or treated just by slowing the infusion rate. Medications such as nonsteroidal anti-inflammatory drugs, antihistamines, or corticosteroids also may be helpful in preventing or treating these common AEs. IgA deficiency with the potential of IgG or IgE antibodies against IgA increases the risk of some AEs but should not be viewed as a contraindication if IgG therapy is needed. Potentially serious AEs include renal dysfunction and/or failure, thromboembolic events, and acute hemolysis. These events usually are multifactorial, related to combinations of constituents in the IgG product as well as risk factors for the recipient. Awareness of these factors should allow minimization of the risks and consequences of these AEs. Subcutaneous IgG is absorbed more slowly into the circulation and has a lower incidence of AEs, but awareness and diligence are necessary whenever IgG is administered. PMID:24565701

  20. Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry

    PubMed Central

    Naik, Subhashchandra; Kumru, Ozan S; Cullom, Melissa; Telikepalli, Srivalli N; Lindboe, Elizabeth; Roop, Taylor L; Joshi, Sangeeta B; Amin, Divya; Gao, Phillip; Middaugh, C Russell; Volkin, David B; Fisher, Mark T

    2014-01-01

    The ability of a GroEL-based bio-layer interferometry (BLI) assay to detect structurally altered and/or aggregated species of pharmaceutically relevant proteins is demonstrated. Assay development included optimizing biotinylated-GroEL immobilization to streptavidin biosensors, combined with biophysical and activity measurements showing native and biotinylated GroEL are both stable and active. First, acidic fibroblast growth factor (FGF-1) was incubated under conditions known to promote (40°C) and inhibit (heparin addition) molten globule formation. Heat exposed (40°C) FGF-1 exhibited binding to GroEL-biosensors, which was significantly diminished in the presence of heparin. Second, a polyclonal human IgG solution containing 6–8% non-native dimer showed an increase in higher molecular weight aggregates upon heating by size exclusion chromatography (SEC). The poly IgG solution displayed binding to GroEL-biosensors initially with progressively increased binding upon heating. Enriched preparations of the IgG dimers or monomers showed significant binding to GroEL-biosensors. Finally, a thermally treated IgG1 monoclonal antibody (mAb) solution also demonstrated increased GroEL-biosensor binding, but with different kinetics. The bound complexes could be partially to fully dissociated after ATP addition (i.e., specific GroEL binding) depending on the protein, environmental stress, and the assay’s experimental conditions. Transmission electron microscopy (TEM) images of GroEL-mAb complexes, released from the biosensor, also confirmed interaction of bound complexes at the GroEL binding site with heat-stressed mAb. Results indicate that the GroEL-biosensor-BLI method can detect conformationally altered and/or early aggregation states of proteins, and may potentially be useful as a rapid, stability-indicating biosensor assay for monitoring the structural integrity and physical stability of therapeutic protein candidates. PMID:25043635

  1. Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry.

    PubMed

    Naik, Subhashchandra; Kumru, Ozan S; Cullom, Melissa; Telikepalli, Srivalli N; Lindboe, Elizabeth; Roop, Taylor L; Joshi, Sangeeta B; Amin, Divya; Gao, Phillip; Middaugh, C Russell; Volkin, David B; Fisher, Mark T

    2014-10-01

    The ability of a GroEL-based bio-layer interferometry (BLI) assay to detect structurally altered and/or aggregated species of pharmaceutically relevant proteins is demonstrated. Assay development included optimizing biotinylated-GroEL immobilization to streptavidin biosensors, combined with biophysical and activity measurements showing native and biotinylated GroEL are both stable and active. First, acidic fibroblast growth factor (FGF-1) was incubated under conditions known to promote (40°C) and inhibit (heparin addition) molten globule formation. Heat exposed (40°C) FGF-1 exhibited binding to GroEL-biosensors, which was significantly diminished in the presence of heparin. Second, a polyclonal human IgG solution containing 6-8% non-native dimer showed an increase in higher molecular weight aggregates upon heating by size exclusion chromatography (SEC). The poly IgG solution displayed binding to GroEL-biosensors initially with progressively increased binding upon heating. Enriched preparations of the IgG dimers or monomers showed significant binding to GroEL-biosensors. Finally, a thermally treated IgG1 monoclonal antibody (mAb) solution also demonstrated increased GroEL-biosensor binding, but with different kinetics. The bound complexes could be partially to fully dissociated after ATP addition (i.e., specific GroEL binding) depending on the protein, environmental stress, and the assay's experimental conditions. Transmission electron microscopy (TEM) images of GroEL-mAb complexes, released from the biosensor, also confirmed interaction of bound complexes at the GroEL binding site with heat-stressed mAb. Results indicate that the GroEL-biosensor-BLI method can detect conformationally altered and/or early aggregation states of proteins, and may potentially be useful as a rapid, stability-indicating biosensor assay for monitoring the structural integrity and physical stability of therapeutic protein candidates. PMID:25043635

  2. Immunogenicity of Recombinant Human Interferon Beta-1b in Immune-Tolerant Transgenic Mice Corresponds with the Biophysical Characteristics of Aggregates.

    PubMed

    Abdolvahab, Mohadeseh Haji; Fazeli, Ahmad; Halim, Andhyk; Sediq, Ahmad S; Fazeli, Mohammad Reza; Schellekens, Huub

    2016-04-01

    Determining to what extent biophysical characteristics of aggregates affect immunogenicity of therapeutic interferon beta-1b. Three recombinant human interferon beta-1b (rhIFNβ-1b) samples with different levels of aggregates generated by copper oxidation, thermal stress, or left untreated, as well as Avonex(®) drug substance and Betaferon(®) drug product, were injected intraperitoneally in nontransgenic and interferon beta transgenic FVB/N mice 5 times per week for 3 weeks. Antibodies against interferon beta were measured using enzyme-linked immunosorbent assay. UV and fluorescence spectroscopy, dynamic light scattering, size exclusion chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC), fluid imaging microscopy, and resonant mass measurement, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, were used to characterize and quantitate aggregates in the 3 rhIFNβ preparations, to correlate biophysical characteristics with immunogenicity. In immune-tolerant interferon beta transgenic FVB/N mice, Betaferon drug product showed the highest immunogenicity, while Avonex drug substance showed the lowest level of immunogenicity. Of the 3 forms of rhIFNβ-1b, copper-oxidized rhIFNβ-1b showed lower immunogenicity than thermally stressed rhIFNβ-1b, despite containing larger aggregates. Both copper-oxidized rhIFNβ-1b and thermally stressed rhIFNβ-1b exhibited changes in protein structure as shown using fluorescence spectroscopy and RP-HPLC. Nontransgenic, nonimmune-tolerant FVB/N mice generated high antibody titers against all interferon beta samples tested. The level of immunogenicity and the breaking of tolerance in FVB/N transgenic mice are not only related to the level of aggregation but also depend on the size and structure of the aggregates. PMID:26835734

  3. n-Dodecyl-β-D-maltoside inhibits aggregation of human interferon-β-1b and reduces its immunogenicity.

    PubMed

    Rifkin, Robert A; Maggio, Edward T; Dike, Sonny; Kerr, Douglas A; Levy, Michael

    2011-03-01

    The development of neutralizing antibodies to the protein drug interferon-β is a significant impediment to its use in the treatment of multiple sclerosis. Neutralizing antibodies to interferon-β arise from aggregation of the peptide during manufacturing and storage. We tested the ability of dodecylmaltoside, a nontoxic alkylsaccharide surfactant, to reduce aggregation of interferon-β in vitro and to reduce its immunogenicity in vivo. Interferon-β, in solution with and without dodecylmaltoside, was periodically evaluated for aggregation by light scatter for 1 month. Interferon-β, with and without dodecylmaltoside, was given 3 days/week for 1 month to mice; the sera of these mice were analyzed for anti-interferon-β antibodies by ELISA. Dodecylmaltoside reduces the aggregation of interferon-β in vitro and its immunogenicity in vivo. Our positive findings warrant additional tests of dodecylmaltoside as a therapeutic adjuvant in rodent models of multiple sclerosis. PMID:20532646

  4. Hsp72 (HSPA1A) Prevents Human Islet Amyloid Polypeptide Aggregation and Toxicity: A New Approach for Type 2 Diabetes Treatment

    PubMed Central

    Rosas, Paola C.; Nagaraja, Ganachari M.; Kaur, Punit; Panossian, Alexander; Wickman, Georg; Garcia, L. Rene; Al-Khamis, Fahd A.; Asea, Alexzander A. A.

    2016-01-01

    Type 2 diabetes is a growing public health concern and accounts for approximately 90% of all the cases of diabetes. Besides insulin resistance, type 2 diabetes is characterized by a deficit in β-cell mass as a result of misfolded human islet amyloid polypeptide (h-IAPP) which forms toxic aggregates that destroy pancreatic β-cells. Heat shock proteins (HSP) play an important role in combating the unwanted self-association of unfolded proteins. We hypothesized that Hsp72 (HSPA1A) prevents h-IAPP aggregation and toxicity. In this study, we demonstrated that thermal stress significantly up-regulates the intracellular expression of Hsp72, and prevents h-IAPP toxicity against pancreatic β-cells. Moreover, Hsp72 (HSPA1A) overexpression in pancreatic β-cells ameliorates h-IAPP toxicity. To test the hypothesis that Hsp72 (HSPA1A) prevents aggregation and fibril formation, we established a novel C. elegans model that expresses the highly amyloidogenic human pro-IAPP (h-proIAPP) that is implicated in amyloid formation and β-cell toxicity. We demonstrated that h-proIAPP expression in body-wall muscles, pharynx and neurons adversely affects C. elegans development. In addition, we demonstrated that h-proIAPP forms insoluble aggregates and that the co-expression of h-Hsp72 in our h-proIAPP C. elegans model, increases h-proIAPP solubility. Furthermore, treatment of transgenic h-proIAPP C. elegans with ADAPT-232, known to induce the expression and release of Hsp72 (HSPA1A), significantly improved the growth retardation phenotype of transgenic worms. Taken together, this study identifies Hsp72 (HSPA1A) as a potential treatment to prevent β-cell mass decline in type 2 diabetic patients and establishes for the first time a novel in vivo model that can be used to select compounds that attenuate h-proIAPP aggregation and toxicity. PMID:26960140

  5. Igg Subclasses Targeting the Flagella of Salmonella enterica Serovar Typhimurium Can Mediate Phagocytosis and Bacterial Killing

    PubMed Central

    Goh, Yun Shan; Armour, Kathryn L; Clark, Michael R; Grant, Andrew J; Mastroeni, Pietro

    2016-01-01

    Invasive non-typhoidal Salmonella are a common cause of invasive disease in immuno-compromised individuals and in children. Multi-drug resistance poses challenges to disease control, with a critical need for effective vaccines. Flagellin is an attractive vaccine candidate due to surface exposure and high epitope copy number, but its potential as a target for opsonophacytic antibodies is unclear. We examined the effect of targeting flagella with different classes of IgG on the interaction between Salmonella Typhimurium and a human phagocyte-like cell line, THP-1. We tagged the FliC flagellar protein with a foreign CD52 mimotope (TSSPSAD) and bacteria were opsonized with a panel of humanised CD52 antibodies with the same antigen-binding V-region, but different constant regions. We found that IgG binding to flagella increases bacterial phagocytosis and reduces viable intracellular bacterial numbers. Opsonisation with IgG3, followed by IgG1, IgG4, and IgG2, resulted in the highest level of bacterial uptake and in the highest reduction in the intracellular load of viable bacteria. Taken together, our data provide proof-of-principle evidence that targeting flagella with antibodies can increase the antibacterial function of host cells, with IgG3 being the most potent subclass. These data will assist the rational design of urgently needed, optimised vaccines against iNTS disease. PMID:27366588

  6. Horse chestnut extract contracts bovine vessels and affects human platelet aggregation through 5-HT(2A) receptors: an in vitro study.

    PubMed

    Felixsson, Emma; Persson, Ingrid A-L; Eriksson, Andreas C; Persson, Karin

    2010-09-01

    Extract from seeds and bark of horse chestnut (Aesculus hippocastanum L) is used as an herbal medicine against chronic venous insufficiency. The effect and mechanism of action on veins, arteries, and platelets are not fully understood. The aim of this study was to investigate the effects and mechanisms of action of horse chestnut on the contraction of bovine mesenteric veins and arteries, and human platelet aggregation. Contraction studies showed that horse chestnut extract dose-dependently contracted both veins and arteries, with the veins being the most sensitive. Contraction of both veins and arteries were significantly inhibited by the 5-HT(2A) receptor antagonist ketanserin. No effect on contraction was seen with the cyclooxygenase inhibitor indomethacin, the alpha(1) receptor antagonist prazosin or the angiotensin AT(1) receptor antagonist saralasin neither in veins nor arteries. ADP-induced human platelet aggregation was significantly reduced by horse chestnut. A further reduction was seen with the extract in the presence of ketanserin. In conclusion, horse chestnut contraction of both veins and arteries is, at least partly, mediated through 5-HT(2A) receptors. Human platelet aggregation is reduced by horse chestnut. The clinical importance of these findings concerning clinical use, possible adverse effects, and drug interactions remains to be investigated. PMID:20148408

  7. Construction aggregates

    USGS Publications Warehouse

    Langer, W.H.; Tepordei, V.V.; Bolen, W.P.

    2000-01-01

    Construction aggregates consist primarily of crushed stone and construction sand and gravel. Total estimated production of construction aggregates increased in 1999 by about 2% to 2.39 Gt (2.64 billion st) compared with 1998. This record production level continued an expansion that began in 1992. By commodities, crushed stone production increased 3.3%, while sand and gravel production increased by about 0.5%.

  8. Construction aggregates

    USGS Publications Warehouse

    Tepordei, V.V.

    1994-01-01

    Part of a special section on industrial minerals in 1993. The 1993 production of construction aggregates increased 6.3 percent over the 1992 figure, to reach 2.01 Gt. This represents the highest estimated annual production of combined crushed stone and construction sand and gravel ever recorded in the U.S. The outlook for construction aggregates and the issues facing the industry are discussed.

  9. Prolonged retention after aggregation into secretory granules of human R183H-growth hormone (GH), a mutant that causes autosomal dominant GH deficiency type II.

    PubMed

    Zhu, Yong Lian; Conway-Campbell, Becky; Waters, Michael J; Dannies, Priscilla S

    2002-11-01

    Human R183H-GH causes autosomal dominant GH deficiency type II. Because we show here that the mutant hormone is fully bioactive, we have sought to locate an impairment in its progress through the secretory pathway as assessed by pulse chase experiments. Newly synthesized wild-type and R183H-GH were stable when expressed transiently in AtT20 cells, and both formed equivalent amounts of Lubrol-insoluble aggregates within 40 min after synthesis. There was no evidence for intermolecular disulfide bond formation in aggregates of wild-type hormone or the R183H mutant. Both wild-type and R183H-GH were packaged into secretory granules, assessed by the ability of 1 mM BaCl2 to stimulate release and by immunocytochemistry. The mutant differed from wild-type hormone in its retention in the cells after packaging into secretory granules; 50% more R183H-GH than wild-type aggregates were retained in AtT20 cells 120 min after synthesis, and stimulated release of R183H-GH or a mixture of R183H-GH and wild-type that had been retained in the cell was reduced. The longer retention of R183H-GH aggregates indicates that a single point mutation in a protein contained in secretory granules affects the rate of secretory granule release. PMID:12399418

  10. Enhanced Efficacy of Human Brain-Derived Neural Stem Cells by Transplantation of Cell Aggregates in a Rat Model of Parkinson's Disease

    PubMed Central

    Shin, Eun Sil; Hwang, Onyou; Hwang, Yu-Shik; Suh, Jun-Kyo Francis; Chun, Young Il

    2014-01-01

    Objective Neural tissue transplantation has been a promising strategy for the treatment of Parkinson's disease (PD). However, transplantation has the disadvantages of low-cell survival and/or development of dyskinesia. Transplantation of cell aggregates has the potential to overcome these problems, because the cells can extend their axons into the host brain and establish synaptic connections with host neurons. In this present study, aggregates of human brain-derived neural stem cells (HB-NSC) were transplanted into a PD animal model and compared to previous report on transplantation of single-cell suspensions. Methods Rats received an injection of 6-OHDA into the right medial forebrain bundle to generate the PD model and followed by injections of PBS only, or HB-NSC aggregates in PBS into the ipsilateral striatum. Behavioral tests, multitracer (2-deoxy-2-[18F]-fluoro-D-glucose ([18F]-FDG) and [18F]-N-(3-fluoropropyl)-2-carbomethoxy-3-(4-iodophenyl)nortropane ([18F]-FP-CIT) microPET scans, as well as immunohistochemical (IHC) and immunofluorescent (IF) staining were conducted to evaluate the results. Results The stepping test showed significant improvement of contralateral forelimb control in the HB-NSC group from 6-10 weeks compared to the control group (p<0.05). [18F]-FP-CIT microPET at 10 weeks posttransplantation demonstrated a significant increase in uptake in the HB-NSC group compared to pretransplantation (p<0.05). In IHC and IF staining, tyrosine hydroxylase and human β2 microglobulin (a human cell marker) positive cells were visualized at the transplant site. Conclusion These results suggest that the HB-NSC aggregates can survive in the striatum and exert therapeutic effects in a PD model by secreting dopamine. PMID:25535514

  11. Selective aggregation of PAMAM dendrimer nanocarriers and PAMAM/ZnPc nanodrugs on human atheromatous carotid tissues: a photodynamic therapy for atherosclerosis.

    PubMed

    Spyropoulos-Antonakakis, Nikolaos; Sarantopoulou, Evangelia; Trohopoulos, Panagiotis N; Stefi, Aikaterina L; Kollia, Zoe; Gavriil, Vassilios E; Bourkoula, Athanasia; Petrou, Panagiota S; Kakabakos, Sotirios; Semashko, Vadim V; Nizamutdinov, Alexey S; Cefalas, Alkiviadis-Constantinos

    2015-01-01

    Photodynamic therapy (PDT) involves the action of photons on photosensitive molecules, where atomic oxygen or OH(-) molecular species are locally released on pathogenic human cells, which are mainly carcinogenic, thus causing cell necrosis. The efficacy of PDT depends on the local nanothermodynamic conditions near the cell/nanodrug system that control both the level of intracellular translocation of nanoparticles in the pathogenic cell and their agglomeration on the cell membrane. Dendrimers are considered one of the most effective and promising drug carriers because of their relatively low toxicity and negligible activation of complementary reactions. Polyamidoamine (PAMAM) dendrite delivery of PDT agents has been investigated in the last few years for tumour selectivity, retention, pharmacokinetics and water solubility. Nevertheless, their use as drug carriers of photosensitizing molecules in PDT for cardiovascular disease, targeting the selective necrosis of macrophage cells responsible for atheromatous plaque growth, has never been investigated. Furthermore, the level of aggregation, translocation and nanodrug delivery efficacy of PAMAM dendrimers or PAMAM/zinc phthalocyanine (ZnPc) conjugates on human atheromatous tissue and endothelial cells is still unknown. In this work, the aggregation of PAMAM zero generation dendrimers (G0) acting as drug delivery carriers, as well as conjugated G0 PAMAM dendrimers with a ZnPc photosensitizer, to symptomatic and asymptomatic human carotid tissues was investigated by using atomic force microscopy (AFM). For the evaluation of the texture characteristics of the AFM images, statistical surface morphological and fractal analytical methodologies and Minkowski functionals were used. All statistical quantities showed that the deposition of nanodrug carriers on healthy tissue has an inverse impact when comparing to the deposition on atheromatous tissue with different aggregation features between G0 and G0/ZnPc nanoparticles and

  12. Strong interactions with polyethylenimine-coated human serum albumin nanoparticles (PEI-HSA NPs) alter α-synuclein conformation and aggregation kinetics

    NASA Astrophysics Data System (ADS)

    Mohammad-Beigi, Hossein; Shojaosadati, Seyed Abbas; Marvian, Amir Tayaranian; Pedersen, Jannik Nedergaard; Klausen, Lasse Hyldgaard; Christiansen, Gunna; Pedersen, Jan Skov; Dong, Mingdong; Morshedi, Dina; Otzen, Daniel E.

    2015-11-01

    The interaction between nanoparticles (NPs) and the small intrinsically disordered protein α-synuclein (αSN), whose aggregation is central in the development of Parkinson's disease, is of great relevance in biomedical applications of NPs as drug carriers. Here we showed using a combination of different techniques that αSN interacts strongly with positively charged polyethylenimine-coated human serum albumin (PEI-HSA) NPs, leading to a significant alteration in the αSN secondary structure. In contrast, the weak interactions of αSN with HSA NPs allowed αSN to remain unfolded. These different levels of interactions had different effects on αSN aggregation. While the weakly interacting HSA NPs did not alter the aggregation kinetic parameters of αSN, the rate of primary nucleation increased in the presence of PEI-HSA NPs. The aggregation rate changed in a PEI-HSA NP-concentration dependent and size independent manner and led to fibrils which were covered with small aggregates. Furthermore, PEI-HSA NPs reduced the level of membrane-perturbing oligomers and reduced oligomer toxicity in cell assays, highlighting a potential role for NPs in reducing αSN pathogenicity in vivo. Collectively, our results highlight the fact that a simple modification of NPs can strongly modulate interactions with target proteins, which may have important and positive implications in NP safety.The interaction between nanoparticles (NPs) and the small intrinsically disordered protein α-synuclein (αSN), whose aggregation is central in the development of Parkinson's disease, is of great relevance in biomedical applications of NPs as drug carriers. Here we showed using a combination of different techniques that αSN interacts strongly with positively charged polyethylenimine-coated human serum albumin (PEI-HSA) NPs, leading to a significant alteration in the αSN secondary structure. In contrast, the weak interactions of αSN with HSA NPs allowed αSN to remain unfolded. These different

  13. Pathologies Associated with Serum IgG4 Elevation

    PubMed Central

    Ebbo, Mikael; Grados, Aurélie; Bernit, Emmanuelle; Vély, Frederic; Boucraut, José; Harlé, Jean-Robert; Daniel, Laurent; Schleinitz, Nicolas

    2012-01-01

    Statement of Purpose. IgG4-related disease (IgG4-RD) is usually associated to an increase of serum IgG4 levels. However other conditions have also been associated to high serum IgG4 levels. Methods. All IgG subclasses analyses performed in our hospital over a one-year period were analyzed. When IgG4 level were over 1.35 g/L, the patient's clinical observation was analyzed and both final diagnosis and reason leading to IgG subclasses analysis were recorded. Only polyclonal increases of IgG4 were considered. Summary of the Results. On 646 IgG subclass analysis performed, 59 patients had serum IgG4 over 1.35 g/L. The final diagnosis associated to serum IgG4 increase was very variable. Most patients (25%) presented with repeated infections, 13.5% with autoimmune diseases, and 10% with IgG4-RD. Other patients presented with cancer, primary immune deficiencies, idiopathic interstitial lung disease, cystic fibrosis, histiocytosis, or systemic vasculitis and 13.5% presented with various pathologies or no diagnosis. Mean IgG4 levels and IgG4/IgG ratio were higher in IgG4-RD than in other pathologies associated to elevated IgG4 levels. Conclusions. Our study confirms that elevation of serum IgG4 is not specific to IgG4-RD. Before retaining IgG4-RD diagnosis in cases of serum IgG4 above 1.35 g/L, several other pathological conditions should be excluded. PMID:22966232

  14. Combined effects of mineral trioxide aggregate and human placental extract on rat pulp tissue and growth, differentiation and angiogenesis in human dental pulp cells.

    PubMed

    Chang, Seok-Woo; Kim, Ji-Youn; Kim, Mi-Joo; Kim, Ga-Hyun; Yi, Jin-Kyu; Lee, Deok-Won; Kum, Kee-Yeon; Kim, Eun-Cheol

    2016-05-01

    Objective The aim of this study was to evaluate the combined effects of mineral trioxide aggregate (MTA) and human placental extract (HPE) on cell growth, differentiation and in vitro angiogenesis of human dental pulp cells (HDPCs) and to identify underlying signal transduction mechanisms. In vivo dental pulp responses in rats for a pulp-capping agent were examined. Materials and methods MTS assay. ALP activity test, alizarin red S staining and RT-PCR for marker genes were carried out to evaluate cell growth and differentiation. HUVEC migration, mRNA expression and capillary tube formation were measured to evaluate angiogenesis. Signal transduction was analysed using Western blotting and confocal microscopy. The pulps of rat maxillary first molars were exposed and capped with either MTA or MTA plus HPE. Histologic observation and scoring were performed. Results Compared to treatment of HDPCs with either HPE or MTA alone, the combination of HPE and MTA increased cell growth, ALP activity, mineralized nodules and expression of marker mRNAs. Combination HPE and MTA increased migration, capillary tube formation and angiogenic gene expression compared with MTA alone. Activation of Akt, mammalian target of rapamycin (mTOR), p38, JNK and ERK MAPK, Akt, and NF-κB were significantly increased by combining HPE and MTA compared with MTA alone. Pulp capping with MTA plus HPE in rats showed superior dentin bridge formation, odontoblastic layers and dentinal tubules and lower inflammatory cell response, compared to the MTA alone group. Conclusions This study demonstrates for the first time that the use of MTA with HPE promotes cell growth, differentiation and angiogenesis in HDPCs, which were associated with mTOR, MAPK and NF-κB pathways. Direct pulp capping with HPE plus MTA showed superior results when compared with MTA alone. Thus, the combination of MTA and HPE may be useful for regenerative endodontics. PMID:26807656

  15. Inhibition of Human Transthyretin Aggregation by Non-Steroidal Anti-Inflammatory Compounds: A Structural and Thermodynamic Analysis

    PubMed Central

    Sant’Anna, Ricardo O.; Braga, Carolina A.; Polikarpov, Igor; Ventura, Salvador; Lima, Luis Mauricio T. R.; Foguel, Debora

    2013-01-01

    Transthyretin (TTR) is a homotetrameric protein that circulates in plasma and cerebral spinal fluid (CSF) whose aggregation into amyloid fibrils has been associated with at least two different amyloid diseases: senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP). In SSA aggregates are composed of WT-TTR, while in FAP more than 100 already-described variants have been found in deposits. Until now, TTR-related diseases have been untreatable, although a new drug called Tafamidis has been approved only in Europe to specifically treat V30M patients. Thus, new strategies are still necessary to treat FAP caused by other variants of TTR. TTR has two channels in the dimer interface that bind to the hormone thyroxin and that have been used to accommodate anti-amyloidogenic compounds. These compounds stabilize the tetramers, rendering TTR less amyloidogenic. Here, we investigated the effects of three non-steroidal anti-inflammatory compounds—sulindac (SUL), indomethacin (IND) and lumiracoxib (LUM)—as tetramer stabilizers and aggregation inhibitors. WT-TTR and the very aggressive TTR variant L55P were used as models. These compounds were able to stabilize TTR against high hydrostatic pressure (HHP), increasing the ΔGf by several kcal. They were also effective in inhibiting WT-TTR and L55P acid- or HHP-induced aggregation; in particular, LUM and IND were very effective, inhibiting almost 100% of the aggregation of both proteins under certain conditions. The species formed when aggregation was performed in the presence of these compounds were much less toxic to cells in culture. The crystal structures of WT-TTR bound to the three compounds were solved at high resolution, allowing the identification of the relevant protein:drug interactions. We discuss here the ligand-binding features of LUM, IND and SUL to TTR, emphasizing the critical interactions that render the protein more stable and less amyloidogenic. PMID:23466880

  16. Investigating the feasibility of scale up and automation of human induced pluripotent stem cells cultured in aggregates in feeder free conditions☆

    PubMed Central

    Soares, Filipa A.C.; Chandra, Amit; Thomas, Robert J.; Pedersen, Roger A.; Vallier, Ludovic; Williams, David J.

    2014-01-01

    The transfer of a laboratory process into a manufacturing facility is one of the most critical steps required for the large scale production of cell-based therapy products. This study describes the first published protocol for scalable automated expansion of human induced pluripotent stem cell lines growing in aggregates in feeder-free and chemically defined medium. Cells were successfully transferred between different sites representative of research and manufacturing settings; and passaged manually and using the CompacT SelecT automation platform. Modified protocols were developed for the automated system and the management of cells aggregates (clumps) was identified as the critical step. Cellular morphology, pluripotency gene expression and differentiation into the three germ layers have been used compare the outcomes of manual and automated processes. PMID:24440272

  17. Pancreatic Islet-Like Three-Dimensional Aggregates Derived From Human Embryonic Stem Cells Ameliorate Hyperglycemia in Streptozotocin-Induced Diabetic Mice.

    PubMed

    Shim, Joong-Hyun; Kim, JongHyun; Han, Jiyou; An, Su Yeon; Jang, Yu Jin; Son, Jeongsang; Woo, Dong-Hun; Kim, Suel-Kee; Kim, Jong-Hoon

    2015-01-01

    We previously reported the in vitro differentiation of human embryonic stem cells (hESCs) into pancreatic endoderm. Here we demonstrate that islet-like three-dimensional (3D) aggregates can be derived from the pancreatic endoderm by optimizing our previous protocol. Sequential treatment with Wnt3a, activin A, and noggin induced a transient upregulation of T and MixL1, followed by increased expression of endodermal genes, including FOXA2, SOX17, and CXCR4. Subsequent treatment with retinoic acid highly upregulated PDX1 expression. We also show that inhibition of sonic hedgehog signaling by bFGF/activin βB and cotreatment with VEGF and FGF7 produced many 3D cellular clusters that express both SOX17 and PDX1. We found for the first time that proteoglycans and vimentin(+) mesenchymal cells were mainly localized in hESC-derived PDX1(+) clusters. Importantly, treatment with chlorate, an inhibitor of proteoglycan sulfation, together with inhibition of Notch signaling significantly increased the expression of Neurog3 and NeuroD1, promoting a transition from PDX1(+) progenitor cells toward mature pancreatic endocrine cells. Purified dithizone(+) 3D aggregates generated by our refined protocol produced pancreatic hormones and released insulin in response to both glucose and pharmacological drugs in vitro. Furthermore, the islet-like 3D aggregates decreased blood glucose levels and continued to exhibit pancreatic features after transplantation into diabetic mice. Generation of islet-like 3D cell aggregates from human pluripotent stem cells may overcome the shortage of cadaveric donor islets for future cases of clinical islet transplantation. PMID:25397866

  18. IgG4-Related Kidney Disease and IgG4-Related Retroperitoneal Fibrosis.

    PubMed

    Kawano, Mitsuhiro; Yamada, Kazunori

    2016-08-01

    Immunoglobulin G4-related kidney disease (IgG4-RKD) is the collective name encompassing renal parenchymal and renal pelvic lesions. The hallmark of renal parenchymal lesions of IgG4-related disease is plasma cell-rich tubulointerstitial nephritis with numerous IgG4-positive plasma cells and characteristic fibrosis. In addition, glomerular lesions are sometimes present, with membranous glomerulonephritis being the most common. Although IgG4-RKD shows good responsiveness to corticosteroid therapy, follow-up imaging studies have revealed that partial cortical scars persist when the start of therapy is delayed. In this review, the authors provide an overview of the latest knowledge of IgG4-RKD, focusing in particular on its pathological and imaging characteristic features. PMID:27466797

  19. The Arg-Gly-Asp-containing peptide, rhodostomin, inhibits in vitro cell adhesion to extracellular matrices and platelet aggregation caused by saos-2 human osteosarcoma cells.

    PubMed Central

    Chiang, H. S.; Yang, R. S.; Huang, T. F.

    1995-01-01

    Saos-2 cells, derived from a primary human osteosarcoma, caused dose-dependent platelet aggregation in heparinised human platelet-rich plasma. Saos-2 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin but unaffected by apyrase. The cell suspension shortened the plasma recalcification times of normal, factor VIII-deficient and factor IX-deficient human plasmas in a dose-dependent manner. However, the cell suspension did not affect the recalcification time of factor VII-deficient plasma. Moreover, a monoclonal antibody (MAb) against human tissue factor completely abolished TCIPA. Flow cytometric analysis using anti-integrin MAbs as the primary binding ligands demonstrated that the integrin receptors alpha v beta 3, alpha 5 beta 1 and alpha 6 beta 1 were present of Saos-2 cells, which might mediate tumour cell adhesion to extracellular matrix. Rhodostomin, an Arg-Gly-Asp (RGD)-containing snake venom peptide which antagonises the binding of fibrinogen to platelet membrane glycoprotein IIb/IIIa, prevented Saos-2 TCIPA as well as tumour cell adhesion to vitronectin, fibronectin and collagen type I. Likewise, the synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) showed a similar effect. On a molar basis, rhodostomin was about 18,000 and 1000 times, respectively, more potent than GRGDS in inhibiting TCIPA and tumour cell adhesion. PMID:7841039

  20. Staphylococci-induced Human Platelet Injury Mediated by Protein A and Immunoglobulin G Fc Fragment Receptor

    PubMed Central

    Hawiger, Jack; Steckley, Sylvia; Hammond, Dianne; Cheng, Charles; Timmons, Sheila; Glick, Alan D.; Des Prez, Roger M.

    1979-01-01

    Bloodstream infections with staphylococci are accompanied by thromboembolic complications. We have studied the mechanism of the interaction of staphylococci with human blood platelets. Staphylococci that possess protein A, a bacterial receptor for the Fc fragment of immunoglobulin G (IgG), caused aggregation of human platelets in whole plasma accompanied by release of [3H]serotonin. These reactions were time and concentration dependent, requiring two or more staphylococci per platelet to give maximal response within 5 min. The interaction between staphylococci and platelets required the presence of cell wall-bound protein A and of IgG with an intact Fc fragment. It did not require an intact complement system. Cell wall-bound protein A (solid phase) was capable of aggregating human platelets in whole plasma. In contrast, free, solubilized protein A (fluid phase) did not cause measurable aggregation, and release of [3H]serotonin was reduced. An excess of free, solubilized protein A blocked aggregation of human platelets induced by staphylococci in whole plasma. The role of the Fc fragment of IgG in the staphylococci-human platelet interaction was demonstrated by an experiment in which free, isolated Fc fragment blocked aggregation of platelets in whole plasma induced by staphylococci. Furthermore, binding of 125I-protein A to human platelets was demonstrated in the presence of complete IgG with intact Fc fragment but not in the presence of the F(ab)2 fragment. Binding of the protein A-IgG complex to the human platelet Fc receptor was paralleled by the release of [3H]serotonin. These results represent a novel example of the interaction of two phylogenetically different Fc receptors, one on prokaryotic staphylococci and the other on human platelets. Their common ligand, IgG, is amplified by one Fc receptor (protein A) to react with another Fc receptor present on human platelets, which results in membrane-mediated aggregation and release reaction occurring in whole

  1. High concentrations of therapeutic IgG1 antibodies are needed to compensate for inhibition of antibody-dependent cellular cytotoxicity by excess endogenous immunoglobulin G.

    PubMed

    Preithner, Susanne; Elm, Stefanie; Lippold, Sandra; Locher, Mathias; Wolf, Andreas; da Silva, Antonio J; Baeuerle, Patrick A; Prang, Nadja S

    2006-03-01

    A common feature of human IgG1 antibodies used for cancer treatment is that their anti-tumour efficacy requires high serum trough levels and continued therapy for several months. Treatment cycles, thereby, consume several grams of IgG1 translating into significant drug needs and costs. The basis for the low in vivo efficacy, which is in contrast to high in vitro antibody-dependent cellular cytotoxicity (ADCC), is not well understood. Here, we have explored factors contributing to this discrepancy using adecatumumab (MT201), a fully human monoclonal IgG1 against epithelial cell adhesion molecule (Ep-CAM) and trastuzumab (Herceptin), a humanized IgG1 with specificity for the human epithelial growth factor receptor type 2 (HER-2) antigen. We found that physiological levels of human sera strongly inhibited ADCC of both IgG1 antibodies. Effects showed some dependence on the density of Ep-CAM and HER-2 targets, the tumour cell line tested and on effector cell and serum donors. Removal of IgG by affinity chromatography abolished the inhibitory effect of a serum pool. Inhibition of ADCC was fully restored by adding back the IgG fraction or by an equal amount of IgG from a commercial source. We further demonstrate that CD56-positive lymphocytes within human PBMC contributed >90% to ADCC and that normal serum levels of IgG effectively competed for in vitro binding of an IgG1 antibody to low-affinity Fcgamma receptor type III (CD16), as is present on natural killer (NK) cells. Competition of serum IgG for binding of therapeutic IgG1 to NK cell may be one important reason why high antibody doses are required in the clinic for treatment of cancer by an ADCC-based mechanism. PMID:16102830

  2. Histopathology of IgG4-Related Autoimmune Hepatitis and IgG4-Related Hepatopathy in IgG4-Related Disease.

    PubMed

    Nakanuma, Yasuni; Ishizu, Yoji; Zen, Yoh; Harada, Kenichi; Umemura, Takeji

    2016-08-01

    Immunoglobulin G4-related disease (IgG4-RD) is a systemic disease involving many organs; it includes IgG4-related sclerosing cholangitis and inflammatory pseudotumor in the hepatobiliary system. Two types of hepatic parenchymal involvement have been reported in IgG4-RD: IgG4-related autoimmune hepatitis (AIH) and IgG4-hepatopathy. Moreover, only three cases of IgG4-related AIH have been reported. Immunoglobulin G4-related AIH is clinicopathologically similar to AIH, except for an elevated serum IgG4 level and heavy infiltration of IgG4-positive plasma cells in the liver tissue. Interestingly, IgG4-related AIH can be complicated by well-known IgG4-RD(s). Immunoglobulin G4-hepatopathy, which includes various histopathological lesions encountered in the liver of patients with type I autoimmune pancreatitis, is classified into five histological categories: portal inflammation, large bile duct damage, portal sclerosis, lobular hepatitis, and cholestasis. Immunoglobulin G4-hepatopathy is currently a collective term covering hepatic lesions primarily or secondarily related to IgG4-related sclerosing cholangitis and type 1 autoimmune pancreatitis. In conclusion, the liver is not immune to IgG4-RD, and at least two types of hepatic involvement in IgG4-RD have been reported: IgG4-related AIH and IgG4-hepatopathy. Additional studies are required to clarify their precise clinical significance with respect to IgG4-RD and inherent liver diseases. PMID:27466793

  3. Metal-dependent hydrolysis of myelin basic protein by IgGs from the sera of patients with multiple sclerosis.

    PubMed

    Polosukhina, Dar'ya I; Kanyshkova, Tat'yana G; Doronin, Boris M; Tyshkevich, Olga B; Buneva, Valentina N; Boiko, Alexey N; Gusev, Evgenii I; Nevinsky, Georgy A; Favorova, Olga O

    2006-02-28

    Homogeneous IgG fractions were obtained by chromatography of the sera of patients with multiple sclerosis (MS) on Protein G-Sepharose under conditions that remove non-specifically bound proteins. These IgGs contained several chelated metals, the relative amount of which decreases in the order: Fe>or=Ca>Cu>or=Zn>or=Mg>or=Mn>or=Pb>or=Co>or=Ni. In contrast to homogeneous IgGs of healthy individuals, Abs of MS patients effectively hydrolyzed human myelin basic protein (MBP). A minor metal-dependent fraction was obtained by chromatography of highly purified IgGs from MS patient on Chelex-100. This IgG fraction did not hydrolyze human MBP in the absence of Me(2+) ions but was activated after addition of Me(2+) ions: Mg(2+)>Mn(2+)>Cu(2+)>Ca(2+). Proteolytic activities of IgGs from other MS patients were also activated by other metal ions (Ni(2+), Fe(2+), Co(2+), Zn(2+), Pb(2+), and Co(2+)) and especially Ni(2+). Ni(2+)-activated IgGs were separated into distinct MBP-hydrolyzing fractions by chromatography on HiTraptrade mark Chelating Sepharose charged with Ni(2+). Detection of Mg(2+)-dependent proteolytic activity in the SDS-PAGE area corresponding only to IgG provided direct evidence that IgG from sera of MS patients possesses metal-dependent human MBP-hydrolyzing activity. Observed properties of MS abzymes distinguish them from other known mammalian metalloproteases and demonstrate their pronounced catalytic diversity. Metal-dependent IgGs from MS patients represent the first example of abzymes with metal-dependent proteolytic activity. PMID:16310860

  4. IgG Subclass Staining in Routine Renal Biopsy Material.

    PubMed

    Hemminger, Jessica; Nadasdy, Gyongyi; Satoskar, Anjali; Brodsky, Sergey V; Nadasdy, Tibor

    2016-05-01

    Immunofluorescence staining plays a vital role in nephropathology, but the panel of antibodies used has not changed for decades. Further classification of immunoglobulin (Ig)G-containing immune-type deposits with IgG subclass staining (IgG1, IgG2, IgG3, and IgG4) has been shown to be of diagnostic utility in glomerular diseases, but their value in the evaluation of renal biopsies has not been addressed systematically in large renal biopsy material. Between January 2007 and June 2014, using direct immunofluorescence, we stained every renal biopsy for the IgG subclasses if there was moderate to prominent glomerular IgG staining and/or IgG-predominant or IgG-codominant glomerular staining. The total number of biopsies stained was 1084, which included 367 cases of membranous glomerulonephritis, 307 cases of lupus nephritis, 74 cases of fibrillary glomerulonephritis, 53 cases of proliferative glomerulonephritis with monoclonal IgG deposits, and 25 cases of antiglomerular basement membrane disease, among others. We found that monoclonality of IgG deposits cannot always be reliably determined on the basis of kappa and lambda light chain staining alone, particularly if concomitant (frequently nonspecific) IgM staining is present. In IgG heavy and heavy and light chain deposition disease (3 cases), subclass staining is very helpful, and in proliferative glomerulonephritis with monoclonal IgG deposits subclass staining is necessary. IgG subclass staining is useful in differentiating primary from secondary membranous glomerulonephritis. In proliferative glomerulonephritis with polyclonal IgG deposition, IgG1 dominance/codominance with concomitant IgG3 and IgG2 but weak or absent IgG4 staining favors an underlying autoimmune disease. IgG subclass staining is a very useful diagnostic method in a selected cohort of renal biopsies, particularly in biopsies with glomerulonephritis with monoclonal IgG deposits. PMID:26848798

  5. IgG Subclasses and Allotypes: From Structure to Effector Functions

    PubMed Central

    Vidarsson, Gestur; Dekkers, Gillian; Rispens, Theo

    2014-01-01

    Of the five immunoglobulin isotypes, immunoglobulin G (IgG) is most abundant in human serum. The four subclasses, IgG1, IgG2, IgG3, and IgG4, which are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. These regions are involved in binding to both IgG-Fc receptors (FcγR) and C1q. As a result, the different subclasses have different effector functions, both in terms of triggering FcγR-expressing cells, resulting in phagocytosis or antibody-dependent cell-mediated cytotoxicity, and activating complement. The Fc-regions also contain a binding epitope for the neonatal Fc receptor (FcRn), responsible for the extended half-life, placental transport, and bidirectional transport of IgG to mucosal surfaces. However, FcRn is also expressed in myeloid cells, where it participates in both phagocytosis and antigen presentation together with classical FcγR and complement. How these properties, IgG-polymorphisms and post-translational modification of the antibodies in the form of glycosylation, affect IgG-function will be the focus of the current review. PMID:25368619

  6. Serum IgG antibodies to C1q in hypocomplementemic urticarial vasculitis syndrome.

    PubMed

    Wisnieski, J J; Naff, G B

    1989-09-01

    Urticaria, angioedema, and arthritis are cardinal features of hypocomplementemic urticarial vasculitis syndrome (HUVS). Considered to be an immune complex-mediated disorder, HUVS has been differentiated from systemic lupus erythematosus (SLE), based on its clinical manifestations and the C1q precipitin (C1q-p) reaction, which is manifested as gel precipitation of C1q by a small percentage of HUVS IgG molecules. This phenomenon has been attributed to an Fc region abnormality, and the responsible IgG molecules are said to possess C1q-p activity. We purified IgG from 4 HUVS patients and confirmed that HUVS IgG contains C1q binding activity. F(ab')2 fragments from these patients also bound to C1q, as measured by 2 different C1q binding methods at physiologic ionic strength; HUVS IgG Fc fragments did not bind to C1q. Preincubation of HUVS F(ab')2 fragments with antibody to human F(ab')2 prevented subsequent binding to C1q. We conclude that IgG antibodies to C1q are present in HUVS serum, and it is likely that these antibodies are C1q-p. Because the clinical manifestations of HUVS and the presence of anti-C1q antibodies have been described in patients with SLE, our findings support the concept that HUVS is an autoimmune syndrome related to SLE. PMID:2528353

  7. Autoimmunoreactive IgGs Against Cardiac Lipid Raft-Associated Proteins in Patients with POTS

    PubMed Central

    Wang, Xiao-Li; Ling, Tian-You; Charlesworth, M. Cristine; Figueroa, Juan J.; Low, Phillip; Shen, Win-Kuang; Lee, Hon-Chi

    2013-01-01

    Lipid rafts are specialized plasma membrane microdomains that serve as platforms for integrating cellular signal transductions. We have recently reported that autoantibodies against cardiac membrane proteins are present in patients with postural orthostatic tachycardia syndrome (POTS). In this study, we examined the presence of autoimmunoreactive IgGs against lipid raft proteins in these patients. IgGs were purified from the sera of 10 patients and 7 normal controls. Cardiac lipid raft preparations were isolated from normal human heart tissue. The lipid raft-associated proteins were resolved by 2DE and immunoblotted against IgGs from each subject. Protein spots that reacted specifically with patient IgGs were identified by nanoLC-MS/MS. Thirty-four such protein spots, and 72 unique proteins were identified. The targets of autoimmunoreactive IgGs include proteins associated with caveolae structure, adrenergic signaling, calcium signaling, cytostructures, chaperone and energy metabolism. Multiple pathways were involved including those that regulate caveolae-mediated signaling, oxidative phosphorylation, fatty acid metabolism, protein ubiquitination, and cardiac β-adrenergic signaling. Our results suggest that cardiac lipid raft-associated proteins are targets of autoimmunoreactive IgGs from patients with POTS. Autoimmunity may play a role in the pathogenesis of POTS. PMID:23562385

  8. Interactions between integrin αIIbβ3 and the serotonin transporter regulate serotonin transport and platelet aggregation in mice and humans

    PubMed Central

    Carneiro, Ana Marin D.; Cook, Edwin H.; Murphy, Dennis L.; Blakely, Randy D.

    2008-01-01

    The essential contribution of the antidepressant-sensitive serotonin (5-HT) transporter SERT (which is encoded by the SLC6A4 gene) to platelet 5-HT stores suggests an important role of this transporter in platelet function. Here, using SERT-deficient mice, we have established a role for constitutive SERT expression in efficient ADP- and thrombin-triggered platelet aggregation. Additionally, using pharmacological blockers of SERT and the vesicular monoamine transporter (VMAT), we have identified a role for ongoing 5-HT release and SERT activity in efficient human platelet aggregation. We have also demonstrated that fibrinogen, an activator of integrin αIIbβ3, enhances SERT activity in human platelets and that integrin αIIbβ3 interacts directly with the C terminus of SERT. Consistent with these findings, knockout mice lacking integrin β3 displayed diminished platelet SERT activity. Conversely, HEK293 cells engineered to express human SERT and an activated form of integrin β3 exhibited enhanced SERT function that coincided with elevated SERT surface expression. Our results support an unsuspected role of αIIbβ3/SERT associations as well as αIIbβ3 activation in control of SERT activity in vivo that may have broad implications for hyperserotonemia, cardiovascular disorders, and autism. PMID:18317590

  9. The non-protein amino acid BMAA is misincorporated into human proteins in place of L-serine causing protein misfolding and aggregation.

    PubMed

    Dunlop, Rachael Anne; Cox, Paul Alan; Banack, Sandra Anne; Rodgers, Kenneth John

    2013-01-01

    Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNA(Ala) with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, β-N-methylamino-L-alanine (BMAA), can be misincorporated in place of L-serine into human proteins. We also report that this misincorporation can be inhibited by L-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS. PMID:24086518

  10. The Non-Protein Amino Acid BMAA Is Misincorporated into Human Proteins in Place of l-Serine Causing Protein Misfolding and Aggregation

    PubMed Central

    Dunlop, Rachael Anne; Cox, Paul Alan; Banack, Sandra Anne; Rodgers, Kenneth John

    2013-01-01

    Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNAAla with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, β-N-methylamino-L-alanine (BMAA), can be misincorporated in place of l-serine into human proteins. We also report that this misincorporation can be inhibited by l-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS. PMID:24086518

  11. Oxidation of the Tryptophan 32 Residue of Human Superoxide Dismutase 1 Caused by Its Bicarbonate-dependent Peroxidase Activity Triggers the Non-amyloid Aggregation of the Enzyme*

    PubMed Central

    Coelho, Fernando R.; Iqbal, Asif; Linares, Edlaine; Silva, Daniel F.; Lima, Filipe S.; Cuccovia, Iolanda M.; Augusto, Ohara

    2014-01-01

    The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1WT and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp32 residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp32 residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1WT and hSOD1G93A mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp32 residue in the process. The results showed that Trp32 residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp32 residue (bovine SOD1 and hSOD1W32F mutant). The results support a role for the oxidation products of the hSOD1-Trp32 residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1. PMID:25237191

  12. Production of aggregation prone human interferon gamma and its mutant in highly soluble and biologically active form by SUMO fusion technology.

    PubMed

    Tileva, M; Krachmarova, E; Ivanov, I; Maskos, K; Nacheva, G

    2016-01-01

    The Escherichia coli expression system is a preferable choice for production of recombinant proteins. A disadvantage of this system is the target protein aggregation in "inclusion bodies" (IBs) that further requires solubilisation and refolding, which is crucial for the properties and the yield of the final product. In order to prevent aggregation, SUMO fusion tag technology has been successfully applied for expression of eukaryotic proteins, including human interferon gamma (hIFNγ) that was reported, however, with no satisfactory biological activity. We modified this methodology for expression and purification of both the wild type hIFNγ and an extremely prone to aggregation mutant hIFNγ-K88Q, whose recovery from IBs showed to be ineffective upon numerous conditions. By expression of the N-terminal His-SUMO fusion proteins in the E. coli strain BL21(DE3)pG-KJE8, co-expressing two chaperone systems, at 24 °C a significant increase in solubility of both target proteins (1.5-fold for hIFNγ and 8-fold for K88Q) was achieved. Two-step chromatography (affinity and ion-exchange) with on-dialysis His-SUMO-tag cleavage was applied for protein purification that yielded 6.0-7.0mg/g wet biomass for both proteins with >95% purity and native N-termini. The optimised protocol led to increased yields from 5.5 times for hIFNγ up to 100 times for K88Q in comparison to their isolation from IBs. Purified hIFNγ showed preserved thermal stability and antiproliferative activity corresponding to that of the native reference sample (3 × 10(7)IU/mg). The developed methodology represents an optimised procedure that can be successfully applied for large scale expression and purification of aggregation-prone proteins in soluble native form. PMID:26407523

  13. Neonatal Fc Receptor-Mediated IgG Transport Across Porcine Intestinal Epithelial Cells: Potentially Provide the Mucosal Protection.

    PubMed

    Guo, Jinyue; Li, Fei; He, Qigai; Jin, Hui; Liu, Mei; Li, Shaowen; Hu, Sishun; Xiao, Yuncai; Bi, Dingren; Li, Zili

    2016-06-01

    It has been well characterized that piglets can absorb colostrum IgG across the intestine to neonatal bloodstream and a certain level of IgG has been found in the mucosal secretions of the porcine intestinal tract. However, little is known about how the maternal IgG transport across the intestinal barrier and how IgG enter the lumen of intestinal tract. In this study, we demonstrated that the porcine neonatal Fc receptor (pFcRn) was expressed in a model of normal porcine intestinal epithelial cells (IPEC-J2) as well as in kidney cells (PK-15), and pFcRn was mainly distributed in the apical side of the polarized IPEC-J2 cells. Analyzing the phylogenetic relatedness of this gene we found that swine and human neonatal Fc receptor (FcRn) amino acid sequence are closer than rodents. We also showed that pFcRn mediated bidirectional IgG transport across polarized IPEC-J2 cells and bound to IgG in a pH-dependent manner. Furthermore, pFcRn-transcytosed viral-specific IgG reduced the transmissible gastroenteritis virus (TGEV) yield from the luminal direction by a 50% tissue culture infective dose (TCID50) assay. Our results indicate that pFcRn-dependent bidirectional IgG transport across the intestinal epithelium plays critical role in the acquisition of humoral immunity in early life and in host defense at mucosal surfaces. PMID:26982157

  14. Two years' performance of an in-house ELISA for diagnosis of Legionnaires' disease: detection of specific IgM and IgG antibodies against Legionella pneumophila serogroup 1, 3 and 6 in human serum.

    PubMed

    Elverdal, P L; Jørgensen, C S; Krogfelt, K A; Uldum, S A

    2013-08-01

    The aim of this study was to evaluate the performance of an in-house ELISA for the diagnosis of Legionnaires' disease (LD) by detection of IgM and IgG antibodies against Legionella (L.) pneumophila serogroups (sg) 1, 3 and 6. The evaluation was done throughout a two-year period in a diagnostic routine laboratory. Furthermore, the sensitivity of four different methods, the in-house L. pneumophila antibody test (ELISA), the urinary antigen test (Binax® EIA), an in-house PCR and culture, both alone and in combination was evaluated. From 2008 to 2010, 12,158 serum samples from 10,503 patients were analysed. During the same period, 361 cases of laboratory-confirmed LD cases were recorded in Denmark, but of these only 113 had a serum sample examined. The positive predictive value of the in-house ELISA was calculated to be 12.8 and the negative predictive value was 99.6, using only the confirmed LD cases as true positives. The sensitivity of the in-house ELISA for the detection of IgM and IgG antibodies in the confirmed LD cases was 61% and 36%, respectively. By combining the two ELISA assays the sensitivity increased to 66%. The sensitivity of the Legionella urinary antigen test (Binax® EIA) was 63%, of the in-house PCR 87% and of culture 69%. When all the different methods were combined, a higher sensitivity was calculated--for in-house ELISA (IgM+IgG) and Binax® EIA 91%, in-house ELISA (IgM+IgG) and in-house PCR 93%, in-house ELISA (IgM+IgG) and culture 93%, Binax® EIA and in-house PCR 79%, Binax® EIA and culture 68% and in-house PCR and culture 94%. This study confirms that the detection of IgG and IgM antibodies by ELISA is an important diagnostic tool, also during the initial phase of the disease. Furthermore, we showed that LD in Denmark with or without serum samples collected exhibits the same age and sex distribution and epidemiology, as in the rest of Europe, i.e., mostly men are infected, infections are mostly community acquired, followed by infection from

  15. Antitumor Efficacy of Anti-GD2 IgG1 Is Enhanced by Fc Glyco-Engineering.

    PubMed

    Xu, Hong; Guo, Hongfen; Cheung, Irene Y; Cheung, Nai-Kong V

    2016-07-01

    The affinity of therapeutic antibodies for Fcγ receptors (FcγRs) strongly influences their antitumor potency. To generate antibodies with optimal binding and immunologic efficacy, we compared the affinities of different versions of an IgG1 Fc region that had an altered peptide backbone, altered glycans, or both. To produce IgG1 with glycans that lacked α1,6-fucose, we used CHO cells that were deficient in the enzyme UDP-N-acetylglucosamine: α-3-d-mannoside-β-1,2-N-acetylglucosaminyltransferase I (GnT1), encoded by the MGAT1 gene. Mature N-linked glycans require this enzyme, and without it, CHO cells synthesize antibodies carrying only Man5-GlcNAc2, which were more effective in antibody-dependent cell-mediated cytotoxicity (ADCC). Our engineered IgG1, hu3F8-IgG1, is specific for GD2, a neuroendocrine tumor ganglioside. Its peptide mutant is IgG1-DEL (S239D/I332E/A330L), both produced in wild-type CHO cells. When produced in GnT1-deficient CHO cells, we refer to them as IgG1n and IgG1n-DEL, respectively. Affinities for human FcγRs were measured using Biacore T-100 (on CD16 and CD32 polymorphic alleles), their immunologic properties compared for ADCC and complement-mediated cytotoxicity (CMC) in vitro, and pharmacokinetics and antitumor effects were compared in vivo in humanized mice. IgG1n and IgG1n-DEL contained only mannose and acetylglucosamine and had preferential affinity for activating CD16s, over inhibitory CD32B, receptors. In vivo, the antitumor effects of IgG1, IgG1-DEL, and IgG1n-DEL were similar but modest, whereas IgG1n was significantly more effective (P < 0.05). Thus, IgG1n antibodies produced in GnT1-deficient CHO cells may have potential as improved anticancer therapeutics. Cancer Immunol Res; 4(7); 631-8. ©2016 AACR. PMID:27197064

  16. MuSK Myasthenia Gravis IgG4 Disrupts the Interaction of LRP4 with MuSK but Both IgG4 and IgG1-3 Can Disperse Preformed Agrin-Independent AChR Clusters

    PubMed Central

    Koneczny, Inga; Cossins, Judith; Waters, Patrick; Beeson, David; Vincent, Angela

    2013-01-01

    A variable proportion of patients with generalized myasthenia gravis (MG) have autoantibodies to muscle specific tyrosine kinase (MuSK). During development agrin, released from the motor nerve, interacts with low density lipoprotein receptor-related protein-4 (LRP4), which then binds to MuSK; MuSK interaction with the intracellular protein Dok7 results in clustering of the acetylcholine receptors (AChRs) on the postsynaptic membrane. In mature muscle, MuSK helps maintain the high density of AChRs at the neuromuscular junction. MuSK antibodies are mainly IgG4 subclass, which does not activate complement and can be monovalent, thus it is not clear how the antibodies cause disruption of AChR numbers or function to cause MG. We hypothesised that MuSK antibodies either reduce surface MuSK expression and/or inhibit the interaction with LRP4. We prepared MuSK IgG, monovalent Fab fragments, IgG1-3 and IgG4 fractions from MuSK-MG plasmas. We asked whether the antibodies caused endocytosis of MuSK in MuSK-transfected cells or if they inhibited binding of LRP4 to MuSK in co-immunoprecipitation experiments. In parallel, we investigated their ability to reduce AChR clusters in C2C12 myotubes induced by a) agrin, reflecting neuromuscular development, and b) by Dok7- overexpression, producing AChR clusters that more closely resemble the adult neuromuscular synapse. Total IgG, IgG4 or IgG1-3 MuSK antibodies were not endocytosed unless cross-linked by divalent anti-human IgG. MuSK IgG, Fab fragments and IgG4 inhibited the binding of LRP4 to MuSK and reduced agrin-induced AChR clustering in C2C12 cells. By contrast, IgG1-3 antibodies did not inhibit LRP4-MuSK binding but, surprisingly, did inhibit agrin-induced clustering. Moreover, both IgG4 and IgG1-3 preparations dispersed agrin-independent AChR clusters in Dok7-overexpressing C2C12 cells. Thus interference by IgG4 antibodies of the LRP4-MuSK interaction will be one pathogenic mechanism of MuSK antibodies, but IgG1-3 Mu

  17. Construction aggregates

    USGS Publications Warehouse

    Tepordei, V.V.

    1993-01-01

    Part of a special section on the market performance of industrial minerals in 1992. Production of construction aggregates increased by 4.6 percent in 1992. This increase was due, in part, to the increased funding for transportation and infrastructure projects. The U.S. produced about 1.05 Gt of crushed stone and an estimated 734 Mt of construction sand and gravel in 1992. Demand is expected to increase by about 5 percent in 1993.

  18. Construction aggregates

    USGS Publications Warehouse

    Nelson, T.I.; Bolen, W.P.

    2007-01-01

    Construction aggregates, primarily stone, sand and gravel, are recovered from widespread naturally occurring mineral deposits and processed for use primarily in the construction industry. They are mined, crushed, sorted by size and sold loose or combined with portland cement or asphaltic cement to make concrete products to build roads, houses, buildings, and other structures. Much smaller quantities are used in agriculture, cement manufacture, chemical and metallurgical processes, glass production and many other products.

  19. Quantification of the Binding Properties of Cu2+ to the Amyloid-Beta Peptide: Coordination Spheres for Human and Rat Peptides and Implication on Cu2+-Induced Aggregation

    PubMed Central

    Hong, Lian; Carducci, Tessa M.; Bush, William D.; Dudzik, Christopher G.; Millhauser, Glenn L.; Simon, John D.

    2010-01-01

    SUMMARY There is no consensus on the coordinating ligands for Cu2+ by A β. Yet the differences in peptide sequence between human and rat have been hypothesized to alter metal ion binding in a manner that alters Cu2+-induced aggregation of A β. Herein, we employ isothermal titration calorimetry (ITC), circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy to examine the Cu2+ coordination spheres to human and rat A β and an extensive set of A β(16) mutants. EPR of the mutant peptides is consistent with a 3N1O binding geometry, like the native human peptide at pH 7.4. The thermodynamic data reveal an equilibrium between three coordination spheres, {NH2, O-, NIm His6, amide N−}, {NH2, O-, NIm His6, NIm His13} and {NH2, O-, NIm His6, NIm His14} for human A β(16) but only one for rat A β(16), { NH2, O-, NIm His6, N−}, at pH 7.4 -6.5. ITC and CD data establish that the mutation R5G is sufficient for reproducing this difference in Cu2+ binding properties at pH 7.4. The substitution of bulky and positively charged Arg by Gly is proposed to stabilize the coordination {NH2, O-, NIm His6, amide N−} that then results in one dominating coordination sphere for the case of the rat peptide. The differences in the coordination geometries for Cu2+ by the human and rat A β are proposed to contribute to the variation in the ability of Cu2+ to induce aggregation of A β peptides. PMID:20690669

  20. Response to platelet-activating factor in human platelets stored and aged in plasma. Decrease in aggregation, phosphoinositide turnover, and receptor affinity

    SciTech Connect

    Shukla, S.D.; Morrison, W.J.; Klachko, D.M.

    1989-07-01

    Human platelet concentrates were stored in polyolefin bags at 22 to 24 degrees C on a horizontal shaker for up to 8 days. At different intervals, aliquots of platelet-rich plasma (PRP) were removed aseptically and five variables, i.e., platelet counts, morphology, platelet-activating factor (PAF)-stimulated aggregation, phosphoinositide turnover, and (3H)PAF binding to platelet receptors, were studied. The number of platelets did not change during the 8 days of storage. Scanning electron microscopy of the platelets revealed a gradual morphologic change from biconcave flat discs to irregular, crenated forms. The PAF-induced aggregation of platelets declined with time of storage. A decrease to 50 percent of the Day 1 aggregatory response to PAF was evident on Day 2, and there was a further decline to about 20 percent by Day 6. Similarly, PAF receptor-coupled phosphoinositide turnover, as monitored by 32P incorporation into individual phosphoinositides, decreased dramatically with storage. After 2 to 3 days of storage, the phosphoinositide turnover was reduced to 50 percent of the original response, and it continued to decline to about 25 percent of original response by Day 5 or 6. The binding of (3H)PAF to washed human platelets indicated subtle changes between Days 2 and 4, which became more noticeable by Day 6. These results have raised the possibility of changes in the number of the receptors and/or their affinity for the ligand during storage. We conclude that although the number of platelets was maintained during storage for 8 days, a general deterioration of their responses to PAF occurred at the levels of cell surface receptor, transmembrane signaling (phosphoinositide turnover), and response (aggregation).

  1. Characterization and screening of IgG binding to the neonatal Fc receptor.

    PubMed

    Neuber, Tobias; Frese, Katrin; Jaehrling, Jan; Jäger, Sebastian; Daubert, Daniela; Felderer, Karin; Linnemann, Mechthild; Höhne, Anne; Kaden, Stefan; Kölln, Johanna; Tiller, Thomas; Brocks, Bodo; Ostendorp, Ralf; Pabst, Stefan

    2014-01-01

    The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from degradation and increases the serum half-life of IgG, thereby contributing to a higher concentration of IgG in the serum. Because altered FcRn binding may result in a reduced or prolonged half-life of IgG molecules, it is advisable to characterize Fc receptor binding of therapeutic antibody lead candidates prior to the start of pre-clinical and clinical studies. In this study, we characterized the interactions between FcRn of different species (human, cynomolgus monkey, mouse and rat) and nine IgG molecules from different species and isotypes with common variable heavy (VH) and variable light chain (VL) domains. Binding was analyzed at acidic and neutral pH using surface plasmon resonance (SPR) and biolayer interferometry (BLI). Furthermore, we transferred the well-accepted, but low throughput SPR-based method for FcRn binding characterization to the BLI-based Octet platform to enable a higher sample throughput allowing the characterization of FcRn binding already during early drug discovery phase. We showed that the BLI-based approach is fit-for-purpose and capable of discriminating between IgG molecules with significant differences in FcRn binding affinities. Using this high-throughput approach we investigated FcRn binding of 36 IgG molecules that represented all VH/VL region combinations available in the fully human, recombinant antibody library Ylanthia®. Our results clearly showed normal FcRn binding profiles for all samples. Hence, the variations among the framework parts, complementarity-determining region (CDR) 1 and CDR2 of the fragment antigen binding (Fab) domain did not significantly change FcRn binding. PMID:24802048

  2. Anticomplementary activity of horse IgG and F(ab')2 antivenoms.

    PubMed

    Squaiella-Baptistão, Carla Cristina; Marcelino, José Roberto; Ribeiro da Cunha, Luiz Eduardo; Gutiérrez, José María; Tambourgi, Denise V

    2014-03-01

    Envenomation by poisonous animals is a neglected condition according to the World Health Organization (WHO). Antivenoms are included in the WHO Essential Medicines List. It has been assumed that immunoglobulin G (IgG) antivenoms could activate the complement system through Fc and induce early adverse reactions (EARs). However, data in the literature indicate that F(ab')2 fragments can also activate the complement system. Herein, we show that several batches of IgG and F(ab')2 antivenoms from the Butantan, Vital Brazil, and Clodomiro Picado Institutes activated the complement classical pathway and induced the production of C3a; however, only those antivenoms from Clodomiro Picado generated C5a. Different protein profiles (IgG heavy chain, protein contaminants, and aggregates) were observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses. Our results show that various antivenoms from different producers are able to activate the classical pathway of the complement system and generate anaphylatoxins, and these findings suggest that factors, such as composition, contaminant proteins, and aggregates, may influence the anticomplementary activity of antivenoms in vitro. Therefore, there is a need to further improve antivenom production methods to reduce their anticomplementary activity and potential to cause EARs. PMID:24445201

  3. Anticomplementary Activity of Horse IgG and F(ab')2 Antivenoms

    PubMed Central

    Squaiella-Baptistão, Carla Cristina; Marcelino, José Roberto; Ribeiro da Cunha, Luiz Eduardo; Gutiérrez, José María; Tambourgi, Denise V.

    2014-01-01

    Envenomation by poisonous animals is a neglected condition according to the World Health Organization (WHO). Antivenoms are included in the WHO Essential Medicines List. It has been assumed that immunoglobulin G (IgG) antivenoms could activate the complement system through Fc and induce early adverse reactions (EARs). However, data in the literature indicate that F(ab')2 fragments can also activate the complement system. Herein, we show that several batches of IgG and F(ab')2 antivenoms from the Butantan, Vital Brazil, and Clodomiro Picado Institutes activated the complement classical pathway and induced the production of C3a; however, only those antivenoms from Clodomiro Picado generated C5a. Different protein profiles (IgG heavy chain, protein contaminants, and aggregates) were observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses. Our results show that various antivenoms from different producers are able to activate the classical pathway of the complement system and generate anaphylatoxins, and these findings suggest that factors, such as composition, contaminant proteins, and aggregates, may influence the anticomplementary activity of antivenoms in vitro. Therefore, there is a need to further improve antivenom production methods to reduce their anticomplementary activity and potential to cause EARs. PMID:24445201

  4. Intravenous IgG (IVIG) and subcutaneous IgG (SCIG) preparations have comparable inhibitory effect on T cell activation, which is not dependent on IgG sialylation, monocytes or B cells.

    PubMed

    Issekutz, Andrew C; Rowter, Derek; Miescher, Sylvia; Käsermann, Fabian

    2015-10-01

    IVIG modulates T cell activation in vitro and inflammatory-autoimmune conditions in vivo. Sialylation of IgG, Fc receptor interactions, modulation of monocyte/macrophage/B cell functions have been implicated in IVIG effects. Subcutaneous IgG (SCIG) therapy is increasingly used for IgG replacement but whether these preparations share the effects of IVIG on T cell modulation is not documented. We compared the potency of SCIG-Hizentra™ (20% IgG preparation) with IVIG-Privigen® (10% IgG) for T cell inhibition, and assessed the involvement of IgG sialylation, monocytes and B cells in this process. Human PBMCs or sorted cells were cultured 3-7 days, and T cells were stimulated with immobilized anti-CD3 mAb or Candida antigen. Thymidine incorporation into DNA was quantitated and cytokines assayed by ELISA/Luminex® assay. IVIG and SCIG both dose-dependently (1-20mg/ml) inhibited (up to >80%) T cell proliferation to anti-CD3 mAb. Response to Candida albicans was comparably inhibited by IVIG and SCIG by 50-80% at 10mg/ml with inhibition even at 3mg/ml (P<0.05). These effects were not affected by depletion of sialic acid containing IgG using neuraminidase treatment or lectin affinity chromatography. With anti-CD3 or Candida stimulation, IL-1β, IL-2, IL-5, IL-6, IL-13, GMCSF, TNF-α, interferon-γ (with anti-CD3) and IL-17 (with Candida) levels were suppressed by IVIG or SCIG, with no effect on IL-4, IL-10, IL-12, IL-15 or TGFβ. Monocytes or B cells were not required for IgG-induced suppression of proliferation, in fact depletion of monocytes potentiated the IgG-induced inhibition. Reconstitution with monocytes restored the original inhibitory effect. These data show that IVIG (Privigen®) and SCIG (Hizentra™) have comparable inhibitory effects on T cell activation, which do not require sialylation of IgG. Inhibition is independent of monocytes or B cells. There is a potent suppression of multiple effector cytokines. Like IVIG, SCIG therapy is expected to show

  5. C-ANCA-positive IgG fraction from patients with Wegener's granulomatosis induces lung vasculitis in rats

    PubMed Central

    WEIDEBACH, W; VIANA, V S T; LEON, E P; BUENO, C; LEME, A S; ARANTES-COSTA, F M; MARTINS, M A; SALDIVA, P H N; BONFA, E

    2002-01-01

    The aim of the present study was to analyse in rats the ability of C-ANCA-positive IgG fraction in triggering inflammatory response on pulmonary tissue. Wistar rats (n = 18) were injected via the the internal jugular vein with 20 mg of total C-ANCA-positive IgG fraction isolated from serum of three different Wegener's granulomatosis patients obtained before therapy. Similarly, control rats were treated with IgG fraction from two rheumatoid arthritis patients (n = 7), IgG from six normal human sera (n = 15) or saline (n = 18), respectively. Animals were sacrificed after 24h of injection for histological analysis of the lungs. Vasculitis and inflammatory infiltrate were consistently absent in rats injected with rheumatoid arthritis IgG or saline and in 14/15 of normal IgG treated animals. In contrast, marked vasculitis was observed in all 18 animals injected with C-ANCA-positive IgG fraction. The histological features were characterized by the presence of a perivascular pleomorphic cellular sheath, particularly around small vessels, endothelial adherence and diapedesis of polymorphonuclear leucocytes and presence of granuloma-like lesions. A dose–response relationship was observed between protein concentration of C-ANCA IgG sample and the intensity of the inflammatory response in the animals. In addition, IgG fraction with undetectable C-ANCA, obtained from one patient in remission after treatment, was not able to reproduce the pulmonary tissue alterations induced by its paired IgG that was positive for C-ANCA taken before therapy. The experimental model described herein may be useful to characterize more effectively the pathogenic mechanism of C-ANCA in Wegener's disease. PMID:12100022

  6. Construction aggregates

    USGS Publications Warehouse

    Bolen, W.P.; Tepordei, V.V.

    2001-01-01

    The estimated production during 2000 of construction aggregates, crushed stone, and construction sand and gravel increased by about 2.6% to 2.7 Gt (3 billion st), compared with 1999. The expansion that started in 1992 continued with record production levels for the ninth consecutive year. By commodity, construction sand and gravel production increased by 4.5% to 1.16 Gt (1.28 billion st), while crushed stone production increased by 1.3% to 1.56 Gt (1.72 billion st).

  7. A novel clinical entity, IgG4-related disease (IgG4RD): general concept and details.

    PubMed

    Umehara, Hisanori; Okazaki, Kazuichi; Masaki, Yasufumi; Kawano, Mitsuhiro; Yamamoto, Motohisa; Saeki, Takako; Matsui, Shoko; Sumida, Takayuki; Mimori, Tsuneyo; Tanaka, Yoshiya; Tsubota, Kazuo; Yoshino, Tadashi; Kawa, Shigeyuki; Suzuki, Ritsuro; Takegami, Tsutomu; Tomosugi, Naohisa; Kurose, Nozomu; Ishigaki, Yasuhito; Azumi, Atsushi; Kojima, Masaru; Nakamura, Shigeo; Inoue, Dai

    2012-02-01

    IgG4-related disease (IgG4RD) is a novel clinical disease entity characterized by elevated serum IgG4 concentration and tumefaction or tissue infiltration by IgG4-positive plasma cells. IgG4RD may be present in a certain proportion of patients with a wide variety of diseases, including Mikulicz's disease, autoimmune pancreatitis, hypophysitis, Riedel thyroiditis, interstitial pneumonitis, interstitial nephritis, prostatitis, lymphadenopathy, retroperitoneal fibrosis, inflammatory aortic aneurysm, and inflammatory pseudotumor. Although IgG4RD forms a distinct, clinically independent disease category and is attracting strong attention as a new clinical entity, many questions and problems still remain to be elucidated, including its pathogenesis, the establishment of diagnostic criteria, and the role of IgG4. Here we describe the concept of IgG4RD and up-to-date information on this emerging disease entity. PMID:21881964

  8. Generation of Islet-like Cell Aggregates from Human Adipose Tissue-derived Stem Cells by Lentiviral Overexpression of PDX-1

    PubMed Central

    Bahrebar, M.; Soleimani, M.; Karimi, M. H.; Vahdati, A.; Yaghobi, R.

    2015-01-01

    Background: Pancreatic duodenal homeobox1 (PDX-1) is a transcription factor that is important in regulating pancreas development and maintaining β-cell function. β-cell replacement is an effective approach for the treatment of type 1 diabetes. Human adipose-mesenchymal stem cells (hAMSCs) are the ideal population cells for differentiating into insulin-producing cells. Objective: To determine if islet-like cell aggregates production could be generated from hAMSCs by lentiviral overexpression of PDX-1. Methods: After isolation of hAMSCs, characteristics of these cells were identified by flow-cytometic analysis and multilineage differentiation studies. PDX-1 gene delivered into hAMSCs through lentiviral vector for differentiating hAMSCs into insulin-producing cells (IPCs) at the utilized protocol for 14 days. Characteristics of IPCs were evaluated by immunocytofluorescence, dithizone staining, and quantitative reverse transcription PCR. In response to high glucose medium, insulin release was detected by chemiluminescence enzyme immunoassay. Results: The islet-like cell aggregates appeared about 10 days after introduction of PDX-1 into hAMSCs. PDX-1 induced its own expression (auto-induction), a number of islet-related genes such as Ngn3, Nkx2-2, and insulin. The insulin-positive cells were detected in the PDX-1 transduced cells. In response to glucose challenge test, secretion of insulin hormone in the medium with high glucose concentration significantly increased in the PDX-1-transduced cells related to medium with low glucose concentration. Conclusion: Introduction of lentiviral PDX-1 significantly induces hAMSCs to differentiate into islet-like cell aggregates, which may provide a source of adipose stem cells-derived insulin-producing cells for cell replacement therapy in type 1 diabetes. PMID:26082830

  9. An atypical IgM class platelet cold agglutinin induces GPVI-dependent aggregation of human platelets.

    PubMed

    Sánchez Guiu, I M; Martínez-Martinez, I; Martínez, C; Navarro-Fernandez, J; García-Candel, F; Ferrer-Marín, F; Vicente, V; Watson, S P; Andrews, R K; Gardiner, E E; Lozano, M L; Rivera, J

    2015-08-01

    Platelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to ex vivo platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin αIIbβ3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A2 production. Patient serum induced temperature- and αIIbβ3-dependent decrease of platelet count in allogeneic donor citrated platelet-rich plasma (PRP), but not in PRP from Glanzmann's thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression, (14)C-serotonin release, and TXA2 production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (FcγRIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding. PMID:25994029

  10. Identification of Anti-Long Chain Saturated Fatty Acid IgG Antibodies in Serum of Patients with Type 2 Diabetes

    PubMed Central

    Nicholas, Dequina A.; Salto, Lorena M.; Boston, Ava M.; Kim, Nan Sun; Larios, Marco; Beeson, W. Lawrence; Firek, Anthony F.; Casiano, Carlos A.; Langridge, William H. R.; Cordero-MacIntyre, Zaida; De Leon, Marino

    2015-01-01

    High levels of serum long chain saturated fatty acids (LCSFAs) have been associated with inflammation in type 2 diabetes. Dietary SFAs can promote inflammation, the secretion of IgG antibodies, and secretion of the proinflammatory cytokine IL-1β. This study characterizes anti-LCSFA IgG antibodies from patients with type 2 diabetes. Serum samples from several cohorts with type 2 diabetes were analyzed for the presence of anti-LCSFA IgG, the cytokine IL-1β, and nonesterified fatty acids. Anti-LCSFA IgG was isolated from patient samples and used for in vitro characterization of avidity and specificity. A cohort participating in En Balance, a diabetes health education program that improved diabetes management, tested positive for anti-LCSFA IgG. Following the 3-month program, the cohort showed a significant reduction in anti-LCSFA IgG levels. Anti-LCSFA antibodies isolated from these patients demonstrated high avidity, were specific for long chain SFAs, and correlated with serum fatty acids in patients with managed type 2 diabetes. Interestingly, anti-LCSFA IgG neutralized PA-induced IL-1β secretion by dendritic cells. Our data shows that nonesterified SFAs are recognized by IgG antibodies present in human blood. The identification of anti-LCSFA IgG antibodies in human sera establishes a basis for further exploration of lipid induced immune responses in diabetic patients. PMID:26633920

  11. The TUBEX typhoid test based on particle-inhibition immunoassay detects IgM but not IgG anti-O9 antibodies.

    PubMed

    Tam, Frankie Chi Hang; Lim, Pak Leong

    2003-11-01

    A serological test kit (TUBEX, IDL Biotech, Sweden) developed recently for the diagnosis of typhoid fever detects antibodies to the Salmonella enterica serovar Typhi lipopolysaccharide (LPS) O9 antigen. The antibodies are detected by their ability to inhibit the interaction between two types of reagent particles: (a). indicator latex microspheres sensitized with an anti-O9 monoclonal antibody, and (b). magnetic microspheres sensitized with S. typhi LPS. Following rapid mixing of the serum with these reagents and sedimentation of the magnetic particles by magnetic force, the concentration of indicator particles left in suspension provides a measure of the inhibition. Whereas it was previously assumed that both IgM and IgG antibodies could inhibit in the system, the present study reveals, surprisingly, that only the IgM antibodies do. It is not clear why IgG anti-O9 antibodies, both of mouse and human origin, do not inhibit, although these can bind to the LPS-sensitized magnetic particles as efficiently as the IgM antibodies. In addition, they can also inhibit very well in another detection system (ELISA) which uses a similar assay format and the same antibody and antigen reagents. Increasing the size of the LPS-sensitized microspheres made no difference; microscopic analysis of the TUBEX reaction mixture revealed that while the indicator particles bound abundantly to the IgG-aggregated LPS-sensitized particles, forming large clumps, these only formed a very light decoration on the IgM-aggregated particles. Thus, the TUBEX system is ideally suited for use in the diagnosis of infections as it allows IgM antibodies to be detected easily and rapidly from whole sera. PMID:14604543

  12. Transfer of IgG in the female genital tract by MHC class I-related neonatal Fc receptor (FcRn) confers protective immunity to vaginal infection

    PubMed Central

    Palaniyandi, Senthilkumar; Zeng, Rongyu; Tuo, Wenbin; Roopenian, Derry C.; Zhu, Xiaoping

    2011-01-01

    IgG is a major Ig subclass in mucosal secretions of the human female genital tract, where it predominates over the IgA isotype. Despite the abundance of IgG, surprisingly little is known about where and how IgG enters the lumen of the genital tract and the exact role local IgG plays in preventing sexually transmitted diseases. We demonstrate here that the neonatal Fc receptor, FcRn, is expressed in female genital tract epithelial cells of humans and mice and binds IgG in a pH-dependent manner. In vitro we show that FcRn mediates bidirectional IgG transport across polarized human endometrial HEC-1-A monolayers and primary human genital epithelial cells. Furthermore, endosomal acidification appears to be a prerequisite for FcRn-mediated IgG transcytosis; IgG transcytosis was demonstrated in vivo by translocation of systemically administered IgG into the genital lumen in WT but not FcRn-KO mice. The biological relevance of FcRn-transported IgG was demonstrated by passive immunization using herpes simplex virus-2 (HSV-2)–specific polyclonal serum, which conferred significantly higher protection against intravaginal challenge infection by the HSV-2 186 strain in WT mice than in FcRn-KO mice. These studies demonstrate that FcRn-mediated transport is a mechanism by which IgG can act locally in the female genital tract in immune surveillance and in host defense against sexually transmitted diseases. PMID:21368166

  13. Interleukin-1beta partially alleviates cyclosporin A-induced suppression of IgG1 isotype response to thyroglobulin in BALB/c mice in vivo.

    PubMed Central

    Dalai, S K; Miriyala, B; Kar, S K

    1998-01-01

    Cyclosporin A (CsA) at 120 mg/kg body weight when injected subcutaneously into BALB/c mice along with thyroglobulin emulsified in incomplete Freund's adjuvant (IFA) was found to suppress antigen-specific IgG titre by 86%. Isotyping revealed that both IgG1 and IgG2a titres were suppressed by 87% and 57%, respectively. But under identical conditions when complete Freund's adjuvant (CFA) was used, the suppression of antigen-specific IgG, IgG1 and IgG2a titres was 50%, 51% and 55%, respectively. Injection of anti-IL-1beta-neutralizing hamster monoclonal antibodies along with thyroglobulin and CsA emulsified in CFA increased the suppression of antigen-specific IgG titre. Under such conditions the IgG1 titre was suppressed more than the IgG2a titre. Recombinant human interleukin-1 receptor antagonist (rhuIL-1ra) also enhanced the suppression caused by CsA in the presence of CFA but control hamster immunoglobulin had no such effect. Recombinant human IL-1beta, when administered along with thyroglobulin and CsA emulsified in IFA, alleviated the suppression of antigen-specific IgG titre and the IgG1 titre was alleviated more than the IgG2a titre. Under identical conditions, rhuIL-1ra did not alleviate CsA-induced suppression. Lymphocytes from the lymph nodes of thyroglobulin-sensitized BALB/c mice when stimulated in vitro by thyroglobulin in the presence of CsA, secreted very little interferon-gamma (IFN-gamma) and IL-4, but on addition of an optimal dose of rhuIL-1beta, IFN-gamma and IL-4 secretion was partially restored. PMID:9767461

  14. Diagnostic performance of serum IgG4 level for IgG4-related disease: a meta-analysis.

    PubMed

    Xu, Wen-Long; Ling, Ying-Chun; Wang, Zhi-Kai; Deng, Fang

    2016-01-01

    An elevated serum IgG4 level is one of the most useful factors in the diagnosis of IgG4-related disease (IgG4-RD). In this study, we performed a meta-analysis of the published articles assessing the diagnostic accuracy of serum IgG4 concentrations for IgG4-RD. The databases of MEDLINE/PubMed, EMBASE and Web of Science were systematically searched for relevant studies. Sensitivities and specificities of serum IgG4 in each study were calculated, and the hierarchical summary receiver operating characteristic (HSROC) model with a random effects model were employed to obtain the individual and pooled estimates of sensitivities and specificities. In total, twenty-three studies comprising 6048 patients with IgG4-RD were included in the meta-analysis. The pooled sensitivity was 85% with a 95% confidence interval (CI) of 78-90%; the pooled specificity was 93% with a 95% CI of 90-95%. The HSROC curve for quantitative serum IgG4 lies closer to the upper left corner of the plot, and the area under the curve (AUC) was 0.95 (95% CI 0.93, 0.97), which suggested a high diagnostic accuracy of serum IgG4 for the entity of IgG4-RD. Our study suggests that serum IgG4 has high sensitivity and specificity in the diagnosis of IgG4-RD. PMID:27558881

  15. Diagnostic performance of serum IgG4 level for IgG4-related disease: a meta-analysis

    PubMed Central

    Xu, Wen-long; Ling, Ying-chun; Wang, Zhi-kai; Deng, Fang

    2016-01-01

    An elevated serum IgG4 level is one of the most useful factors in the diagnosis of IgG4-related disease (IgG4-RD). In this study, we performed a meta-analysis of the published articles assessing the diagnostic accuracy of serum IgG4 concentrations for IgG4-RD. The databases of MEDLINE/PubMed, EMBASE and Web of Science were systematically searched for relevant studies. Sensitivities and specificities of serum IgG4 in each study were calculated, and the hierarchical summary receiver operating characteristic (HSROC) model with a random effects model were employed to obtain the individual and pooled estimates of sensitivities and specificities. In total, twenty-three studies comprising 6048 patients with IgG4-RD were included in the meta-analysis. The pooled sensitivity was 85% with a 95% confidence interval (CI) of 78–90%; the pooled specificity was 93% with a 95% CI of 90–95%. The HSROC curve for quantitative serum IgG4 lies closer to the upper left corner of the plot, and the area under the curve (AUC) was 0.95 (95% CI 0.93, 0.97), which suggested a high diagnostic accuracy of serum IgG4 for the entity of IgG4-RD. Our study suggests that serum IgG4 has high sensitivity and specificity in the diagnosis of IgG4-RD. PMID:27558881

  16. The role of plasma membrane STIM1 and Ca2 +entry in platelet aggregation. STIM1 binds to novel proteins in human platelets

    PubMed Central

    Ambily, A.; Kaiser, W.J.; Pierro, C.; Chamberlain, E.V.; Li, Z.; Jones, C.I.; Kassouf, N.; Gibbins, J.M.; Authi, K.S.

    2014-01-01

    Ca2 + elevation is essential to platelet activation. STIM1 senses Ca2 + in the endoplasmic reticulum and activates Orai channels allowing store-operated Ca2 + entry (SOCE). STIM1 has also been reported to be present in the plasma membrane (PM) with its N-terminal region exposed to the outside medium but its role is not fully understood. We have examined the effects of the antibody GOK/STIM1, which recognises the N-terminal region of STIM1, on SOCE, agonist-stimulated Ca2 + entry, surface exposure, in vitro thrombus formation and aggregation in human platelets. We also determined novel binding partners of STIM1 using proteomics. The dialysed GOK/STIM1 antibody failed to reduced thapsigargin- and agonist-mediated Ca2 + entry in Fura2-labelled cells. Using flow cytometry we detect a portion of STIM1 to be surface-exposed. The dialysed GOK/STIM1 antibody reduced thrombus formation by whole blood on collagen-coated capillaries under flow and platelet aggregation induced by collagen. In immunoprecipitation experiments followed by proteomic analysis, STIM1 was found to extract a number of proteins including myosin, DOCK10, thrombospondin-1 and actin. These studies suggest that PM STIM1 may facilitate platelet activation by collagen through novel interactions at the plasma membrane while the essential Ca2 +-sensing role of STIM1 is served by the protein in the ER. PMID:24308967

  17. Comparison of Mineral Trioxide Aggregate and Diluted Formocresol in Pulpotomized Human Primary Molars: 42-month Follow-up and Survival Analysis

    PubMed Central

    Mettlach, Sarah E.; Zealand, Cameron M.; Botero, Tatiana M.; Boynton, James R.; Majewski, Robert F.; Hu, Jan ChingChun

    2015-01-01

    Purpose The purpose of this study was to test the hypothesis that there is no significant difference in the clinical and radiographic outcomes of diluted formocresol (DFC) compared to gray mineral trioxide aggregate (GMTA) pulpotomy in human primary molars. Methods A total of 152 children with 252 primary molars met selection criteria. Of those, 119 and 133 teeth were randomly assigned to the GMTA and DFC groups, respectively. Periapical radiographs, taken pre- and/or postoperatively and at each 6-month follow-up, were digitized and evaluated by three blinded and calibrated examiners. Results Over a 42-month period, a total of 865 clinical and radiographic evaluations were conducted. There was no significant difference in clinical success, with the cumulative proportion of GMTA-treated teeth surviving at 0.98 vs DFC-treated teeth at 0.95 (P>.05). Radiographic success, however, was significantly greater for GMTA vs DFC, with the cumulative proportion of GMTA-treated teeth surviving at 0.90 vs DFC-treated teeth at 0.47 (P<.001). Overall, DFC-treated teeth were 5.1 times more likely to fail than GMTA-treated teeth. Radiographic pathologies were observed more frequently in the DFC-treated teeth (P<.05). Conclusion Gray mineral trioxide aggregate can be considered an acceptable replacement for diluted formocresol when used as a medicament for primary molar pulpotomies. PMID:23756301

  18. Elastin peptides prepared from piscine and mammalian elastic tissues inhibit collagen-induced platelet aggregation and stimulate migration and proliferation of human skin fibroblasts.

    PubMed

    Shiratsuchi, Eri; Ura, Megumi; Nakaba, Misako; Maeda, Iori; Okamoto, Kouji

    2010-11-01

    We obtained pure elastin peptides from bovine ligamentum nuchae, porcine aorta, and bonito bulbus arteriosus. The inhibitory activity of these elastin peptides on platelet aggregation induced by collagen and the migratory and proliferative responsivenesses of human skin fibroblasts to these elastin peptides were examined. All of bonito, bovine, and porcine elastin peptides found to inhibit platelet aggregation, but bonito elastin peptides showed a higher inhibitory activity than bovine and porcine elastin peptides did. All elastin peptides enhanced the proliferation of fibroblasts 3.5- to 4.5-fold at a concentration of 10 µg/ml. Bovine and porcine elastin peptides stimulated the migration of fibroblasts, with the optimal response occurring at 10(-1) µg/ml, while maximal response was at 10(2) µg/ml for bonito elastin peptides. Furthermore, pretreatment of fibroblasts by lactose depressed their ability to migrate in response to all elastin peptides, suggesting the involvement of elastin receptor in cell response. These results suggest that both mammalian and piscine elastin peptides can be applied as useful biomaterials in which elasticity, antithrombotic property, and the enhancement of cell migration and proliferation are required. PMID:20853312

  19. Controlled Dual Growth Factor Delivery From Microparticles Incorporated Within Human Bone Marrow-Derived Mesenchymal Stem Cell Aggregates for Enhanced Bone Tissue Engineering via Endochondral Ossification

    PubMed Central

    Dang, Phuong N.; Dwivedi, Neha; Phillips, Lauren M.; Yu, Xiaohua; Herberg, Samuel; Bowerman, Caitlin; Solorio, Loran D.; Murphy, William L.

    2016-01-01

    Bone tissue engineering via endochondral ossification has been explored by chondrogenically priming cells using soluble mediators for at least 3 weeks to produce a hypertrophic cartilage template. Although recapitulation of endochondral ossification has been achieved, long-term in vitro culture is required for priming cells through repeated supplementation of inductive factors in the media. To address this challenge, a microparticle-based growth factor delivery system was engineered to drive endochondral ossification within human bone marrow-derived mesenchymal stem cell (hMSC) aggregates. Sequential exogenous presentation of soluble transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) at various defined time courses resulted in varying degrees of chondrogenesis and osteogenesis as demonstrated by glycosaminoglycan and calcium content. The time course that best induced endochondral ossification was used to guide the development of the microparticle-based controlled delivery system for TGF-β1 and BMP-2. Gelatin microparticles capable of relatively rapid release of TGF-β1 and mineral-coated hydroxyapatite microparticles permitting more sustained release of BMP-2 were then incorporated within hMSC aggregates and cultured for 5 weeks following the predetermined time course for sequential presentation of bioactive signals. Compared with cell-only aggregates treated with exogenous growth factors, aggregates with incorporated TGF-β1- and BMP-2-loaded microparticles exhibited enhanced chondrogenesis and alkaline phosphatase activity at week 2 and a greater degree of mineralization by week 5. Staining for types I and II collagen, osteopontin, and osteocalcin revealed the presence of cartilage and bone. This microparticle-incorporated system has potential as a readily implantable therapy for healing bone defects without the need for long-term in vitro chondrogenic priming. Significance This study demonstrates the regulation of chondrogenesis

  20. Controlled Dual Growth Factor Delivery From Microparticles Incorporated Within Human Bone Marrow-Derived Mesenchymal Stem Cell Aggregates for Enhanced Bone Tissue Engineering via Endochondral Ossification.

    PubMed

    Dang, Phuong N; Dwivedi, Neha; Phillips, Lauren M; Yu, Xiaohua; Herberg, Samuel; Bowerman, Caitlin; Solorio, Loran D; Murphy, William L; Alsberg, Eben

    2016-02-01

    Bone tissue engineering via endochondral ossification has been explored by chondrogenically priming cells using soluble mediators for at least 3 weeks to produce a hypertrophic cartilage template. Although recapitulation of endochondral ossification has been achieved, long-term in vitro culture is required for priming cells through repeated supplementation of inductive factors in the media. To address this challenge, a microparticle-based growth factor delivery system was engineered to drive endochondral ossification within human bone marrow-derived mesenchymal stem cell (hMSC) aggregates. Sequential exogenous presentation of soluble transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) at various defined time courses resulted in varying degrees of chondrogenesis and osteogenesis as demonstrated by glycosaminoglycan and calcium content. The time course that best induced endochondral ossification was used to guide the development of the microparticle-based controlled delivery system for TGF-β1 and BMP-2. Gelatin microparticles capable of relatively rapid release of TGF-β1 and mineral-coated hydroxyapatite microparticles permitting more sustained release of BMP-2 were then incorporated within hMSC aggregates and cultured for 5 weeks following the predetermined time course for sequential presentation of bioactive signals. Compared with cell-only aggregates treated with exogenous growth factors, aggregates with incorporated TGF-β1- and BMP-2-loaded microparticles exhibited enhanced chondrogenesis and alkaline phosphatase activity at week 2 and a greater degree of mineralization by week 5. Staining for types I and II collagen, osteopontin, and osteocalcin revealed the presence of cartilage and bone. This microparticle-incorporated system has potential as a readily implantable therapy for healing bone defects without the need for long-term in vitro chondrogenic priming. Significance: This study demonstrates the regulation of chondrogenesis

  1. Label-free Raman mapping of surface distribution of protein a and IgG biomolecules.

    PubMed

    Combs, Zachary A; Chang, Sehoon; Clark, Tolecia; Singamaneni, Srikanth; Anderson, Kyle D; Tsukruk, Vladimir V

    2011-03-15

    We have demonstrated a nanoengineered substrate composed of micropatterned silver nanoparticles to be used for the label-free mapping of adsorbed biomolecules. We utilized surface-enhanced Raman scattering (SERS) phenomenon to monitor the known bioanalytes, protein A and human immunoglobulin G (IgG). The SERS substrate was composed of a poly(alylamine hydrochloride) (PAH)/poly(styrenesulfonate) (PSS) layer-by-layer (LbL) nanocoating micropatterned with silver nanoparticles confined to microscopic stripes. Selective adsorption of biomacromolecules is facilitated by the amine-terminated LbL nanocoating, which prevents the surface adsorption of positively charged protein A across the surface except on the patterned regions containing negatively charged silver nanoparticles. Furthermore, adsorption of IgG on predetermined regions is facilitated by the selective binding of the Fc region of IgG to protein A. This label-free SERS approach provides accurate, selective, and fast detection of protein A and IgG solutions with a nanomolar concentration, down to below 1 nM for IgG in solution. This method could also be utilized for the facile detection of proteins in field conditions as well as in clinical, forensic, industrial, and environmental laboratories. PMID:21294559

  2. 1,4-Benzenediboronic-Acid-Induced Aggregation of Gold Nanoparticles: Application to Hydrogen Peroxide Detection and Biotin-Avidin-Mediated Immunoassay with Naked-Eye Detection.

    PubMed

    Yang, Ya-Chun; Tseng, Wei-Lung

    2016-05-17

    Hydrogen-peroxide (H2O2)-induced growth of small-sized gold nanoparticles (AuNPs) is often implemented for H2O2 sensing and plasmonic immunoassay. In contrast, there is little-to-no information in the literature regarding the application of H2O2-inhibited aggregation of citrate-capped AuNPs. This study discloses that benzene-1,4-diboronic acid (BDBA) was effective in driving the aggregation of citrate-capped AuNPs through an interaction between α-hydroxycarboxylate of citrate and boronic acids of BDBA. The H2O2-mediated oxidation of BDBA resulted in the conversion of boronic acid groups to phenol groups. The oxidized BDBA was incapable of triggering the aggregation of citrate-capped AuNPs. Thus, the presence of H2O2 prohibited BDBA-induced aggregation of citrate-capped AuNPs. The BDBA-induced aggregation of citrate-capped AuNPs can be paired with the glucose oxidase (GOx)-glucose system to design a colorimetric probe for glucose. Moreover, a H2O2·BDBA·AuNP probe was integrated with sandwich immunoassay, biotinylated antibody, and avidin-conjugated GOx for the selective naked-eye detection of rabbit immunoglobulin G (IgG) and human-prostate-specific antigen (PSA). The lowest detectable concentrations of rabbit IgG and human PSA by the naked eye were down to 0.1 and 4 ng/mL, respectively. More importantly, the proposed plasmonic immunoassay allowed the naked-eye quantification of 0-10 ng/mL PSA at an interval of 2 ng/mL in plasma samples. PMID:27091002

  3. Voltage-driven translocation behaviors of IgG molecule through nanopore arrays

    PubMed Central

    2013-01-01

    Nanopore-based biosensing has attracted more and more interests in the past years, which is also regarded as an emerging field with major impact on bio-analysis and fundamental understanding of nanoscale interactions down to single-molecule level. In this work, the voltage-driven translocation properties of goat antibody to human immunoglobulin G (IgG) are investigated using nanopore arrays in polycarbonate membranes. Obviously, the background ionic currents are modulated by IgG molecules for their physical place-holding effect. However, the detected ionic currents do ‘not’ continuously decrease as conceived; the currents first decrease, then increase, and finally stabilize with increasing IgG concentration. To understand this phenomenon, a simplified model is suggested, and the calculated results contribute to the understanding of the abnormal phenomenon in the actual ionic current changing tendency. PMID:23676116

  4. Imaging of inflammatory arthritis with technetium-99m-labeled IgG

    SciTech Connect

    Breedveld, F.C.; van Kroonenburgh, M.J.; Camps, J.A.; Feitsma, H.I.; Markusse, H.M.; Pauwels, E.K. )

    1989-12-01

    The accumulation of nonspecific polyclonal human immunoglobulin G (IgG) radiolabeled with 99mTc was compared to that of (99mTc)albumin and (99mTc)nanocolloid in rats with collagen induced arthritis. Serial scintigrams were acquired directly, 4 and 24 hr after injection. A clearly discernable image of the site of synovitis was seen with (99mTc)IgG as early as 4 hr postinjection. The relative intensity of the inflammatory lesion was maximal at 24 hr. Discrimination between arthritic and nonarthritic joints as well as correlations between the relative intensity of the arthritic joint and clinical indices of joint inflammation were superior with IgG compared to albumin or nanocolloid. These studies show that localization and severity of inflammatory joint disease can be detected with radiolabeled nonspecific IgG.

  5. Voltage-driven translocation behaviors of IgG molecule through nanopore arrays

    NASA Astrophysics Data System (ADS)

    Liu, Lei; Wang, Bing; Sha, Jingjie; Yang, Yue; Hou, Yaozong; Ni, Zhonghua; Chen, Yunfei

    2013-05-01

    Nanopore-based biosensing has attracted more and more interests in the past years, which is also regarded as an emerging field with major impact on bio-analysis and fundamental understanding of nanoscale interactions down to single-molecule level. In this work, the voltage-driven translocation properties of goat antibody to human immunoglobulin G (IgG) are investigated using nanopore arrays in polycarbonate membranes. Obviously, the background ionic currents are modulated by IgG molecules for their physical place-holding effect. However, the detected ionic currents do `not' continuously decrease as conceived; the currents first decrease, then increase, and finally stabilize with increasing IgG concentration. To understand this phenomenon, a simplified model is suggested, and the calculated results contribute to the understanding of the abnormal phenomenon in the actual ionic current changing tendency.

  6. Detection of histidine oxidation in a monoclonal immunoglobulin gamma (IgG) 1 antibody.

    PubMed

    Amano, Masato; Kobayashi, Naoki; Yabuta, Masayuki; Uchiyama, Susumu; Fukui, Kiichi

    2014-08-01

    Although oxidation of methionine and tryptophan are known as popular chemical modifications that occur in monoclonal antibody (mAb) molecules, oxidation of other amino acids in mAbs has not been reported to date. In this study, oxidation of the histidine residue in a human immunoglobulin gamma (IgG) 1 molecule was discovered for the first time by mass spectrometry. The oxidation of a specific histidine located at the CH2 domain of IgG1 occurred under light stress, but it was not observed under heat stress. With the forced degradation study using several reactive oxygen species, the singlet oxygen was attributed to a reactive source of the histidine oxidation. The reaction mechanism of the histidine oxidation was proposed on the basis of the mass spectrometric analysis of IgG1 oxidized in deuterium oxide and hydrogen heavy oxide. PMID:24940720

  7. Micro-arthropod communities under human disturbance: is taxonomic aggregation a valuable tool for detecting multivariate change? Evidence from Mediterranean soil oribatid coenoses

    NASA Astrophysics Data System (ADS)

    Caruso, Tancredi; Migliorini, Massimo

    2006-07-01

    Animal communities are sensitive to environmental disturbance, and several multivariate methods have recently been developed to detect changes in community structure. The complex taxonomy of soil invertebrates constrains the use of the community level in monitoring environmental changes, since species identification requires expertise and time. However, recent literature data on marine communities indicate that little multivariate information is lost in the taxonomic aggregation of species data to high rank taxa. In the present paper, this hypothesis was tested on two oribatid mite (Oribatida, Acari) assemblages under two different kinds of disturbance: metal pollution and fires. Results indicate that data sets built at the genus and family systematic rank can detect the effects of disturbance with little loss of information. This is an encouraging result in view of the use of the community level as a preliminary tool for describing patterns of human-disturbed soil ecosystems.

  8. Calcium bioaccessibility and uptake by human intestinal like cells following in vitro digestion of casein phosphopeptide-calcium aggregates.

    PubMed

    Perego, Silvia; Del Favero, Elena; De Luca, Paola; Dal Piaz, Fabrizio; Fiorilli, Amelia; Cantu', Laura; Ferraretto, Anita

    2015-06-01

    Casein phosphopeptides (CPPs), derived by casein proteolysis, can bind calcium ions and keep them in solution. In vitro studies have demonstrated CPP-induced cell calcium uptake, depending on the formation of (CPP + calcium) complexes and on the degree of differentiation of the intestinal cells. With the present study, we address the persistence of the complexes and of the CPP-induced calcium uptake in intestinal like cells after the digestion process, thus examining their eligibility to serve as nutraceuticals. A calcium-preloaded CPP preparation of commercial origin (Ca-CPPs) was subjected to in vitro digestion. The evolution of the supramolecular structure of the Ca-CPP complexes was studied using laser-light and X-ray scattering. The bioactivity of the pre- and post-digestion Ca-CPPs was determined in differentiated Caco2 and HT-29 cells by video imaging experiments using Fura-2. We found that Ca-CPP aggregates keep a complex supramolecular organization upon digestion, despite getting smaller in size and increasing internal calcium dispersion. Concomitantly and most interestingly, digested Ca-CPPs clearly enhance the uptake of calcium ions, especially in Caco2 cells. In contrast, digestion depletes the ability of post-loaded decalcified-CPPs (Ca-dekCPPs), with a weaker internal structure, to induce calcium uptake. The enhanced bioactivity reached upon digestion strongly suggests a recognized role of Ca-CPPs, in the form used here, as nutraceuticals. PMID:25927875

  9. In situ characterization of protein aggregates in human tissues affected by light chain amyloidosis: a FTIR microspectroscopy study.

    PubMed

    Ami, Diletta; Lavatelli, Francesca; Rognoni, Paola; Palladini, Giovanni; Raimondi, Sara; Giorgetti, Sofia; Monti, Luca; Doglia, Silvia Maria; Natalello, Antonino; Merlini, Giampaolo

    2016-01-01

    Light chain (AL) amyloidosis, caused by deposition of amyloidogenic immunoglobulin light chains (LCs), is the most common systemic form in industrialized countries. Still open questions, and premises for developing targeted therapies, concern the mechanisms of amyloid formation in vivo and the bases of organ targeting and dysfunction. Investigating amyloid material in its natural environment is crucial to obtain new insights on the molecular features of fibrillar deposits at individual level. To this aim, we used Fourier transform infrared (FTIR) microspectroscopy for studying in situ unfixed tissues (heart and subcutaneous abdominal fat) from patients affected by AL amyloidosis. We compared the infrared response of affected tissues with that of ex vivo and in vitro fibrils obtained from the pathogenic LC derived from one patient, as well as with that of non amyloid-affected tissues. We demonstrated that the IR marker band of intermolecular β-sheets, typical of protein aggregates, can be detected in situ in LC amyloid-affected tissues, and that FTIR microspectroscopy allows exploring the inter- and intra-sample heterogeneity. We extended the infrared analysis to the characterization of other biomolecules embedded within the amyloid deposits, finding an IR pattern that discloses a possible role of lipids, collagen and glycosaminoglycans in amyloid deposition in vivo. PMID:27373200

  10. Anti-Pseudomonas aeruginosa IgY Antibodies Induce Specific Bacterial Aggregation and Internalization in Human Polymorphonuclear Neutrophils

    PubMed Central

    Thomsen, K.; Christophersen, L.; Bjarnsholt, T.; Jensen, P. Ø.; Moser, C.

    2015-01-01

    Polymorphonuclear neutrophils (PMNs) are essential cellular constituents in the innate host response, and their recruitment to the lungs and subsequent ubiquitous phagocytosis controls primary respiratory infection. Cystic fibrosis pulmonary disease is characterized by progressive pulmonary decline governed by a persistent, exaggerated inflammatory response dominated by PMNs. The principal contributor is chronic Pseudomonas aeruginosa biofilm infection, which attracts and activates PMNs and thereby is responsible for the continuing inflammation. Strategies to prevent initial airway colonization with P. aeruginosa by augmenting the phagocytic competence of PMNs may postpone the deteriorating chronic biofilm infection. Anti-P. aeruginosa IgY antibodies significantly increase the PMN-mediated respiratory burst and subsequent bacterial killing of P. aeruginosa in vitro. The mode of action is attributed to IgY-facilitated formation of immobilized bacteria in aggregates, as visualized by fluorescence microscopy and the induction of increased bacterial hydrophobicity. Thus, the present study demonstrates that avian egg yolk immunoglobulins (IgY) targeting P. aeruginosa modify bacterial fitness, which enhances bacterial killing by PMN-mediated phagocytosis and thereby may facilitate a rapid bacterial clearance in airways of people with cystic fibrosis. PMID:25895968

  11. In situ characterization of protein aggregates in human tissues affected by light chain amyloidosis: a FTIR microspectroscopy study

    PubMed Central

    Ami, Diletta; Lavatelli, Francesca; Rognoni, Paola; Palladini, Giovanni; Raimondi, Sara; Giorgetti, Sofia; Monti, Luca; Doglia, Silvia Maria; Natalello, Antonino; Merlini, Giampaolo

    2016-01-01

    Light chain (AL) amyloidosis, caused by deposition of amyloidogenic immunoglobulin light chains (LCs), is the most common systemic form in industrialized countries. Still open questions, and premises for developing targeted therapies, concern the mechanisms of amyloid formation in vivo and the bases of organ targeting and dysfunction. Investigating amyloid material in its natural environment is crucial to obtain new insights on the molecular features of fibrillar deposits at individual level. To this aim, we used Fourier transform infrared (FTIR) microspectroscopy for studying in situ unfixed tissues (heart and subcutaneous abdominal fat) from patients affected by AL amyloidosis. We compared the infrared response of affected tissues with that of ex vivo and in vitro fibrils obtained from the pathogenic LC derived from one patient, as well as with that of non amyloid-affected tissues. We demonstrated that the IR marker band of intermolecular β-sheets, typical of protein aggregates, can be detected in situ in LC amyloid-affected tissues, and that FTIR microspectroscopy allows exploring the inter- and intra-sample heterogeneity. We extended the infrared analysis to the characterization of other biomolecules embedded within the amyloid deposits, finding an IR pattern that discloses a possible role of lipids, collagen and glycosaminoglycans in amyloid deposition in vivo. PMID:27373200

  12. Modifiers of mutant huntingtin aggregation

    PubMed Central

    Teuling, Eva; Bourgonje, Annika; Veenje, Sven; Thijssen, Karen; de Boer, Jelle; van der Velde, Joeri; Swertz, Morris; Nollen, Ellen

    2011-01-01

    Protein aggregation is a common hallmark of a number of age-related neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and polyglutamine-expansion disorders such as Huntington’s disease, but how aggregation-prone proteins lead to pathology is not known. Using a genome-wide RNAi screen in a C. elegans-model for polyglutamine aggregation, we previously identified 186 genes that suppress aggregation. Using an RNAi screen for human orthologs of these genes, we here present 26 human genes that suppress aggregation of mutant huntingtin in a human cell line. Among these are genes that have not been previously linked to mutant huntingtin aggregation. They include those encoding eukaryotic translation initiation, elongation and translation factors, and genes that have been previously associated with other neurodegenerative diseases, like the ATP-ase family gene 3-like 2 (AFG3L2) and ubiquitin-like modifier activating enzyme 1 (UBA1). Unravelling the role of these genes will broaden our understanding of the pathogenesis of Huntington’s disease. PMID:21915392

  13. Diagnostic Value of Serum IgG4 for IgG4-Related Disease

    PubMed Central

    Hao, Mingju; Liu, Min; Fan, Gaowei; Yang, Xin; Li, Jinming

    2016-01-01

    Abstract Many studies about serum IgG4 for the diagnosis of IgG4-related disease (IgG4-RD) have been reported. However, these studies had relatively small sample sizes and the diagnostic accuracy values varied much between them. The aim of this study was to perform a meta-analysis to evaluate the diagnostic value of serum IgG4 for IgG4-RD. We conducted a search of relevant articles using MEDLINE, EMBASE, Web of Science, SCOPUS, and Cochrane Library databases published before December 2015. Studies those assessed the diagnostic accuracy of serum IgG4 for IgG4-RD and those provided the cut-off value for serum IgG4 were included. Data were synthesized using the random-effect model. Statistical analysis was performed using STATA with the MIDAS module and Meta-DiSc 1.4 software. A total of 9 case-control studies were analyzed, which included 1235 patients with IgG4-RD and 5696 overall controls. The pooled estimate, for a cut-off value ranged from 135 to 144 mg/dL, produced a sensitivity of 87.2% (95% CI, 85.2–89.0%) and a specificity of 82.6% (95% CI, 81.6–83.6%). The positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were 6.48 (95% CI, 3.98–10.57), 0.14 (95% CI, 0.09–0.21), and 45.15 (95% CI, 23.41–87.06), respectively. The area under the curve (AUC) of the summary receiver operating characteristic curve (SROC) was 0.94 (0.92–0.96). When a cut-off value of 2-fold the upper limit of normal was used (ranged from 270 to 280 mg/dL), the pooled sensitivity was 63% (95% CI, 60.0–66.0%), and the specificity was 94.8% (95% CI, 94.1–95.4%). The PLR, NLR, and DOR were 13.3 (95% CI, 7.39–24.0), 0.41 (95% CI, 0.29–0.58) and 33.42 (95% CI, 13.88–80.43), respectively. The AUC of the SROC was 0.92 (0.90–0.94). Only a relatively small number of studies were included, and significant heterogeneity was observed in this meta-analysis. Serum IgG4 is a modestly effective marker to diagnose IgG4-RD. Doubling the cut

  14. Seroprevalence of IgG1 and IgG4 class antibodies against Mycobacterium avium subsp. paratuberculosis in Japanese population.

    PubMed

    Otsubo, Shigeru; Cossu, Davide; Eda, Shigetoshi; Otsubo, Yuriko; Sechi, Leonardo Antonio; Suzuki, Tsuyoshi; Iwao, Yumiko; Yamamoto, Shizuo; Kuribayashi, Takashi; Momotani, Eiichi

    2015-10-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the established causative agent of Johne's disease in cattle and other ruminants, and it has also been speculated to be a putative etiological agent of several human autoimmune diseases. It is acknowledged that dairy products deriving from infected animals play a role (could be vehicles) in exposing humans to MAP. MAP could stimulate the human immune system by means of their complex antigen (in the case of lipids, multivalent antigens) and may modulate it, acting as adjuvant molecules such as Freund's complete adjuvant. The immune system might be abnormally stimulated by the constant presence of MAP antigens (for example, in the dairy products), and this might be particularly relevant in genetically predisposed individuals. However, there is limited understanding about the current human exposure to MAP. The present study analyzed the antibody recognition profile of MAP lipophilic antigens in a cohort of 126 healthy Japanese. We measured the serum levels of total immunoglobulin G (IgG) and subclasses targeting MAP surface antigens through ethanol vortex indirect enzyme-linked immunosorbent assay (EVELISA) by using serum absorbed with Mycobacterium phlei. Elevated IgG (especially IgG1 and IgG4) responses were observed in 14% of the sera. To assess the specificity of EVELISA, the same samples were analyzed by means of a commercially available Johnelisa II kit. It was noteworthy that a high degree of correlation was observed when comparing the two methodologies (rs=0.7, p<0.0001). Moreover, in order to investigate the specificity of the binding, inhibition assay experiments were carried out also searching for antibodies against Bacillus Calmette-Guérin antigens, but no cross-reaction was observed. The result obtained represents the first evidence implying that the Japanese population is exposed to MAP, and additionally the existence of a foodborne chain of exposure that transmits MAP antigens to humans. PMID

  15. Comprehensive Analysis of the Therapeutic IgG4 Antibody Pembrolizumab: Hinge Modification Blocks Half Molecule Exchange In Vitro and In Vivo.

    PubMed

    Yang, Xiaoyu; Wang, Fengqiang; Zhang, Ying; Wang, Larry; Antonenko, Svetlana; Zhang, Shuli; Zhang, Yi Wei; Tabrizifard, Mohammad; Ermakov, Grigori; Wiswell, Derek; Beaumont, Maribel; Liu, Liming; Richardson, Daisy; Shameem, Mohammed; Ambrogelly, Alexandre

    2015-12-01

    IgG4 antibodies are evolving as an important class of cancer immunotherapies. However, human IgG4 can undergo Fab arm (half molecule) exchange with other IgG4 molecules in vivo. The hinge modification by a point mutation (S228P) prevents half molecule exchange of IgG4. However, the experimental confirmation is still expected by regulatory agencies. Here, we report for the first time the extensive analysis of half molecule exchange for a hinge-modified therapeutic IgG4 molecule, pembrolizumab (Keytruda) targeting programmed death 1 (PD1) receptor that was approved for advanced melanoma. Studies were performed in buffer or human serum using multiple exchange partners including natalizumab (Tysabri) and human IgG4 pool. Formation of bispecific antibodies was monitored by fluorescence resonance energy transfer, exchange with Fc fragments, mixed mode chromatography, immunoassays, and liquid chromatography-mass spectrometry. The half molecule exchange was also examined in vivo in SCID (severe combined immunodeficiency) mice. Both in vitro and in vivo results indicate that the hinge modification in pembrolizumab prevented half molecule exchange, whereas the unmodified counterpart anti-PD1 wt showed active exchange activity with other IgG4 antibodies or self-exchange activity with its own molecules. Our work, as an example expected for meeting regulatory requirements, contributes to establish without ambiguity that hinge-modified IgG4 antibodies are suitable for biotherapeutic applications. PMID:26308749

  16. Evaluating the Human Damage of Tsunami at Each Time Frame in Aggregate Units Based on GPS data

    NASA Astrophysics Data System (ADS)

    Ogawa, Y.; Akiyama, Y.; Kanasugi, H.; Shibasaki, R.; Kaneda, H.

    2016-06-01

    Assessments of the human damage caused by the tsunami are required in order to consider disaster prevention at such a regional level. Hence, there is an increasing need for the assessments of human damage caused by earthquakes. However, damage assessments in japan currently usually rely on static population distribution data, such as statistical night time population data obtained from national census surveys. Therefore, human damage estimation that take into consideration time frames have not been assessed yet. With these backgrounds, the objectives of this study are: to develop a method for estimating the population distribution of the for each time frame, based on location positioning data observed with mass GPS loggers of mobile phones, to use a evacuation and casualties models for evaluating human damage due to the tsunami, and evaluate each time frame by using the data developed in the first objective, and 3) to discuss the factors which cause the differences in human damage for each time frame. By visualizing the results, we clarified the differences in damage depending on time frame, day and area. As this study enables us to assess damage for any time frame in and high resolution, it will be useful to consider provision for various situations when an earthquake may hit, such as during commuting hours or working hours and week day or holiday.

  17. Affinity and catalytic heterogeneity of polyclonal myelin basic protein-hydrolyzing IgGs from sera of patients with multiple sclerosis

    PubMed Central

    Legostaeva, Galina A; Polosukhina, Dar’ya I; Bezuglova, Anna M; Doronin, Boris M; Buneva, Valentina N; Nevinsky, Georgy A

    2010-01-01

    Abstract Human myelin basic protein (hMBP)-hydrolyzing activity was recently shown to be an intrinsic property of antibodies (Abs) from multiple sclerosis (MS) patients. Here, we present the first evidence demonstrating a significant diversity of different fractions of polyclonal IgGs (pIgGs) from MS patients in their affinity for hMBP and in the ability of pIgGs to hydrolyze hBMP at different optimal pHs (3–10.5). IgGs containing λ- and κ-types of light chains demonstrated comparable relative activities in the hydrolysis of hMBP. IgGs of IgG1–IgG4 sub-classes were analyzed for catalytic activity. IgGs of all four sub-classes were catalytically active, with their contribution to the total activity of Abzs in the hydrolysis of hMBP and its 19-mer oligopeptide increasing in the order: IgG1 (1.5–2.1%) < IgG2 (4.9–12.8%) < IgG3 (14.7–25.0%) < IgG4 (71–78%). Our findings suggest that the immune systems of individual MS patients generate a variety of anti-hMBP abzymes with different catalytic properties, which can attack hMBP of myelin-proteolipid shell of axons, playing an important role in MS pathogenesis. PMID:19438809

  18. Aggregation of Antibody Drug Conjugates at Room Temperature: SAXS and Light Scattering Evidence for Colloidal Instability of a Specific Subpopulation.

    PubMed

    Frka-Petesic, B; Zanchi, D; Martin, N; Carayon, S; Huille, S; Tribet, C

    2016-05-17

    Coupling a hydrophobic drug onto monoclonal antibodies via lysine residues is a common route to prepare antibody-drug conjugates (ADC), a promising class of biotherapeutics. But a few chemical modifications on protein surface often increase aggregation propensity, without a clear understanding of the aggregation mechanisms at stake (loss of colloidal stability, self-assemblies, denaturation, etc.), and the statistical nature of conjugation introduces polydispersity in the ADC population, which raises questions on whether the whole ADC population becomes unstable. To characterize the average interactions between ADC, we monitored small-angle X-ray scattering in solutions of monoclonal IgG1 human antibody drug conjugate, with average degree of conjugation of 0, 2, or 3 drug molecules per protein. To characterize stability, we studied the kinetics of aggregation at room temperature. The intrinsic Fuchs stability ratio of the ADC was estimated from the variation over time of scattered light intensity and hydrodynamic radius, in buffers of varying pH, and at diverse sucrose (0% or 10%) and NaCl (0 or 100 mM) concentrations. We show that stable ADC stock solutions became unstable upon pH shift, well below the pH of maximum average attraction between IgGs. Data indicate that aggregation can be ascribed to a fraction of ADC population usually representing less than 30 mol % of the sample. In contrast to the case of (monodisperse) monoclonal antibodies, our results suggest that a poor correlation between stability and average interaction parameters should be expected as a corollary of dispersity of ADC conjugation. In practice, the most unstable fraction of the ADC population can be removed by filtration, which affects remarkably the apparent stability of the samples. Finally, the lack of correlation between the kinetic stability and variations of the average inter-ADC interactions is tentatively attributed to the uneven nature of charge distributions and the presence of

  19. Colostrogenesis: IgG1 transcytosis mechanisms.

    PubMed

    Baumrucker, Craig R; Bruckmaier, Rupert M

    2014-03-01

    Biological transport of intact proteins across epithelial cells has been documented for many absorptive and secretory tissues. Immunoglobulins were some of the earliest studied proteins in this category. The transcellular transport (transcytosis) of immunoglobulins in neonatal health and development has been recognized; the process is especially significant with ungulates because they do not transcytose immunoglobulins across the placenta to the neonate. Rather, they depend upon mammary secretion of colostrum and intestinal absorption of immunoglobulins in order to provide intestinal and systemic defense until the young ungulate develops its own humoral defense mechanisms. The neonatal dairy calf's ability to absorb immunoglobulins from colostrum is assisted by a ~24 h "open gut" phenomenon where large proteins pass the intestinal epithelial cells and enter the systemic system. However, a critical problem recognized for newborn dairy calves is that an optimum mass of colostrum Immunoglobulin G (IgG) needs to be absorbed within that 24 h window in order to provide maximal resistance to disease. Many calves do not achieve the optimum because of poor quality colostrum. While many studies have focused on calf absorption, the principal cause of the problem resides with the extreme variation (g to kg) in the mammary gland's capacity to transfer blood IgG1 into colostrum. Colostrum is a unique mammary secretory product that is formed during late pregnancy when mammary cells are proliferating and differentiating in preparation for lactation. In addition to the transcytosis of immunoglobulins, the mammary gland also concentrates a number of circulating hormones into colostrum. Remarkably, the mechanisms in the formation of colostrum in ungulates have been rather modestly studied. The mechanisms and causes of this variation in mammary gland transcytosis of IgG1 are examined, evaluated, and in some cases, explained. PMID:24474529

  20. Rapid assessment of human amylin aggregation and its inhibition by copper(II) ions by laser ablation electrospray ionization mass spectrometry with ion mobility separation.

    PubMed

    Li, Hang; Ha, Emmeline; Donaldson, Robert P; Jeremic, Aleksandar M; Vertes, Akos

    2015-10-01

    Native electrospray ionization (ESI) mass spectrometry (MS) is often used to monitor noncovalent complex formation between peptides and ligands. The relatively low throughput of this technique, however, is not compatible with extensive screening. Laser ablation electrospray ionization (LAESI) MS combined with ion mobility separation (IMS) can analyze complex formation and provide conformation information within a matter of seconds. Islet amyloid polypeptide (IAPP) or amylin, a 37-amino acid residue peptide, is produced in pancreatic beta-cells through proteolytic cleavage of its prohormone. Both amylin and its precursor can aggregate and produce toxic oligomers and fibrils leading to cell death in the pancreas that can eventually contribute to the development of type 2 diabetes mellitus. The inhibitory effect of the copper(II) ion on amylin aggregation has been recently discovered, but details of the interaction remain unknown. Finding other more physiologically tolerated approaches requires large scale screening of potential inhibitors. Here, we demonstrate that LAESI-IMS-MS can reveal the binding stoichiometry, copper oxidation state, and the dissociation constant of human amylin-copper(II) complex. The conformations of hIAPP in the presence of copper(II) ions were also analyzed by IMS, and preferential association between the β-hairpin amylin monomer and the metal ion was found. The copper(II) ion exhibited strong association with the -HSSNN- residues of the amylin. In the absence of copper(II), amylin dimers were detected with collision cross sections consistent with monomers of β-hairpin conformation. When copper(II) was present in the solution, no dimers were detected. Thus, the copper(II) ions disrupt the association pathway to the formation of β-sheet rich amylin fibrils. Using LAESI-IMS-MS for the assessment of amylin-copper(II) interactions demonstrates the utility of this technique for the high-throughput screening of potential inhibitors of amylin

  1. Rapid Assessment of Human Amylin Aggregation and Its Inhibition by Copper(II) Ions by Laser Ablation Electrospray Ionization Mass Spectrometry with Ion Mobility Separation

    PubMed Central

    Donaldson, Robert P.; Jeremic, Aleksandar M.; Vertes, Akos

    2015-01-01

    Native electrospray ionization (ESI) mass spectrometry (MS) is often used to monitor noncovalent complex formation between peptides and ligands. The relatively low throughput of this technique, however, is not compatible with extensive screening. Laser ablation electrospray ionization (LAESI) MS combined with ion mobility separation (IMS) can analyze complex formation and provide conformation information within a matter of seconds. Islet amyloid polypeptide (IAPP) or amylin, a 37-amino acid residue peptide, is produced in pancreatic beta-cells through proteolytic cleavage of its prohormone. Both amylin and its precursor can aggregate and produce toxic oligomers and fibrils leading to cell death in the pancreas that can eventually contribute to the development of type 2 diabetes mellitus. The inhibitory effect of the copper(II) ion on amylin aggregation has been recently discovered, but details of the interaction remain unknown. Finding other more physiologically tolerated approaches requires large scale screening of potential inhibitors. Here, we demonstrate that LAESI-IMS-MS can reveal the binding stoichiometry, copper oxidation state, and the dissociation constant of human amylin–copper(II) complex. The conformations of hIAPP in the presence of copper(II) ions were also analyzed by IMS, and preferential association between the β-hairpin amylin monomer and the metal ion was found. The copper(II) ion exhibited strong association with the –HSSNN– residues of the amylin. In the absence of copper(II), amylin dimers were detected with collision cross sections consistent with monomers of β-hairpin conformation. When copper(II) was present in the solution, no dimers were detected. Thus, the copper(II) ions disrupt the association pathway to the formation of β-sheet rich amylin fibrils. Using LAESI-IMS-MS for the assessment of amylin–copper(II) interactions demonstrates the utility of this technique for the high-throughput screening of potential inhibitors of

  2. LpMab-12 Established by CasMab Technology Specifically Detects Sialylated O-Glycan on Thr52 of Platelet Aggregation-Stimulating Domain of Human Podoplanin

    PubMed Central

    Kato, Yukinari; Ogasawara, Satoshi; Oki, Hiroharu; Goichberg, Polina; Honma, Ryusuke; Fujii, Yuki; Kaneko, Mika K.

    2016-01-01

    Podoplanin (PDPN), also known as Aggrus, possesses three tandem repeat of platelet aggregation-stimulating (PLAG) domains in its N-terminus. Among the PLAG domains, sialylated O-glycan on Thr52 of PLAG3 is essential for the binding to C-type lectin-like receptor-2 (CLEC-2) and the platelet-aggregating activity of human PDPN (hPDPN). Although various anti-hPDPN monoclonal antibodies (mAbs) have been generated, no specific mAb has been reported to target the epitope containing glycosylated Thr52. We recently established CasMab technology to develop mAbs against glycosylated membrane proteins. Herein, we report the development of a novel anti-glycopeptide mAb (GpMab), LpMab-12. LpMab-12 detected endogenous hPDPN by flow cytometry. Immunohistochemical analyses also showed that hPDPN-expressing lymphatic endothelial and cancer cells were clearly labeled by LpMab-12. The minimal epitope of LpMab-12 was identified as Asp49–Pro53 of hPDPN. Furthermore, LpMab-12 reacted with the synthetic glycopeptide of hPDPN, corresponding to 38–54 amino acids (hpp3854: 38-EGGVAMPGAEDDVVTPG-54), which carries α2–6 sialylated N-acetyl-D-galactosamine (GalNAc) on Thr52. LpMab-12 did not recognize non-sialylated GalNAc-attached glycopeptide, indicating that sialylated GalNAc on Thr52 is necessary for the binding of LpMab-12 to hPDPN. Thus, LpMab-12 could serve as a new diagnostic tool for determining whether hPDPN possesses the sialylation on Thr52, a site-specific post-translational modification critical for the hPDPN association with CLEC-2. PMID:27031228

  3. Wild-type human TDP-43 expression causes TDP-43 phosphorylation, mitochondrial aggregation, motor deficits and early mortality in transgenic mice

    PubMed Central

    Xu, Ya-Fei; Gendron, Tania F.; Zhang, Yong-Jie; Lin, Wen-Lang; D’Alton, Simon; Sheng, Hong; Casey, Monica Castanedes; Tong, Jimei; Knight, Joshua; Yu, Xin; Rademakers, Rosa; Boylan, Kevin; Hutton, Mike; McGowan, Eileen; Dickson, Dennis W.; Lewis, Jada; Petrucelli, Leonard

    2011-01-01

    Transactivation response DNA-binding protein 43 (TDP-43) is a principal component of ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis (ALS). Mutations in TARDBP, the gene encoding TDP-43, are associated with sporadic and familial ALS, yet multiple neurodegenerative diseases exhibit TDP-43 pathology without known TARDBP mutations. While TDP-43 has been ascribed a number of roles in normal biology, including mRNA splicing and transcription regulation, elucidating disease mechanisms associated with this protein is hindered by the lack of models to dissect such functions. We have generated transgenic (TDP-43PrP) mice expressing full-length human TDP-43 (hTDP-43) driven by the mouse prion promoter to provide a tool to analyze the role of wild-type hTDP-43 in the brain and spinal cord. Expression of hTDP-43 caused a dose dependant down regulation of mouse TDP-43 RNA and protein. Moderate overexpression of hTDP-43 resulted in TDP-43 truncation, increased cytoplasmic and nuclear ubiquitin levels, as well as intranuclear and cytoplasmic aggregates that were immunopositive for phosphorylated TDP-43. Of note, abnormal juxtanuclear aggregates of mitochondria were observed, accompanied by enhanced levels of Fis1 and phosphorylated DLP1, key components of the mitochondrial fission machinery. Conversely, a marked reduction in mitofusin 1 expression, which plays an essential role in mitochondrial fusion, was observed in TDP-43PrP mice. Finally, TDP-43PrP mice showed reactive gliosis, axonal and myelin degeneration, gait abnormalities and early lethality. This TDP-43 transgenic line provides a valuable tool for identifying potential roles of wild-type TDP-43 within the central nervous system and for studying TDP-43-associated neurotoxicity. PMID:20702714

  4. Fabrication and detection of tissue engineered bone aggregates based on encapsulated human ADSCs within hybrid calcium alginate/bone powder gel-beads in a spinner flask.

    PubMed

    Song, Kedong; Yang, Yanfei; Xu, Lili; Tian, Jiaxin; Fan, Jiangli; Jiao, Zeren; Feng, Shihao; Wang, Hong; Wang, Yiwei; Wang, Ling; Liu, Tianqing

    2016-05-01

    Traditional treatment for bone diseases limits their clinical application due to undesirable host immune rejection, limited donator sources and severe pain and suffering for patients. Bone tissue engineering therefore is expected to be a more effective way in treating bone diseases. In the present study, hybrid calcium alginate/bone powder gel-beads with a uniform size distribution, good biocompatibility and osteoinductive capability, were prepared to be used as an in-vitro niche-like matrix. The beads were optimized using 2.5% (w/v) sodium alginate solution, 4.5% (w/v) CaCl2 solution and 5.0mg/mL bone powder using an easy-to-use method. Human ADSCs were cultured and induced into chondrocytes and osteoblasts, respectively. The cells were characterized by histological staining showing the ADSCs were able to maintain their characteristic morphology with multipotent differentiation ability. ADSCs at density of 5 × 10(6)cells/mL were encapsulated into the gel-beads aiming to explore cell expansion under different conditions and the osteogenic induction of ADSCs was verified by specific staining. Results demonstrated that the encapsulated ADSCs expanded 5.6 folds in 10 days under dynamic condition via spinner flask, and were able to differentiate into osteoblasts (OBs) with extensive mineralized nodules forming the bone aggregates over 3 weeks postosteogenic induction. In summary, hybrid gel-beads encapsulating ADSCs are proved to be feasible as a new method to fabricate tissue engineered bone aggregation with potential to treat skeletal injury in the near future. PMID:26952485

  5. Driving Cartilage Formation in High-Density Human Adipose-Derived Stem Cell Aggregate and Sheet Constructs Without Exogenous Growth Factor Delivery

    PubMed Central

    Dang, Phuong N.; Solorio, Loran D.

    2014-01-01

    An attractive cell source for cartilage tissue engineering, human adipose-derived stem cells (hASCs) can be easily expanded and signaled to differentiate into chondrocytes. This study explores the influence of growth factor distribution and release kinetics on cartilage formation within 3D hASC constructs incorporated with transforming growth factor-β1 (TGF-β1)-loaded gelatin microspheres. The amounts of microspheres, TGF-β1 concentration, and polymer degradation rate were varied within hASC aggregates. Microsphere and TGF-β1 loading concentrations were identified that resulted in glycosaminoglycan (GAG) production comparable to those of control aggregates cultured in TGF-β1-containing medium. Self-assembling hASC sheets were then engineered for the production of larger, more clinically relevant constructs. Chondrogenesis was observed in hASC-only sheets cultured with exogenous TGF-β1 at 3 weeks. Importantly, sheets with incorporated TGF-β1-loaded microspheres achieved GAG production similar to sheets treated with exogenous TGF-β1. Cartilage formation was confirmed histologically via observation of cartilage-like morphology and GAG staining. This is the first demonstration of the self-assembly of hASCs into high-density cell sheets capable of forming cartilage in the presence of exogenous TGF-β1 or with TGF-β1-releasing microspheres. Microsphere incorporation may bypass the need for extended in vitro culture, potentially enabling hASC sheets to be implanted more rapidly into defects to regenerate cartilage in vivo. PMID:24873753

  6. Overlapping IgG4 Responses to Self- and Environmental Antigens in Endemic Pemphigus Foliaceus.

    PubMed

    Qian, Ye; Jeong, Joseph S; Ye, Jian; Dang, Bim; Abdeladhim, Maha; Aoki, Valeria; Hans-Filhio, Gunter; Rivitti, Evandro A; Valenzuela, Jesus G; Diaz, Luis A

    2016-03-01

    The etiology of human autoimmune diseases in general remains largely unknown, although the genetic and environmental interplay may be relevant. This applies to the autoimmune diseases of the skin such as the pemphigus phenotypes and others. In this group, there is an endemic form of pemphigus foliaceus (also known as fogo selvagem [FS]) in which the pathogenic IgG4 autoantibody response to the self-antigen desmoglein 1 (Dsg1) cross-reacts with the LJM11 sand fly salivary gland Ag. In this investigation, we dissected the IgG4 autoantibody repertoires used by FS patients in response to endogenous self-Dsg1 and exogenous LJM11 sand fly Ag. Based on analyses of the genetic clonal signatures of these Abs, our results indicate that there is a significant overlap between these two responses, as all identified IgG4 mAbs cross-react to both Dsg1 and LJM11 Ags. Germline H- and L-chain V gene Abs generated according to mutated cross-reactive mAbs preserved their reactivity to both Ags. Our findings suggest that both Dsg1 autoantigen and LJM11 environmental Ag could be the initial antigenic stimulants for the IgG4 autoimmune responses in FS. These results support our hypothesis that LJM11 Ag plays a substantial role in triggering the IgG4 autoantibody development in FS and provide new insights on how noninfectious environmental Ag(s) may drive the generation of autoantibodies in IgG4-related autoimmune diseases. PMID:26826247

  7. Selective aggregation of PAMAM dendrimer nanocarriers and PAMAM/ZnPc nanodrugs on human atheromatous carotid tissues: a photodynamic therapy for atherosclerosis

    NASA Astrophysics Data System (ADS)

    Spyropoulos-Antonakakis, Nikolaos; Sarantopoulou, Evangelia; Trohopoulos, Panagiotis N.; Stefi, Aikaterina L.; Kollia, Zoe; Gavriil, Vassilios E.; Bourkoula, Athanasia; Petrou, Panagiota S.; Kakabakos, Sotirios; Semashko, Vadim V.; Nizamutdinov, Alexey S.; Cefalas, Alkiviadis-Constantinos

    2015-05-01

    Photodynamic therapy (PDT) involves the action of photons on photosensitive molecules, where atomic oxygen or OH- molecular species are locally released on pathogenic human cells, which are mainly carcinogenic, thus causing cell necrosis. The efficacy of PDT depends on the local nanothermodynamic conditions near the cell/nanodrug system that control both the level of intracellular translocation of nanoparticles in the pathogenic cell and their agglomeration on the cell membrane. Dendrimers are considered one of the most effective and promising drug carriers because of their relatively low toxicity and negligible activation of complementary reactions. Polyamidoamine (PAMAM) dendrite delivery of PDT agents has been investigated in the last few years for tumour selectivity, retention, pharmacokinetics and water solubility. Nevertheless, their use as drug carriers of photosensitizing molecules in PDT for cardiovascular disease, targeting the selective necrosis of macrophage cells responsible for atheromatous plaque growth, has never been investigated. Furthermore, the level of aggregation, translocation and nanodrug delivery efficacy of PAMAM dendrimers or PAMAM/zinc phthalocyanine (ZnPc) conjugates on human atheromatous tissue and endothelial cells is still unknown.

  8. Insights into the potential aggregation liabilities of the b12 Fab fragment via elevated temperature molecular dynamics.

    PubMed

    Buck, Patrick M; Kumar, Sandeep; Singh, Satish K

    2013-03-01

    Aggregation is a common hurdle faced during the development of antibody therapeutics. In this study, we explore the potential aggregation liabilities of the Fab (fragment antigen-binding) from a human IgG1κ antibody via multiple elevated temperature molecular dynamic simulations, analogous to accelerated stability studies performed during formulation development. Deformation and solvent exposure changes in response to thermal stress were monitored for individual structural domains (V(H), V(L), C(H)1 and C(L)), their interfaces (V(H):V(L) and C(H)1:C(L)), edge beta-strands and sequence-predicted aggregation-prone regions (APRs). During simulations, domain interfaces deformed prior to the unfolding of individual domains. However, interfacial beta-strands retained their secondary structure and remained solvent protected longer than all other strands or loops. Thus, APRs located in interfacial beta-strands are effectively blocked from self-association. Structural deformations were also observed in complementarity-determining regions, edge beta-strands and adjoining framework beta-strands, which increased their solvent-accessible surface area and exposed APRs in these regions. From the analysis of these structural changes, two potential aggregation liabilities were identified in the V(H) domain of this Fab. Insights gained from this investigation should be useful in devising a rational structure-based strategy for the design and selection of antibody candidates with high potency and improved developability. PMID:23188804

  9. Aggregation kinetics and structure of cryoimmunoglobulins clusters

    NASA Astrophysics Data System (ADS)

    Spirito, M. De; Chiappini, R.; Bassi, F. Andreasi; Stasio, E. Di; Giardina, B.; Arcovito, G.

    2002-02-01

    Cryoimmunoglobulins are pathological antibodies characterized by a temperature-dependent reversible insolubility. Rheumatoid factors (RF) are immunoglobulins possessing anti-immunoglobulin activity and usually consist of an IgM antibody that recognizes IgG as antigen. These proteins are present in sera of patients affected by a large variety of different pathologies, such as HCV infection, neoplastic and autoimmune diseases. Aggregation and precipitation of cryoimmunoglobulins, leading to vasculiti, are physical phenomena behind such pathologies. A deep knowledge of the physico-chemical mechanisms regulating such phenomena plays a fundamental role in biological and clinical applications. In this work, a preliminary investigation of the aggregation kinetics and of the final macromolecular structure of the aggregates is presented. Through static light scattering techniques, the gyration radius Rg and the fractal dimension Dm of the growing clusters have been determined. However, while the initial aggregation mechanism could be described using the universal reaction-limited cluster-cluster aggregation (RLCCA) theory, at longest times from the beginning of the process, the RLCCA theory fails and a restructuring of clusters is observed together with an increase of the cluster fractal dimension Dm up to a value Dm∼3. The time tn, at which the restructuring takes place, and the final cluster size can be modulated by varying the quenching temperature.

  10. Removal of terminal alpha-galactosyl residues from xenogeneic porcine endothelial cells. Decrease in complement-mediated cytotoxicity but persistence of IgG1-mediated antibody-dependent cell-mediated cytotoxicity.

    PubMed

    Watier, H; Guillaumin, J M; Piller, F; Lacord, M; Thibault, G; Lebranchu, Y; Monsigny, M; Bardos, P

    1996-07-15

    To determine the role of the terminal alpha-galactosyl residue in the endothelial damage mediated by human xenoreactive natural antibodies (IgM and IgG), we treated porcine endothelial cells in culture with green coffee bean alpha-galactosidase. A practically complete removal of terminal alpha-Gal residues (as evaluated by flow cytometry with Bandeiraea simplicifolia isolectin B4) and concomitant exposure of N-acetyllactosamine were obtained without altering cell viability. A dramatic decrease in IgM and IgG binding (from a pool of human sera) was observed, confirming the key role of the alpha-galactosyl residues. The enzyme treatment did not induce any nonspecific immunoglobulin binding sites, but led to the exposure of new epitopes for a minor fraction of IgM. The main residual IgM and IgG binding could be due to xenoantigens other than the alpha-galactosyl residues. When alpha-galactosidase-treated endothelial cells were used as targets in cytotoxicity experiments, they were less susceptible than untreated cells to complement-mediated cytotoxicity induced by fresh human serum. In contrast, they did not acquire resistance to human IgG-dependent cellular cytotoxicity, despite the decrease in IgG binding. Because it is known that antibody-dependent cytotoxicity mediated by CD16+ NK cells is dependent on IgG1 and IgG3, and not on IgG2 or IgG4, which was confirmed by blocking experiments, we studied the binding of all four subclasses to intact and alpha-galactosidase-treated endothelial cells. Two major subclasses, IgG1 and IgG2, bound to untreated endothelial cells, whereas IgG3 binding was low and IgG4 binding was negligible. A decrease in IgG1, IgG2, and IgG3 binding was observed upon alpha-galactosidase treatment, indicating that antibodies belonging to these three subclasses recognize alpha-galactosyl residues. The decrease in IgG2 binding was more pronounced than the decrease in IgG1 binding. Collectively, these data indicate that IgG1 xenoreactive natural

  11. Interaction of antibody-aggregated C4 and guinea-pig red cells: coagglutination phenomenon of Bordet and Gengou.

    PubMed Central

    Davies, K; Wilson, A B; Coombs, R R

    1981-01-01

    The component of bovine serum (coagglutinogen) responsible for coagglutination of guinea-pig red cells has been shown to be c4, as anticipated from our previously reported findings on human serum. To effect coagglutination, the C4 needs to be aggregated by antibody; thereby probably increasing the avidity of the C4 for the receptors on guinea-pig red cells. The coagglutinating activity is lost if univalent Fab anti-C4 is used, but it can be restored by adding IgG antibody to the Fab. A procedure is described for producing antibody reagents to human or bovine C4 by injecting guinea-pigs with well-washed coagglutinated guinea-pig red cells. PMID:7275174

  12. Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin.

    PubMed

    Hjorth, Christian Fogt; Norrman, Mathias; Wahlund, Per-Olof; Benie, Andrew J; Petersen, Bent O; Jessen, Christian M; Pedersen, Thomas Å; Vestergaard, Kirsten; Steensgaard, Dorte B; Pedersen, Jan Skov; Naver, Helle; Hubálek, František; Poulsen, Christian; Otzen, Daniel

    2016-04-01

    A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation. PMID:26921119

  13. Albumin quotient, IgG concentration, and IgG index determinations in cerebrospinal fluid of neonatal foals.

    PubMed

    Andrews, F M; Geiser, D R; Sommardahl, C S; Green, E M; Provenza, M

    1994-06-01

    Total protein (TP), albumin, and IgG concentrations were measured in CSF from the atlanto-occipital (AO) and lumbosacral (LS) sites and in serum of 15 clinically normal neonatal foals < or = 10 days old (mean, 7.0 days). The albumin quotient (AQ; CSF albumin/serum albumin x 100) and IgG index ([CSF IgG/serum IgG] x [serum albumin/CSF albumin]), indicators of blood-brain barrier permeability and intrathecal IgG production, respectively, were then calculated. Mean +/- SD values obtained from the foals of this study were: serum albumin, 2,900 +/- 240 mg/dl; serum IgG, 1,325 +/- 686 mg/dl; AO CSF total protein (TP), 82.8 +/- 19.2 mg/dl; LS CSF TP, 83.6 +/- 16.1 mg/dl; AO CSF albumin, 52.0 +/- 8.6 mg/dl; LS CSF albumin, 53.8 +/- 15.7 mg/dl; AO CSF IgG, 10.2 +/- 5.5 mg/dl; LS CSF IgG, 9.9 +/- 5.7 mg/dl; AO AQ, 1.86 +/- 0.29; LS AQ, 1.85 +/- 0.51, AO IgG index, 0.52 +/- 0.28; and LS IgG index, 0.48 +/- 0.27. Significant difference between values for the AO and LS sites was not found. A CSF albumin concentration > 85.2 mg/dl or AQ > 2.4, as determined by mean +/- 2 SD, may indicate increased blood-brain barrier permeability. An IgG index value > 1.0 may indicate intrathecal IgG production. Values obtained for foals of this study should serve as baseline for comparison in the evaluation of blood-brain barrier permeability and intrathecal IgG production in neonatal foals with neurologic disease. PMID:7944008

  14. 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. PMID:25970007

  15. The association between naturally acquired IgG subclass specific antibodies to the PfRH5 invasion complex and protection from Plasmodium falciparum malaria

    PubMed Central

    Weaver, Rupert; Reiling, Linda; Feng, Gaoqian; Drew, Damien R.; Mueller, Ivo; Siba, Peter M.; Tsuboi, Takafumi; Richards, Jack S.; Fowkes, Freya J. I.; Beeson, James G.

    2016-01-01

    Understanding the targets and mechanisms of human immunity to malaria is important for advancing the development of highly efficacious vaccines and serological tools for malaria surveillance. The PfRH5 and PfRipr proteins form a complex on the surface of P. falciparum merozoites that is essential for invasion of erythrocytes and are vaccine candidates. We determined IgG subclass responses to these proteins among malaria-exposed individuals in Papua New Guinea and their association with protection from malaria in a longitudinal cohort of children. Cytophilic subclasses, IgG1 and IgG3, were predominant with limited IgG2 and IgG4, and IgG subclass-specific responses were higher in older children and those with active infection. High IgG3 to PfRH5 and PfRipr were significantly and strongly associated with reduced risk of malaria after adjusting for potential confounding factors, whereas associations for IgG1 responses were generally weaker and not statistically significant. Results further indicated that malaria exposure leads to the co-acquisition of IgG1 and IgG3 to PfRH5 and PfRipr, as well as to other PfRH invasion ligands, PfRH2 and PfRH4. These findings suggest that IgG3 responses to PfRH5 and PfRipr may play a significant role in mediating naturally-acquired immunity and support their potential as vaccine candidates and their use as antibody biomarkers of immunity. PMID:27604417

  16. The association between naturally acquired IgG subclass specific antibodies to the PfRH5 invasion complex and protection from Plasmodium falciparum malaria.

    PubMed

    Weaver, Rupert; Reiling, Linda; Feng, Gaoqian; Drew, Damien R; Mueller, Ivo; Siba, Peter M; Tsuboi, Takafumi; Richards, Jack S; Fowkes, Freya J I; Beeson, James G

    2016-01-01

    Understanding the targets and mechanisms of human immunity to malaria is important for advancing the development of highly efficacious vaccines and serological tools for malaria surveillance. The PfRH5 and PfRipr proteins form a complex on the surface of P. falciparum merozoites that is essential for invasion of erythrocytes and are vaccine candidates. We determined IgG subclass responses to these proteins among malaria-exposed individuals in Papua New Guinea and their association with protection from malaria in a longitudinal cohort of children. Cytophilic subclasses, IgG1 and IgG3, were predominant with limited IgG2 and IgG4, and IgG subclass-specific responses were higher in older children and those with active infection. High IgG3 to PfRH5 and PfRipr were significantly and strongly associated with reduced risk of malaria after adjusting for potential confounding factors, whereas associations for IgG1 responses were generally weaker and not statistically significant. Results further indicated that malaria exposure leads to the co-acquisition of IgG1 and IgG3 to PfRH5 and PfRipr, as well as to other PfRH invasion ligands, PfRH2 and PfRH4. These findings suggest that IgG3 responses to PfRH5 and PfRipr may play a significant role in mediating naturally-acquired immunity and support their potential as vaccine candidates and their use as antibody biomarkers of immunity. PMID:27604417

  17. Transport across the rabbit foetal yolk-sac of fractions of IgG from several mammalian species.

    PubMed Central

    Hemmings, W A; Jones, R E; Faulk, W P

    1975-01-01

    Electrophoretically migrating fast and slow IgG fractions of rabbit, mouse, human, guinea-pig, dog, horse and cow sera were prepared by DEAE-cellulose chromatography, labelled with either 131I or 125I, and tested in passage across yolk-sac splanchnopleur of rabbits. In foetal sera, slow IgG generally reached higher concentrations than fast IgG, but even with mouse Ig, which has a high ratio between the two fractions, the difference was only four-fold. This differential between fast and slow IgG was not sufficient to explain the absorptive selectivity between IgG of different species. The capacity of isotopically labelled rabbit and equine IgG to be transported across the rabbit foetal yolk sac was also studied using fractions prepared by isoelectric focusing. Marked differences in concentration quotient (CQ) values (CQ equals (concentration in foetal serum)/(concentration in injected material)) between individual pI peaks for these species were found. Images FIG. 1 FIG. 2 PMID:1168622

  18. The global landscape of cognition: hierarchical aggregation as an organizational principle of human cortical networks and functions

    PubMed Central

    Taylor, P.; Hobbs, J. N.; Burroni, J.; Siegelmann, H. T.

    2015-01-01

    Though widely hypothesized, limited evidence exists that human brain functions organize in global gradients of abstraction starting from sensory cortical inputs. Hierarchical representation is accepted in computational networks, and tentatively in visual neuroscience, yet no direct holistic demonstrations exist in vivo. Our methods developed network models enriched with tiered directionality, by including input locations, a critical feature for localizing representation in networks generally. Grouped primary sensory cortices defined network inputs, displaying global connectivity to fused inputs. Depth-oriented networks guided analyses of fMRI databases (~17,000 experiments;~1/4 of fMRI literature). Formally, we tested whether network depth predicted localization of abstract versus concrete behaviors over the whole set of studied brain regions. For our results, new cortical graph metrics, termed network-depth, ranked all databased cognitive function activations by network-depth. Thus, we objectively sorted stratified landscapes of cognition, starting from grouped sensory inputs in parallel, progressing deeper into cortex. This exposed escalating amalgamation of function or abstraction with increasing network-depth, globally. Nearly 500 new participants confirmed our results. In conclusion, data-driven analyses defined a hierarchically ordered connectome, revealing a related continuum of cognitive function. Progressive functional abstraction over network depth may be a fundamental feature of brains, and is observed in artificial networks. PMID:26669858

  19. The global landscape of cognition: hierarchical aggregation as an organizational principle of human cortical networks and functions.

    PubMed

    Taylor, P; Hobbs, J N; Burroni, J; Siegelmann, H T

    2015-01-01

    Though widely hypothesized, limited evidence exists that human brain functions organize in global gradients of abstraction starting from sensory cortical inputs. Hierarchical representation is accepted in computational networks, and tentatively in visual neuroscience, yet no direct holistic demonstrations exist in vivo. Our methods developed network models enriched with tiered directionality, by including input locations, a critical feature for localizing representation in networks generally. Grouped primary sensory cortices defined network inputs, displaying global connectivity to fused inputs. Depth-oriented networks guided analyses of fMRI databases (~17,000 experiments;~1/4 of fMRI literature). Formally, we tested whether network depth predicted localization of abstract versus concrete behaviors over the whole set of studied brain regions. For our results, new cortical graph metrics, termed network-depth, ranked all databased cognitive function activations by network-depth. Thus, we objectively sorted stratified landscapes of cognition, starting from grouped sensory inputs in parallel, progressing deeper into cortex. This exposed escalating amalgamation of function or abstraction with increasing network-depth, globally. Nearly 500 new participants confirmed our results. In conclusion, data-driven analyses defined a hierarchically ordered connectome, revealing a related continuum of cognitive function. Progressive functional abstraction over network depth may be a fundamental feature of brains, and is observed in artificial networks. PMID:26669858

  20. Antigen Reversal Identifies Targets of Opsonizing IgGs against Pregnancy-Associated Malaria

    PubMed Central

    Lambert, Lester H.; Bullock, Jeanee L.; Cook, Sharma T.; Miura, Kazutoyo; Garboczi, David N.; Diakite, Mahamadou; Fairhurst, Rick M.; Singh, Kavita

    2014-01-01

    Clinical immunity to pregnancy associated-malaria (PAM) in multigravida women has been attributed to antibod