Science.gov

Sample records for aggregated human igg

  1. Elucidation of Acid-induced Unfolding and Aggregation of Human Immunoglobulin IgG1 and IgG2 Fc

    PubMed Central

    Latypov, Ramil F.; Hogan, Sabine; Lau, Hollis; Gadgil, Himanshu; Liu, Dingjiang

    2012-01-01

    Understanding the underlying mechanisms of Fc aggregation is an important prerequisite for developing stable and efficacious antibody-based therapeutics. In our study, high resolution two-dimensional nuclear magnetic resonance (NMR) was employed to probe structural changes in the IgG1 Fc. A series of 1H-15N heteronuclear single-quantum correlation NMR spectra were collected between pH 2.5 and 4.7 to assess whether unfolding of CH2 domains precedes that of CH3 domains. The same pH range was subsequently screened in Fc aggregation experiments that utilized molecules of IgG1 and IgG2 subclasses with varying levels of CH2 glycosylation. In addition, differential scanning calorimetry data were collected over a pH range of 3–7 to assess changes in CH2 and CH3 thermostability. As a result, compelling evidence was gathered that emphasizes the importance of CH2 stability in determining the rate and extent of Fc aggregation. In particular, we found that Fc domains of the IgG1 subclass have a lower propensity to aggregate compared with those of the IgG2 subclass. Our data for glycosylated, partially deglycosylated, and fully deglycosylated molecules further revealed the criticality of CH2 glycans in modulating Fc aggregation. These findings provide important insights into the stability of Fc-based therapeutics and promote better understanding of their acid-induced aggregation process. PMID:22084250

  2. Perturbation of thermal unfolding and aggregation of human IgG1 Fc fragment by Hofmeister anions.

    PubMed

    Zhang-van Enk, Jian; Mason, Bruce D; Yu, Lei; Zhang, Le; Hamouda, Wael; Huang, Gang; Liu, Dingjiang; Remmele, Richard L; Zhang, Jifeng

    2013-02-04

    The thermal unfolding and subsequent aggregation of the unglycosylated Fc fragment of a human IgG1 antibody (Fc) were studied in the salt solutions of Na(2)SO(4), KF, KCl and KSCN at pH 4.8 and 7.2 below and at its pI of 7.2, respectively, using differential scanning calorimetry (DSC), far ultraviolet circular dichroism (far-UV CD), size exclusion chromatography (SE-HPLC) and light scattering. First, our experimental results demonstrated that the thermal unfolding of the C(H)2 domain of the Fc was sufficient to induce aggregation. Second, at both pH conditions, the anions (except F(-)) destabilized the C(H)2 domain where the effectiveness of SO(4)(2-) > SCN(-) > Cl(-) > F(-) was more apparent at pH 4.8. In addition, the thermal stability of the C(H)2 domain was less sensitive to the change in salt concentration at pH 7.2 than at pH 4.8. Third, at pH 4.8 when the Fc had a net positive charge, the anions accelerated the aggregation reaction with SO(4)(2-) > SCN(-) > Cl(-) > F(-) in effectiveness. But these anions slowed down the aggregation kinetics at pH 7.2 with similar effectiveness when the Fc was net charge neutral. We hypothesize that the effectiveness of the anion on destabilizing the C(H)2 domain could be attributed to its ability to perturb the free energy for both of the native and unfolded states. The effect of the anions on the kinetics of the aggregation reaction could be interpreted based on the modulation of the electrostatic protein-protein interactions by the anions.

  3. N-Terminal Truncation of an Isolated Human IgG1 CH2 Domain Significantly Increases its Stability and Aggregation Resistance

    PubMed Central

    Gong, Rui; Wang, Yanping; Ying, Tianlei; Feng, Yang; Streaker, Emily; Prabakaran, Ponraj; Dimitrov, Dimiter S.

    2013-01-01

    Isolated human immunoglobulin G (IgG) CH2 domains are promising scaffolds for novel candidate therapeutics. Unlike other human IgG domains, CH2 is not involved in strong interchain interactions and isolated CH2 is relatively stable. However, isolated single CH2 is prone to aggregation. In native IgG and Fc molecules, the N-terminal residues of CH2 from the two heavy chains interact with each other and form hinge regions. By contrast, the N-terminal residues are highly disordered in isolated CH2. We have hypothesized that removal of the CH2 N-terminal residues may not only increase its stability but also its aggregation resistance. To test this hypothesis we constructed a shortened variant of IgG1 CH2 (CH2s) where the first seven residues of the N-terminus were deleted. We found that the thermal stability of CH2s was increased by 5°C compared to CH2. Importantly, we demonstrated that CH2s is significantly less prone to aggregation than CH2 as measured by Thioflavin T (ThT) fluorescence, turbidity and light scattering. We also found that the CH2s exhibited pH-dependent binding to a soluble single-chain human neonatal Fc receptor (shFcRn) which was significantly stronger than the very weak shFcRn binding to CH2 as measured by flow cytometry. Computer modeling suggested a possible mode of CH2 aggregation involving its N-terminal residues. Therefore, deletion of the N-terminal residues could increase drugability of CH2-based therapeutic candidates. This strategy to increase stability and aggregation resistance could also be applicable to other Ig-related proteins. PMID:23641816

  4. 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

  5. Binding kinetics of monomeric and aggregated IgG to Kupffer cells and hepatocytes of mice.

    PubMed Central

    Sancho, J; González, E; Escanero, J F; Egido, J

    1984-01-01

    The binding kinetics of human monomeric IgG and stable heat-aggregated IgG (A-IgG) to Fc receptors of hepatocytes and Kupffer cells isolated from mice was studied. After injection of radiolabelled proteins the 60-70% of hepatic uptake was recovered in parenchymal cells (hepatocytes). In experiments in vitro the A-IgG bound in larger amounts to hepatocytes and Kupffer cells than monomeric IgG. The association rate constants of aggregates were somewhat higher for Kupffer cells than for hepatocytes whereas the percentage uptake of aggregates by Kupffer cells was only 5-15% of that of hepatocytes. The equilibrium constants of aggregates binding to both cells amounted to 0.4-1 X 10(8) M-1 for A-IgG compared with an equilibrium constant for monomeric IgG of 1-2 X 10(7)M-1. The maximum number of IgG and A-IgG molecules bound per cell was higher on hepatocytes (mean 14 X 10(6)) than on Kupffer cells (mean 2 X 10(5)) which is in agreement with the higher binding capacity of hepatocytes for these proteins observed in vivo and in vitro experiments. The ability to compete for receptor binding seemed to reside exclusively in the Fc portion of IgG since F(ab')2 fragments of IgG failed to inhibit labelled monomeric IgG or A-IgG. The receptor seems to be specific for IgG since unlabelled monomeric IgA demonstrated no binding inhibition of labelled IgG or A-IgG on hepatocytes and Kupffer cells. The overall results further suggest that hepatocytes might through Fc receptors play a collaborative role with the mononuclear phagocytic system in the clearance of circulating immune complexes. PMID:6237982

  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. Phase transitions in human IgG solutions.

    PubMed

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

    2013-09-28

    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

  8. Enhancement of platelet response to immune complexes and IgG aggregates by lipid A-rich bacterial lipopolysaccharides

    PubMed Central

    1978-01-01

    The effect of the common lipid moiety of bacterial LPS on secretion from washed human platelets has been studied. The lipid A-rich LPS of S. minnesota R595 and a lipid A preparation both potentiated platelet serotonin secretion in response to IgG aggregates or immune complexes up to 50-fold but had little effect in the absence of IgG. Lipid A has been shown to bind immune aggregates, raising the possibility that its mechanism of action involved effective enlargement or insolubilization of the aggregates. IgG aggregates of dimer to tetramer size were shown to be platelet simuli, equivalent on a weight basis to larger soluble aggregates. The effect of both sizes of aggregates on platelets were equally enhanced by the LPS, indicating that increased size of aggregates alone could not account for the effect of LPS. Similarly, because lipid A-rich LPS enhanced platelet response to already insoluble immune complexes, its mechanism of action cannot simply be insolubilization of immune aggregates. These LPS did not enhance platelet stimulation by antiplatelet antibody, monosodium urate crystals, or thrombin and only slightly enhanced stimulation by insoluble human skin collagen. This indicates some stimulus specificity in the ability of LPS to increase platelet secretion. The enhancement of cell response to immune complexes by the common lipid region of LPS may represent a mechanism for the diverse effects of LPS in vivo and in vitro. PMID:627836

  9. 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).

  10. Effects of human IgG on locomotion of human neutrophils related to IgG binding of a hydrophobic probe.

    PubMed Central

    Wilkinson, P C

    1980-01-01

    Heat-denatured (63 degrees) human IgG had complex effects on locomotion of human neutrophils. At concentrations of 1 mg/ml and below, it stimulated chemotactic locomotion into filters judged by the leading front assay, however, pre-treatment of the cells or of the filters with denatured IgG caused a reduction in the number of locomoting cells, compared to cells locomoting in a medium containing albumin. These effects took place in complement-free media. Native IgG was not chemotactic. The chemotactic activity of denatured IgG correlated well with increased binding by the same IgG preparations of the hydrophobic probe, 1-anilinonaphthalene-8-sulphonate (ANS), and it is suggested that heating induces a conformational change in the IgG molecule which allows recognition of the altered molecule by neutrophils and activation of a chemotactic response. The integrity of the Fc fragment is required for this activity. As well as a direct chemoattractant activity of IgG, evidence for release of chemotactic factors by cells in contact with aggregated IgG was also obtained. It is suggested that the contrary effects of denatured IgG on neutrophil locomotion are explicable if the protein, like other denatured proteins, activates the sensory chemotactic mechanism in the neutrophil, while at the same time causing a modification of adhesion of cell to substratum which may impair the locomotor capacity of the cells. PMID:7439936

  11. Aggregation and pH-temperature phase behavior for aggregates of an IgG2 antibody.

    PubMed

    Sahin, Erinc; Weiss, William F; Kroetsch, Andrew M; King, Kevin R; Kessler, R Kendall; Das, Tapan K; Roberts, Christopher J

    2012-05-01

    Monomer unfolding and thermally accelerated aggregation kinetics to produce soluble oligomers or insoluble macroscopic aggregates were characterized as a function of pH for an IgG2 antibody using differential scanning calorimetry (DSC) and size-exclusion chromatography (SEC). Aggregate size was quantified via laser light scattering, and aggregate solubility via turbidity and visual inspection. Interestingly, nonnative oligomers were soluble at pH 5.5 above approximately 15°C, but converted reversibly to visible/insoluble particles at lower temperatures. Lower pH values yielded only soluble aggregates, whereas higher pH resulted in insoluble aggregates, regardless of the solution temperature. Unlike the growing body of literature that supports the three-endotherm model of IgG1 unfolding in DSC, the results here also illustrate limitations of that model for other monoclonal antibodies. Comparison of DSC with monomer loss (via SEC) from samples during thermal scanning indicates that the least conformationally stable domain is not the most aggregation prone, and that a number of the domains remain intact within the constituent monomers of the resulting aggregates. This highlights continued challenges with predicting a priori which domain(s) or thermal transition(s) is(are) most relevant for product stability with respect to aggregation.

  12. Immunogenicity of Isogenic IgG in Aggregates and Immune Complexes.

    PubMed

    St Clair, J Benjamin; Detanico, Thiago; Aviszus, Katja; Kirchenbaum, Greg A; Christie, Merry; Carpenter, John F; Wysocki, Lawrence J

    2017-01-01

    A paradox in monoclonal antibody (mAb) therapy is that despite the well-documented tolerogenic properties of deaggregated IgG, most therapeutic IgG mAb induce anti-mAb responses. To analyze CD4 T cell reactions against IgG in various physical states, we developed an adoptive transfer model using CD4+ T cells specific for a Vκ region-derived peptide in the hapten-specific IgG mAb 36-71. We found that heat-aggregated or immune complexes (IC) of mAb 36-71 elicited anti-idiotypic (anti-Id) antibodies, while the deaggregated form was tolerogenic. All 3 forms of mAb 36-71 induced proliferation of cognate CD4+ T cells, but the aggregated and immune complex forms drove more division cycles and induced T follicular helper cells (TFH) development more effectively than did the deaggregated form. These responses occurred despite no adjuvant and no or only trace levels of endotoxin in the preparations. Physical analyses revealed large differences in micron- and nanometer-sized particles between the aggregated and IC forms. These differences may be functionally relevant, as CD4+ T cell proliferation to aggregated, but not IC mAb 36-71, was nearly ablated upon peritoneal injection of B cell-depleting antibody. Our results imply that, in addition to denatured aggregates, immune complexes formed in vivo between therapeutic mAb and their intended targets can be immunogenic.

  13. Comparison of the role of tyrosine residues in human IgG and rabbit IgG in binding of complement subcomponent C1q.

    PubMed Central

    McCall, M N; Easterbrook-Smith, S B

    1989-01-01

    Treatment of covalently cross-linked or heat-aggregated oligomers of human IgG with 4 mM-tetranitromethane abrogated their C1q-binding activity. In contrast, tetranitromethane modification of rabbit IgG oligomers, under identical conditions, had no effect upon their C1q-binding activity. The tetranitromethane treatment led to nitration of about ten tyrosine residues per IgG molecule in both species, and the modification was specific for tyrosine residues. Reduction of the nitrated protein with Na2S2O4 did not lead to recovery of C1q-binding activity in human IgG oligomers or to loss of activity in rabbit IgG oligomers. Tryptic peptides from the nitrated proteins were isolated and a peptide containing nitrotyrosine-319 was recovered from human IgG, as well as peptides from both species corresponding to the region around nitrotyrosine-278. These data are consistent with the inactivation of C1q-binding activity in human IgG being the result of nitration of tyrosine-319; the rabbit IgG is unaffected by nitration because position 319 is phenylalanine. The evidence supports the C1q-receptor site proposed by Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek [(1980) Nature (London) 288, 338-344]: residues 316-338. PMID:2784672

  14. 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

  15. Immunogenicity of Isogenic IgG in Aggregates and Immune Complexes

    PubMed Central

    St. Clair, J. Benjamin; Detanico, Thiago; Aviszus, Katja; Kirchenbaum, Greg A.; Christie, Merry; Carpenter, John F.

    2017-01-01

    A paradox in monoclonal antibody (mAb) therapy is that despite the well-documented tolerogenic properties of deaggregated IgG, most therapeutic IgG mAb induce anti-mAb responses. To analyze CD4 T cell reactions against IgG in various physical states, we developed an adoptive transfer model using CD4+ T cells specific for a Vκ region-derived peptide in the hapten-specific IgG mAb 36–71. We found that heat-aggregated or immune complexes (IC) of mAb 36–71 elicited anti-idiotypic (anti-Id) antibodies, while the deaggregated form was tolerogenic. All 3 forms of mAb 36–71 induced proliferation of cognate CD4+ T cells, but the aggregated and immune complex forms drove more division cycles and induced T follicular helper cells (TFH) development more effectively than did the deaggregated form. These responses occurred despite no adjuvant and no or only trace levels of endotoxin in the preparations. Physical analyses revealed large differences in micron- and nanometer-sized particles between the aggregated and IC forms. These differences may be functionally relevant, as CD4+ T cell proliferation to aggregated, but not IC mAb 36–71, was nearly ablated upon peritoneal injection of B cell-depleting antibody. Our results imply that, in addition to denatured aggregates, immune complexes formed in vivo between therapeutic mAb and their intended targets can be immunogenic. PMID:28114383

  16. 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.

  17. 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.

  18. Reformatting Rituximab into Human IgG2 and IgG4 Isotypes Dramatically Improves Apoptosis Induction In Vitro

    PubMed Central

    Könitzer, Jennifer D.; Sieron, Annette; Wacker, Angelika; Enenkel, Barbara

    2015-01-01

    The direct induction of cell death, or apoptosis, in target cells is one of the effector mechanisms for the anti CD20 antibody Rituximab. Here we provide evidence that Rituximab’s apoptotic ability is linked to the antibody IgG isotype. Reformatting Rituximab from the standard human IgG1 heavy chain into IgG2 or IgG4 boosted in vitro apoptosis induction in the Burkitt’s lymphoma B cell line Ramos five and four-fold respectively. The determinants for this behavior are located in the hinge region and CH1 domain of the heavy chain. By transplanting individual IgG2 or IgG4 specific amino acid residues onto otherwise IgG1 like backbones, thereby creating hybrid antibodies, the same enhancement of apoptosis induction could be achieved. The cysteines at position 131 of the CH1 domain and 219 in the hinge region, involved in IgG2 and IgG4 disulfide formation, were found to be of particular structural importance. Our data indicates that the hybrid antibodies possess a different CD20 binding mode than standard Rituximab, which appears to be key in enhancing apoptotic ability. The presented work opens up an interesting engineering route for enhancing the direct cytotoxic ability of therapeutic antibodies. PMID:26713448

  19. Human/mouse chimeric monoclonal antibodies with human IgG1, IgG2, IgG3 and IgG4 constant domains: electron microscopic and hydrodynamic characterization.

    PubMed

    Phillips, M L; Tao, M H; Morrison, S L; Schumaker, V N

    1994-10-01

    The unique structure of the human IgG3 constant region with its greatly extended hinge can clearly be seen in electron micrographs, which compare a series of recombinant proteins with the same murine anti-dansyl variable domain but constant domains from human IgG1, IgG2, IgG3 and IgG4. The hinge region of IgG3 was found to be very long, with some measurements extending to 100 A. It exhibited considerable flexibility allowing the Fc to be displaced far toward either side. Upon addition of bivalent hapten, all of the monoclonal antibodies formed complexes. IgG1, IgG3 and IgG4 formed circular dimers, composed of two antibodies forming a ring-shaped complex, presumably through the binding of two bivalent haptens. IgG2, on the other hand, showed a distribution of complexes which was noticeably different from the other subclasses. Some circular dimers, some linear dimers and a large amount of monomer were seen. This was interpreted in terms of an energy barrier to ring closure arising from the orientation of the Fab arms of IgG2 probably leading to linear dimers as the predominate complex seen with the analytical ultracentrifuge. A substantial number of these dimers probably dissociated upon dilution for examination in the electron microscope. The distribution of the angles between the Fab arms of the monoclonal antibodies forming the circular dimers has been measured for the different subclasses. Most were open at wide angles (> 100 degrees) but some formed very shallow angles, with the Fab arms being nearly parallel to each other. The free energy for this transition was calculated from the ratio of open/closed angles, and it was found to be proportional to the length of the upper hinge of the monoclonal antibody, in agreement with previous nanosecond depolarization results (Dangl et al., Eur. molec. Biol. Org. J. 7, 1989-1994, 1988).

  20. 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.

  1. The placental transfer of IgG subclasses in human pregnancy.

    PubMed Central

    Pitcher-Wilmott, R W; Hindocha, P; Wood, C B

    1980-01-01

    Total IgG concentrations and the concentrations of the four subclasses of IgG were estimated in thirty-four pairs of maternal and foetal sera from pregnancies of various gestations ranging from 28 to 42 weeks using the method of radial immunodiffusion. It was found that: (1) all subclasses of IgG cross the human placenta freely, (2) foetal levels of IgG and each subclass of IgG exceed maternal levels in full-term pregnancies and (3) there was a close linear relationship between gestational age and the placental transfer of IgG and each of its subclasses. PMID:7438556

  2. [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.

  3. Design and Evaluation of the Highly Concentrated Human IgG Formulation Using Cyclodextrin Polypseudorotaxane Hydrogels.

    PubMed

    Higashi, Taishi; Tajima, Anna; Ohshita, Naoko; Hirotsu, Tatsunori; Abu Hashim, Irhan Ibrahim; Motoyama, Keiichi; Koyama, Sawako; Iibuchi, Ruriko; Mieda, Shiuhei; Handa, Kenji; Kimoto, Tomoaki; Arima, Hidetoshi

    2015-12-01

    To achieve the potent therapeutic effects of human immunoglobulin G (IgG), highly concentrated formulations are required. However, the stabilization for highly concentrated human IgG is laborious work. In the present study, to investigate the potentials of polypseudorotaxane (PPRX) hydrogels consisting of polyethylene glycol (PEG) and α- or γ-cyclodextrin (α- or γ-CyD) as pharmaceutical materials for highly concentrated human IgG, we designed the PPRX hydrogels including human IgG and evaluated their pharmaceutical properties. The α- and γ-CyDs formed PPRX hydrogels with PEG (M.W. 20,000) even in the presence of highly concentrated human IgG (>100 mg/mL). According to the results of (1)H-NMR, powder X-ray diffraction, and Raman microscopy, the formation of human IgG/CyD PPRX hydrogels was based on physical cross-linking arising from their columnar structures. The release profiles of human IgG from the hydrogels were in accordance with the non-Fickian diffusion model. Importantly, the stabilities of human IgG included into the hydrogels against thermal and shaking stresses were markedly improved. These findings suggest that PEG/CyD PPRX hydrogels are useful to prepare the formulation for highly concentrated human IgG.

  4. 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

  5. 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

  6. 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.

  7. IL-27 induces the production of IgG1 by human B cells.

    PubMed

    Boumendjel, Amel; Tawk, Lina; Malefijt, René de Waal; Boulay, Vera; Yssel, Hans; Pène, Jérôme

    2006-12-01

    It has been reported that IL-27 specifically induces the production of IgG2a by mouse B cells and inhibits IL-4-induced IgG1 synthesis. Here, we show that human naïve cord blood expresses a functional IL-27 receptor, consisting of the TCCR and gp130 subunits, although at lower levels as compared to naïve and memory splenic B cells. IL-27 does not induce proliferative responses and does not increase IgG1 production by CD19(+)CD27(+) memory B cells. However, it induces a low, but significant production of IgG1 by naïve CD19(+)CD27(-)IgD(+)IgG(-) spleen and cord blood B cells, activated via CD40, whereas it has no effect on the production of the other IgG subclasses. In addition, IL-27 induces the differentiation of a population of B cells that express high levels of CD38, in association with a down-regulation of surface IgD expression, and that are surface IgG(+/int), CD20(low), CD27(high), indicating that IL-27 promotes isotype switching and plasma cell differentiation of naive B cells. However, as compared to the effects of IL-21 and IL-10, both switch factors for human IgG1 and IgG3, those of IL-27 are modest and regulate exclusively the production of IgG1. Finally, although IL-27 has no effect on IL-4 and anti-CD40-induced Cepsilon germline promoter activity, it up-regulates IL-4-induced IgE production by naive B cells. These results point to a partial redundancy of switch factors regulating the production of IgG1 in humans, and furthermore indicate the existence of a common regulation of the human IgG1and murine IgG2a isotypes by IL-27.

  8. Highly individual patterns of virus-immune IgG effector responses in humans.

    PubMed

    Corrales-Aguilar, Eugenia; Trilling, Mirko; Reinhard, Henrike; Falcone, Valeria; Zimmermann, Albert; Adams, Ortwin; Santibanez, Sabine; Hengel, Hartmut

    2016-10-01

    IgG responses are fundamental to adaptive immunity and document immunological memory of previous pathogen encounter. While specific antigen recognition is mediated by the variable F(ab')2 domain of IgG, various effector functions become activated via the constant Fcγ part bridging IgG-opsonized targets to FcγR-expressing immune effector cells. Traditionally, neutralizing IgG is considered the most appropriate correlate of protective humoral immunity to viruses. However, evidence is increasing that antiviral IgG mediates protection to viruses via activation of FcγRs. Using a test system allowing quantitative detection of virus-immune IgG able to activate FcγRs, sera of healthy individuals and vaccinees were assessed with regard to two prototypical human pathogenic viruses: measles and human cytomegalovirus. Marked differences in the capacity of individuals to generate FcγRI-, FcγRII- and FcγRIII-activating responses were noted. Comparison of FcγR-activating IgG with neutralizing and ELISA IgG concentrations did not correlate for HCMV and only very poorly for MV. Since neither neutralizing IgG nor overall IgG responses faithfully predict the activation of FcγRs, only the simultaneous quantification of IgGs activating defined FcγRs will aid to delineate individual "immunograms" of virus IgG immunity. Such new multiparametric assessment of antiviral IgG qualities could be instrumental in defining correlates of protection and disease progression.

  9. Half molecular exchange of IgGs in the blood of healthy humans: chimeric lambda-kappa-immunoglobulins containing HL fragments of antibodies of different subclasses (IgG1-IgG4).

    PubMed

    Sedykh, Sergey E; Lekchnov, Evgenii A; Prince, Viktor V; Buneva, Valentina N; Nevinsky, Georgy A

    2016-10-20

    In the classic paradigm, immunoglobulins represent products of clonal B cell populations, each producing antibodies recognizing a single antigen (monospecific). There is a common belief that IgGs in mammalian biological fluids are monospecific molecules having stable structures and two identical antigen-binding sites. But the issue concerning the possibility of exchange by HL-fragments between the antibody molecules in human blood is still unexplored. Different physico-chemical and immunological methods for analysis of half-molecule exchange between human blood IgGs were used. Using eighteen blood samples of healthy humans we have shown unexpected results for the first time: blood antibodies undergo extensive post-transcriptional half-molecule exchange and IgG pools on average consist of 62.4 ± 6.5% IgGs containing kappa light chains (kappa-kappa-IgGs), 29.8.6 ± 5.4% lambda light chains (lambda-lambda-IgGs), and 8.8 ± 2.7% (range 2.6-16.8%) IgGs containing both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained on average (%): IgG1 (36.0 and 32.3), IgG2 (50.9 and 51.4), IgG3 (9.7 and 9.9), and IgG4 (6.5 and 5.7), while chimeric kappa-lambda-IgGs consisted of (%): 25.5 ± 4.2 IgG1, 50.8 ± 3.9 IgG2, 9.1 ± 2.1 IgG3, and 14.5 ± 2.2 IgG4. Our unexpected data are indicative of the possibility of half-molecule exchange between blood IgGs of various subclasses, raised against different antigens. The existence of blood chimeric bifunctional IgGs with different binding sites destroys the classic paradigm. Due to the phenomenon of polyspecificity and cross-reactivity of bifunctional IgGs containing HL-fragments of different types to different antigens, such IgGs may be important in human blood for widening their different biological functions.

  10. Human salivary aggregation in Streptococcus intermedius type g strains: relationship with IgA.

    PubMed

    Yamaguchi, Taihei

    2004-06-01

    Bacterial aggregation is an important step in elimination from the human body to protect against infection. Streptococcus intermedius K1K aggregates in human saliva. In this study, the salivary agglutinin was identified. The aggregation level was very strong in sonic-treated saliva and 1-microm filtrate. Preincubation of human saliva with anti-human alpha chain serum or anti-human whole saliva serum completely inhibited aggregation, but preincubation with anti-human micro chain serum or anti-Fc fragment of human IgG serum had no effect. Agglutinin of human saliva that could aggregate the strain K1K was purified using DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Sephacryl S200HR gel filtration. Purified salivary agglutinin was characterized with electrophoresis and immunological techniques, indicating that purified material was IgA. Bacterial aggregation was dependent on the presence of calcium. Saliva filtrate specimens from eight healthy men and eight women showed different aggregation activities. Three men and one woman had little activity. These data show that the present bacterial aggregation was an immunoreaction between IgA in saliva and the bacteria dependent on the levels of calcium. In addition, the IgA in human saliva related with possible calcium-dependent antigen(s) on the surface of strain K1K.

  11. Clearance of soluble aggregates of human immunoglobulin G in healthy volunteers and chimpanzees.

    PubMed

    Lobatto, S; Daha, M R; Voetman, A A; Evers-Schouten, J H; Van Es, A A; Pauwels, E K; Van Es, L A

    1987-07-01

    Using aggregates of IgG (AIgG) obtained by heat aggregation of a 16 g% human IgG solution, we sought a method for measuring the function of the mononuclear phagocyte system with a probe that bears more resemblance to soluble immune complexes than erythrocytes coated with anti-rhesus IgG (EIgG). It was found that intravenous administration of 10 micrograms AIgG/kg body weight did not cause any detectable side effects in chimpanzees. In nine healthy volunteers, a dose of 10 micrograms AIgG/kg body weight was used without any adverse reactions. AIgG is cleared from the human circulation with a t1/2 of 26 +/- 8 min (mean +/- SD). The site of clearance is predominantly the liver, as shown by an average liver spleen uptake ratio of 230:100. In whole blood obtained from the volunteers, it was found that erythrocytes bound significant amounts of AIgG, suggesting that CR1 on erythrocytes is involved in the clearance of complement activating immune complexes in humans. In five of the volunteers, clearance studies with EIgG had been done in a previous study. EIgG was cleared from the circulation with a t1/2 of 30 +/- 6.2 min (mean +/- SD). The predominant site of clearance of EIgG was the spleen. These data indicate that sensitized red blood cells are cleared from the circulation differently from soluble IgG aggregates.

  12. Protein aggregation profile of the human kinome

    PubMed Central

    Graña-Montes, Ricardo; Sant'Anna de Oliveira, Ricardo; Ventura, Salvador

    2012-01-01

    Protein aggregation into amyloid fibrils is associated with the onset of an increasing number of human disorders, including Alzheimer's disease, diabetes, and some types of cancer. The ability to form toxic amyloids appears to be a property of most polypeptides. Accordingly, it has been proposed that reducing aggregation and its effect in cell fitness is a driving force in the evolution of proteins sequences. This control of protein solubility should be especially important for regulatory hubs in biological networks, like protein kinases. These enzymes are implicated in practically all processes in normal and abnormal cell physiology, and phosphorylation is one of the most frequent protein modifications used to control protein activity. Here, we use the AGGRESCAN algorithm to study the aggregation propensity of kinase sequences. We compared them with the rest of globular proteins to decipher whether they display differential aggregation properties. In addition, we compared the human kinase complement with the kinomes of other organisms to see if we can identify any evolutionary trend in the aggregational properties of this protein superfamily. Our analysis indicates that kinase domains display significant aggregation propensity, a property that decreases with increasing organism complexity. PMID:23181023

  13. 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

  14. 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.

  15. 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

  16. Immunoglobulin-sulfated polysaccharide interactions. Binding of agaropectin and heparin by human IgG proteins

    PubMed Central

    1981-01-01

    The interaction of immunoglobulins with certain acidic polysaccharides was demonstrated by the binding of the sulfated glycans agaropectin and heparin by certain human IgG proteins. Heparin-binding IgG proteins can distinguish between the molecular forms of heparin derived from porcine intestine, bovine lung, and rat skin. The major specificity of these proteins is for native and certain high molecular weight subunit components of rat skin heparin. The interactions with multi-chain and single chain rat skin heparin are stable under physiological conditions and involve the Fab and, more specifically, the Fv region of the IgG molecule. These reactions occur as a result of an electrostatic interaction between cationic sites on certain IgG proteins and anionic sulfate resides of agaropectin or heparin. The characteristics of heparin-IgG interaction resemble those of heparin with other plasma proteins, the interactions of which have biological significance. PMID:7252414

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

    PubMed

    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.

  18. 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

  19. Conversion of a Mouse Fab into a Whole Humanized IgG Antibody for Detecting Botulinum Toxin

    DTIC Science & Technology

    2006-04-01

    pentavalent toxoid; Fab, antibody fragment; HRP, horseradish peroxidase; LCκ, kappa light chain ; scFv, single- chain antibody fragments; VL, variable light ...The variable regions from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells. Purified...from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells. Purified humanized IgG demonstrated

  20. Aggregates of ultrafine particles impair phagocytosis of microorganisms by human alveolar macrophages.

    PubMed

    Lundborg, Margot; Dahlén, Sven-Erik; Johard, Urban; Gerde, Per; Jarstrand, Connie; Camner, Per; Låstbom, Lena

    2006-02-01

    We investigated whether exposure of alveolar macrophages to aggregates of ultrafine carbon particles affected subsequent phagocytosis of microorganisms. Human alveolar macrophages were obtained by bronchoalveolar lavage and exposed to aggregates of ultrafine carbon particles or diesel exhaust particles (DEP) for 20 h before measurements of phagocytosis. The particle loads were estimated to be comparable to those of air pollution exposure with established health effects in humans. Phagocytotic activity was measured as attachment and ingestion of four different test particles (amorphous silica particles, yeast cells from Candida albicans, and Cryptococcus neoformans opsonized with specific IgG or fresh serum) that bind to scavenger, mannose, Fc, and complement receptors, respectively. Carbon preloading significantly impaired the attachment and ingestion process (P<0.01) for all particles, except for yeast cells from C. neoformans opsonized with specific IgG. On the average, the accumulated attachment decreased by 30% and the ingested fraction decreased by 10%. Loading of alveolar macrophages with either aggregates of ultrafine DEP or carbon particles impaired the phagocytosis of silica test particles in a similar way. Exposure of human alveolar macrophages to aggregates of carbon or DEP, in concentrations relevant to human environmental exposures, caused significant impairment of phagocytosis of silica particles and microorganisms. The inhibitory effect on particle phagocytosis mediated by four different receptors suggests that air pollution particles cause a general inhibition of macrophage phagocytosis. Such an effect may contribute to increased susceptibility to infections and, for example, result in more exacerbations of asthma and chronic obstructive pulmonary disease.

  1. Fc gamma receptor IIb participates in maternal IgG trafficking of human placental endothelial cells

    PubMed Central

    ISHIKAWA, TOMOKO; TAKIZAWA, TAKAMI; IWAKI, JUN; MISHIMA, TAKUYA; UI-TEI, KUMIKO; TAKESHITA, TOSHIYUKI; MATSUBARA, SHIGEKI; TAKIZAWA, TOSHIHIRO

    2015-01-01

    The human placental transfer of maternal IgG is crucial for fetal and newborn immunity. Low-affinity immunoglobulin gamma Fc region receptor IIb2 (FCGR2B2 or FcγRIIb2) is exclusively expressed in an IgG-containing, vesicle-like organelle (the FCGR2B2 compartment) in human placental endothelial cells; thus, we hypothesized that the FCGR2B2 compartment functions as an IgG transporter. In this study, to examine this hypothesis, we performed in vitro bio-imaging analysis of IgG trafficking by FCGR2B2 compartments using human umbilical vein endothelial cells transfected with a plasmid vector containing enhanced GFP-tagged FCGR2B2 (pFCGR2B2-EGFP). FCGR2B2-EGFP signals were detected as intracellular vesicular structures similar to FCGR2B2 compartments in vivo. The internalization and transcytosis of IgG was significantly higher in the pFCGR2B2-EGFP-transfected cells than in the mock-transfected cells, and the majority of the internalized IgG was co-localized with the FCGR2B2-EGFP signals. Furthermore, we isolated FCGR2B2 compartments from the human placenta and found that the Rab family of proteins [RAS-related protein Rab family (RABs)] were associated with FCGR2B2 compartments. Among the RABs, RAB3D was expressed predominantly in placental endothelial cells. The downregulation of RAB3D by small interfering RNA (siRNA) resulted in a marked reduction in the FCGR2B2-EGFP signals at the cell periphery. Taken together, these findings suggest that FCGR2B2 compartments participate in the transcytosis of maternal IgG across the human placental endothelium and that RAB3D plays a role in regulating the intracellular dynamics of FCGR2B2 compartments. PMID:25778799

  2. Distribution of the IgG Fc Receptor, FcRn, in the Human Fetal Intestine

    PubMed Central

    Shah, Uzma; Dickinson, Bonny L.; Blumberg, Richard S.; Simister, Neil E.; Lencer, Wayne I.; Walker, W. Allan

    2010-01-01

    The intestinal Fc receptor, FcRn, functions in the maternofetal transfer of gamma globulin (IgG) in the neonatal rodent. In humans, most of this transfer is presumed to occur in utero via the placenta. Although the fetus swallows amniotic fluid that contains immunoglobulin, it is unknown whether this transfer also occurs via the fetal intestine. A human FcRn has been identified in the syncytiotrophoblast that mediates the maternofetal transfer of antibody. It has also been identified in human fetal intestine and is postulated to function in IgG transport. We hypothesize that the human fetal intestinal FcRn may play a role in IgG transport from the amniotic fluid into the fetal circulation. The aim of this study was to characterize the distribution of the FcRn along the human fetal intestine. Lysates prepared from human fetal intestine and from a nonmalignant human fetal intestinal epithelial cell line (H4) were subjected to Western blot analysis and probed using anti-FcRn antibodies. A 42-kD band, consistent with the known molecular weight of the FcRn, was detected along the human fetal intestine and in H4 cells. Expression of the human FcRn was confirmed with immunohistochemistry. Our study demonstrates the expression of FcRn along the human fetal intestine and in a human nonmalignant fetal intestinal epithelial cell line (H4), which by location indicates that FcRn could play a role in the uptake and transport of IgG in the human fetus. PMID:12538789

  3. Cutting edge: IL-21 is a switch factor for the production of IgG1 and IgG3 by human B cells.

    PubMed

    Pène, Jérôme; Gauchat, Jean-François; Lécart, Sandrine; Drouet, Elodie; Guglielmi, Paul; Boulay, Vera; Delwail, Adriana; Foster, Don; Lecron, Jean-Claude; Yssel, Hans

    2004-05-01

    IL-21 is a cytokine that regulates the activation of T and NK cells and promotes the proliferation of B cells activated via CD40. In this study, we show that rIL-21 strongly induces the production of all IgG isotypes by purified CD19(+) human spleen or peripheral blood B cells stimulated with anti-CD40 mAb. Moreover, it was found to specifically induce the production of IgG(1) and IgG(3) by CD40-activated CD19(+)CD27(-) naive human B cells. Although stimulation of CD19(+) B cells via CD40 alone induced gamma 1 and gamma 3 germline transcripts, as well as the expression of activation-induced cytidine deaminase, only stimulation with both anti-CD40 mAb and rIL-21 resulted in the production of S gamma/S mu switch circular DNA. These results show that IL-21, in addition to promoting growth and differentiation of committed B cells, is a specific switch factor for the production of IgG(1) and IgG(3).

  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. Adsorption of human serum proteins onto TREN-agarose: purification of human IgG by negative chromatography.

    PubMed

    Bresolin, Igor Tadeu Lazzarotto; Borsoi-Ribeiro, Mariana; Caro, Juliana Rodrigues; dos Santos, Francine Petit; de Castro, Marina Polesi; Bueno, Sonia Maria Alves

    2009-01-01

    Tris(2-aminoethyl)amine (TREN) - a chelating agent used in IMAC - immobilized onto agarose gel was evaluated for the purification of IgG from human serum by negative chromatography. A one-step purification process allowed the recovery of 73.3% of the loaded IgG in the nonretained fractions with purity of 90-95% (based on total protein concentration and nephelometric analysis of albumin, transferrin, and immunoglobulins A, G, and M). The binding capacity was relatively high (66.63 mg of human serum protein/mL). These results suggest that this negative chromatography is a potential technique for purification of IgG from human serum.

  6. Two-step chromatography purification of IgGs possessing sialidase activity from human blood serum.

    PubMed

    Kit, Yury; Bilyy, Rostyslav; Korniy, Nataliya; Tomin, Andriy; Chop'yak, Valentyna; Tolstyak, Yaroslav; Antonyuk, Volodymyr; Stoika, Rostyslav

    2015-03-01

    Sialation of cell surface is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential and clearance of aged cells, while sialation of immunoglobulin G (IgG) molecules determines their anti-inflammatory properties. Recently, we have found for the first time IgG-antibodies possessing sialidase-like activity (sialylic abzyme) in blood serum of multiple myeloma and systemic lupus erythematosis patients. This abzyme was detected in a pool of IgGs purified by a typical procedure including immunoglobulin's precipitation with ammonium sulfate and following chromatography on protein G-Sepharose column. Here we describe a novel matrix for affinity purification of sialylic abzyme that is based on using bovine submandibular gland mucin conjugated to Sepharose matrix (mucin-Sepharose). This matrix preferentially binds sialidase-like IgGs from a pool of sialidase-active fraction of proteins precipitated with 50% ammonium sulfate from blood serum of the systemic lupus erythematosis patients. That allowed us to develop a new scheme of double-step chromatography purification of sialidase-like IgGs from human blood serum.

  7. 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

  8. [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.

  9. The high-affinity receptor for IgG, FcγRI, of humans and non-human primates.

    PubMed

    Chenoweth, Alicia M; Trist, Halina M; Tan, Peck-Szee; Wines, Bruce D; Hogarth, P Mark

    2015-11-01

    Non-human primate (NHP) models, especially involving macaques, are considered important models of human immunity and have been essential in preclinical testing for vaccines and therapeutics. Despite this, much less characterization of macaque Fc receptors has occurred compared to humans or mice. Much of the characterization of macaque Fc receptors so far has focused on the low-affinity Fc receptors, particularly FcγRIIIa. From these studies, it is clear that there are distinct differences between the human and macaque low-affinity receptors and their interaction with human IgG. Relatively little work has been performed on the high-affinity IgG receptor, FcγRI, especially in NHPs. This review will focus on what is currently known of how FcγRI interacts with IgG, from mutation studies and recent crystallographic studies of human FcγRI, and how amino acid sequence differences in the macaque FcγRI may affect this interaction. Additionally, this review will look at the functional consequences of differences in the amino acid sequences between humans and macaques.

  10. Serum or breast milk immunoglobulins mask the self-reactivity of human natural IgG antibodies.

    PubMed

    Djoumerska-Alexieva, Iglika; Manoylov, Iliyan; Dimitrov, Jordan D; Tchorbanov, Andrey

    2014-04-01

    B cells producing IgG antibodies specific to a variety of self- or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein-destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab')2 fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self-reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients.

  11. A C1q Domain Containing Protein from Scallop Chlamys farreri Serving as Pattern Recognition Receptor with Heat-Aggregated IgG Binding Activity

    PubMed Central

    Wang, Leilei; Wang, Lingling; Zhang, Huan; Zhou, Zhi; Siva, Vinu S.; Song, Linsheng

    2012-01-01

    Background The C1q domain containing (C1qDC) proteins refer to a family of all proteins that contain the globular C1q (gC1q) domain, and participate in a series of immune responses depending on their gC1q domains to bind a variety of self and non-self binding ligands. Methodology In the present study, the mRNA expression patterns, localization, and activities of a C1qDC protein from scallop Chlamys farreri (CfC1qDC) were investigated to understand its possible functions in innate immunity. The relative expression levels of CfC1qDC mRNA in hemocytes were all significantly up-regulated after four typical PAMPs (LPS, PGN, β-glucan and polyI:C) stimulation. During the embryonic development of scallop, the mRNA transcripts of CfC1qDC were detected in all the stages, and the expression level was up-regulated from D-hinged larva and reached the highest at eye-spot larva. The endogenous CfC1qDC was dominantly located in the hepatopancreas, gill, kidney and gonad of adult scallop through immunofluorescence. The recombinant protein of CfC1qDC (rCfC1qDC) could not only bind various PAMPs, such as LPS, PGN, β-glucan as well as polyI:C, but also enhance the phagocytic activity of scallop hemocytes towards Escherichia coli. Meanwhile, rCfC1qDC could interact with human heat-aggregated IgG, and this interaction could be inhibited by LPS. Conclusions All these results indicated that CfC1qDC in C. farreri not only served as a PRR involved in the PAMPs recognition, but also an opsonin participating in the clearance of invaders in innate immunity. Moreover, the ability of CfC1qDC to interact with immunoglobulins provided a clue to understand the evolution of classical pathway in complement system. PMID:22905248

  12. 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

  13. 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.

  14. Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

    NASA Astrophysics Data System (ADS)

    Liu, Zhenbao; Zhou, Bo; Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang

    2013-09-01

    A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 μm in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL-1 to 10 ng mL-1. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples.

  15. In Vivo Crystallization of Human IgG in the Endoplasmic Reticulum of Engineered Chinese Hamster Ovary (CHO) Cells

    PubMed Central

    Hasegawa, Haruki; Wendling, John; He, Feng; Trilisky, Egor; Stevenson, Riki; Franey, Heather; Kinderman, Francis; Li, Gary; Piedmonte, Deirdre Murphy; Osslund, Timothy; Shen, Min; Ketchem, Randal R.

    2011-01-01

    Protein synthesis and secretion are essential to cellular life. Although secretory activities may vary in different cell types, what determines the maximum secretory capacity is inherently difficult to study. Increasing protein synthesis until reaching the limit of secretory capacity is one strategy to address this key issue. Under highly optimized growth conditions, recombinant CHO cells engineered to produce a model human IgG clone started housing rod-shaped crystals in the endoplasmic reticulum (ER) lumen. The intra-ER crystal growth was accompanied by cell enlargement and multinucleation and continued until crystals outgrew cell size to breach membrane integrity. The intra-ER crystals were composed of correctly folded, endoglycosidase H-sensitive IgG. Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG, and the crystallization was reproduced in vitro by exposing a high concentration of IgG to a near neutral pH. The striking cellular phenotype implicated the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated progressively in the ER lumen until a threshold concentration was reached to nucleate crystals. Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-to-Golgi transport steps became rate-limiting in cells with high secretory activity. PMID:21464137

  16. Ag(I)-cysteamine complex based electrochemical stripping immunoassay: ultrasensitive human IgG detection.

    PubMed

    Noh, Hui-Bog; Rahman, Md Aminur; Yang, Jee Eun; Shim, Yoon-Bo

    2011-07-15

    An ultrasensitive electrochemical immunosensor for a protein using a Ag (I)-cysteamine complex (Ag-Cys) as a label was fabricated. The low detection of a protein was based on the electrochemical stripping of Ag from the adsorbed Ag-Cys complex on the gold nanoparticles (AuNPs) conjugated human immunoglobulin G (anti-IgG) antibody (AuNPs-anti-IgG). The electrochemical immunosensor was fabricated by immobilizing anti-IgG antibody on a poly-5,2':5',2''-terthiophene-3'-carboxylic acid (polyTTCA) film grown on the glassy carbon electrode through the covalent bond formation between amine groups of anti-IgG and carboxylic acid groups of polyTTCA. The target protein, IgG was sandwiched between the anti-IgG antibody that covalently attached onto the polyTTCA layer and AuNPs-anti-IgG. Using square wave voltammetry, well defined Ag stripping voltammograms were obtained for the each target concentration. Various experimental parameters were optimized and interference effects from other proteins were checked out. The immunosensor exhibited a wide dynamic range with the detection limit of 0.4 ± 0.05 fg/mL. To evaluate the analytical reliability, the proposed immunosensor was applied to human IgG spiked serum samples and acceptable results were obtained indicating that the method can be readily extended to other bioaffinity assays of clinical or environmental significance.

  17. 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.

  18. Aggregation kinetics of human mesenchymal stem cells under wave motion.

    PubMed

    Tsai, Ang-Chen; Liu, Yijun; Yuan, Xuegang; Chella, Ravindran; Ma, Teng

    2016-12-20

    Human mesenchymal stem cells (hMSCs) are primary candidates in cell therapy and regenerative medicine but preserving their therapeutic potency following culture expansion is a significant challenge. hMSCs can spontaneously assemble into three-dimensional (3D) aggregates that enhance their regenerative properties. The present study investigated the impact of hydrodynamics conditions on hMSC aggregation kinetics under controlled rocking motion. While various laboratory methods have been developed for hMSC aggregate production, the rocking platform provides gentle mixing and can be scaled up using large bags as in wave motion bioreactors. The results show that the hMSC aggregation is mediated by cell adhesion molecules and that aggregate size distribution is influenced by seeding density, culture time, and hydrodynamic conditions. The analysis of fluid shear stress by COMSOL indicated that aggregate size distribution is inversely correlated with shear stress and that the rocking angle had a more pronounced effect on aggregate size distribution than the rocking speed due to its impact on shear stress. hMSC aggregates obtained from the bioreactor exhibit increased stemness, migratory properties, and expression of angiogenic factors. The results demonstrate the potential of the rocking platform to produce hMSC aggregates with controlled size distribution for therapeutic application.

  19. Heteroantibody-mediated cytotoxicity: antibody to the high affinity Fc receptor for IgG mediates cytotoxicity by human monocytes that is enhanced by interferon-gamma and is not blocked by human IgG.

    PubMed

    Shen, L; Guyre, P M; Anderson, C L; Fanger, M W

    1986-12-01

    An IgG1 monoclonal antibody, 32.2, raised against the 72,000 dalton monocyte high affinity Fc receptor, was used to examine the role of this receptor in ADCC. This antibody did not inhibit the binding of human IgG1 to monocytes or to the U937 cell line, nor did it block or stimulate their killing of IgG-coated chicken erythrocytes (CE). Whole 32.2 or its Fab fragments were cross-linked to Fab fragments of rabbit anti-CE by using the agent SPDP. The resulting heteroantibodies (32.2 X Fab anti-CE) mediated monocyte and U937 cytotoxicity against CE, whereas an anti-HLA X anti-CE reagent did not. Both FcR expression and heteroantibody-mediated cytotoxicity were increased by culturing monocytes or U937 with IFN-gamma. Although IgG-mediated ADCC was significantly inhibited by 40 micrograms/ml human IgG1, cytotoxicity mediated by 32.2 X Fab anti-CE was not blocked by 2 mg/ml human IgG1, suggesting that such cytotoxicity might not be blocked by IgG in vivo. These data indicate the potential of 32.2 heteroantibodies in analysis of FcR function and in therapy.

  20. Human polyreactive IgM monoclonal antibodies with blocking activity against self-reactive IgG.

    PubMed

    Melero, J; Tarragó, D; Núñez-Roldán, A; Sánchez, B

    1997-04-01

    Natural IgM antibodies have been found to be involved in the control of IgG reactivity in normal serum. The authors investigated the blocking activity of four human IgM monoclonal antibodies (BY-2, BY-7, BY-10 and IRM-7) derived from B-cells from blood samples of three renal dialysis patients, which had shown multispecific properties similar to those observed for natural polyreactive autoantibodies. To achieve this, competitive inhibition assays were performed with these MoAbs on the binding of IgG purified from a healthy control, three patients with SLE, and two patients with autoimmune thyroiditis, to histone, dsDNA, RNP and thyroglobulin. MoAbs inhibited binding of self-reactive IgG to histone and dsDNA, but not to thyroglobulin or RNP, of natural and active or inactive phase disease-associated autoreactive IgG. The inhibitory effect of the MoAbs was mediated by V-region dependent interactions with autoreactive IgG, as shown by the ability of these MoAbs to block the binding of F(ab')2 fragments of autoreactive IgG to antigens (histone and dsDNA). The blocking of autoantibody activity was dose-dependent with maximal inhibition occurring at a specific molar ratio between the patient's IgG and a given MoAb. In contrast, MoAbs did not inhibit binding of IgG alloantibodies present in the sera of four polytransfused renal dialysis patients to target antigens on the surface of different cells. These results support the concept of a functional idiotypic network regulating autoimmune responses, and suggest that the IgM MoAbs under study may be natural polyreactive antibodies belonging to the physiological network of autoantibodies with highly connected V-regions, capable of binding and functionally neutralizing V-regions of natural and pathologic autoantibodies.

  1. Human IgG1 monoclonal antibody against human collagen 17 noncollagenous 16A domain induces blisters via complement activation in experimental bullous pemphigoid model.

    PubMed

    Li, Qiang; Ujiie, Hideyuki; Shibaki, Akihiko; Wang, Gang; Moriuchi, Reine; Qiao, Hong-jiang; Morioka, Hiroshi; Shinkuma, Satoru; Natsuga, Ken; Long, Heather A; Nishie, Wataru; Shimizu, Hiroshi

    2010-12-15

    Bullous pemphigoid (BP) is an autoimmune blistering disease caused by IgG autoantibodies targeting the noncollagenous 16A (NC16A) domain of human collagen 17 (hCOL17), which triggers blister formation via complement activation. Previous in vitro analysis demonstrated that IgG1 autoantibodies showed much stronger pathogenic activity than IgG4 autoantibodies; however, the exact pathogenic role of IgG1 autoantibodies has not been fully demonstrated in vivo. We constructed a recombinant IgG1 mAb against hCOL17 NC16A from BP patients. In COL17-humanized mice, this mAb effectively reproduced a BP phenotype that included subepidermal blisters, deposition of IgG1, C1q and C3, neutrophil infiltration, and mast cell degranulation. Subsequently, alanine substitutions at various C1q binding sites were separately introduced to the Fc region of the IgG1 mAb. Among these mutated mAbs, the one that was mutated at the P331 residue completely failed to activate the complement in vitro and drastically lost pathogenic activity in COL17-humanized mice. These findings indicate that P331 is a key residue required for complement activation and that IgG1-dependent complement activation is essential for blister formation in BP. This study is, to our knowledge, the first direct evidence that IgG1 Abs to hCOL17 NC16A can induce blister formation in vivo, and it raises the possibility that IgG1 mAbs with Fc modification may be used to block pathogenic epitopes in autoimmune diseases.

  2. Anti-Lipid IgG Antibodies Are Produced via Germinal Centers in a Murine Model Resembling Human Lupus

    PubMed Central

    Wong-Baeza, Carlos; Reséndiz-Mora, Albany; Donis-Maturano, Luis; Wong-Baeza, Isabel; Zárate-Neira, Luz; Yam-Puc, Juan Carlos; Calderón-Amador, Juana; Medina, Yolanda; Wong, Carlos; Baeza, Isabel; Flores-Romo, Leopoldo

    2016-01-01

    Anti-lipid IgG antibodies are produced in some mycobacterial infections and in certain autoimmune diseases [such as anti-phospholipid syndrome, systemic lupus erythematosus (SLE)]. However, few studies have addressed the B cell responses underlying the production of these immunoglobulins. Anti-lipid IgG antibodies are consistently found in a murine model resembling human lupus induced by chlorpromazine-stabilized non-bilayer phospholipid arrangements (NPA). NPA are transitory lipid associations found in the membranes of most cells; when NPA are stabilized they can become immunogenic and induce specific IgG antibodies, which appear to be involved in the development of the mouse model of lupus. Of note, anti-NPA antibodies are also detected in patients with SLE and leprosy. We used this model of lupus to investigate in vivo the cellular mechanisms that lead to the production of anti-lipid, class-switched IgG antibodies. In this murine lupus model, we found plasma cells (Gr1−, CD19−, CD138+) producing NPA-specific IgGs in the draining lymph nodes, the spleen, and the bone marrow. We also found a significant number of germinal center B cells (IgD−, CD19+, PNA+) specific for NPA in the draining lymph nodes and the spleen, and we identified in situ the presence of NPA in these germinal centers. By contrast, very few NPA-specific, extrafollicular reaction B cells (B220+, Blimp1+) were found. Moreover, when assessing the anti-NPA IgG antibodies produced during the experimental protocol, we found that the affinity of these antibodies progressively increased over time. Altogether, our data indicate that, in this murine model resembling human lupus, B cells produce anti-NPA IgG antibodies mainly via germinal centers. PMID:27746783

  3. Neutralizing IgG at the portal of infection mediates protection against vaginal simian/human immunodeficiency virus challenge.

    PubMed

    Klein, Katja; Veazey, Ronald S; Warrier, Ranjit; Hraber, Peter; Doyle-Meyers, Lara A; Buffa, Viviana; Liao, Hua-Xin; Haynes, Barton F; Shaw, George M; Shattock, Robin J

    2013-11-01

    Neutralizing antibodies may have critical importance in immunity against human immunodeficiency virus type 1 (HIV-1) infection. However, the amount of protective antibody needed at mucosal surfaces has not been fully established. Here, we evaluated systemic and mucosal pharmacokinetics (PK) and pharmacodynamics (PD) of 2F5 IgG and 2F5 Fab fragments with respect to protection against vaginal challenge with simian-human immunodeficiency virus-BaL in macaques. Antibody assessment demonstrated that 2F5 IgG was more potent than polymeric forms (IgM and IgA) across a range of cellular and tissue models. Vaginal challenge studies demonstrated a dose-dependent protection for 2F5 IgG and no protection with 2F5 Fab despite higher vaginal Fab levels at the time of challenge. Animals receiving 50 or 25 mg/kg of body weight 2F5 IgG were completely protected, while 3/5 animals receiving 5 mg/kg were protected. In the control animals, infection was established by a minimum of 1 to 4 transmitted/founder (T/F) variants, similar to natural human infection by this mucosal route; in the two infected animals that had received 5 mg 2F5 IgG, infection was established by a single T/F variant. Serum levels of 2F5 IgG were more predictive of sterilizing protection than measured vaginal levels. Fc-mediated antiviral activity did not appear to influence infection of primary target cells in cervical explants. However, PK studies highlighted the importance of the Fc portion in tissue biodistribution. Data presented in this study may be important in modeling serum levels of neutralizing antibodies that need to be achieved by either vaccination or passive infusion to prevent mucosal acquisition of HIV-1 infection in humans.

  4. Antigen binding of human IgG Fabs mediate ERK-associated proliferation of human breast cancer cells.

    PubMed

    Wen, Yue-Jin; Mancino, Anne; Pashov, Anastas; Whitehead, Tracy; Stanley, Joseph; Kieber-Emmons, Thomas

    2005-02-01

    Serum-circulating antibody can be linked to poor outcomes in some cancer patients. To investigate the role of human antibodies in regulating tumor cell growth, we constructed a recombinant cDNA expression library of human IgG Fab from a patient with breast cancer. Clones were screened from the library with breast tumor cell lysate. Sequence analysis of the clones showed somatic hypermutations when compared to their closest VH/VL germ-line genes. Initial characterizations focused on five clones. All tested clones displayed stronger binding to antigen derived from primary breast cancers and established breast cancer cell lines than to normal breast tissues. In vitro functional studies showed that four out of five tested clones could stimulate the growth of MDA-MB-231 breast cancer cell lines, and one out of five was able to promote MCF-7 cell growth as well. Involvement of ERK2 pathway was observed. By 1H-NMR spectra and Western blot analysis, it was evident that two tested antibody Fabs are capable of interacting with sialic acid. Our study suggests a possible role for human antibody in promoting tumor cell growth by direct binding of IgG Fab to breast tumor antigen. Such studies prompt speculation regarding the role of serum antibodies in mediating tumor growth as well as their contribution to disease progression.

  5. 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

  6. Anti-Human Herpesvirus 6A/B IgG Correlates with Relapses and Progression in Multiple Sclerosis

    PubMed Central

    Ortega-Madueño, Isabel; Garcia-Montojo, Marta; Dominguez-Mozo, Maria Inmaculada; Garcia-Martinez, Angel; Arias-Leal, Ana Maria; Casanova, Ignacio

    2014-01-01

    Objective To analyze the titers of the IgG and IgM antibodies against human herpesvirus 6A/B (HHV-6A/B) in multiple sclerosis (MS) patients treated with different disease modified therapies (DMTs) along two-years of follow-up. Methods We collected 2163 serum samples from 596 MS; for 301 MS patients a 2-years follow-up was performed. Serum samples of 337 healthy controls were also analyzed. Anti-HHV-6A/B IgG and IgM were analyzed by ELISA (Panbio). Results We found that 129/187 (69.0%) MS patients with a decrease of the anti-HHV-6A/B IgG titers after 2-years with DMTs were free of relapses and progression vs. 46/113 (40.7%) of MS patients with an increase of the anti-HHV-6A/B IgG titers (p = 0.0000015); the higher significance was found for natalizumab. Furthermore, we found that anti-HHV-6A/B IgG titers reached their highest value two weeks before the relapse (p = 0.0142), while the anti-HHV-6A/B IgM titers reached their highest value one month before the relapse (p = 0.0344). Conclusion The measurement of the anti-HHV-6A/B IgG titers could be a good biomarker of clinical response to the different DMTs. The increase of the anti-HHV-6A/B IgG and IgM titers predicts the upcoming clinical relapses. However, further longitudinal studies are needed to validate these results. PMID:25110949

  7. 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.

  8. IgG Fc variant cross-reactivity between human and rhesus macaque FcγRs.

    PubMed

    Boesch, Austin W; Miles, Adam R; Chan, Ying N; Osei-Owusu, Nana Y; Ackerman, Margaret E

    2017-01-05

    Non-human primate (NHP) studies are often an essential component of antibody development efforts before human trials. Because the efficacy or toxicity of candidate antibodies may depend on their interactions with Fcγ receptors (FcγR) and their resulting ability to induce FcγR-mediated effector functions such as antibody-dependent cell-meditated cytotoxicity and phagocytosis (ADCP), the evaluation of human IgG variants with modulated affinity toward human FcγR is becoming more prevalent in both infectious disease and oncology studies in NHP. Reliable translation of these results necessitates analysis of the cross-reactivity of these human Fc variants with NHP FcγR. We report evaluation of the binding affinities of a panel of human IgG subclasses, Fc amino acid point mutants and Fc glycosylation variants against the common allotypes of human and rhesus macaque FcγR by applying a high-throughput array-based surface plasmon resonance platform. The resulting data indicate that amino acid variation present in rhesus FcγRs can result in disrupted, matched, or even increased affinity of IgG Fc variants compared with human FcγR orthologs. These observations emphasize the importance of evaluating species cross-reactivity and developing an understanding of the potential limitations or suitability of representative in vitro and in vivo models before human clinical studies when either efficacy or toxicity may be associated with FcγR engagement.

  9. Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange.

    PubMed

    van der Neut Kolfschoten, Marijn; Schuurman, Janine; Losen, Mario; Bleeker, Wim K; Martínez-Martínez, Pilar; Vermeulen, Ellen; den Bleker, Tamara H; Wiegman, Luus; Vink, Tom; Aarden, Lucien A; De Baets, Marc H; van de Winkel, Jan G J; Aalberse, Rob C; Parren, Paul W H I

    2007-09-14

    Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe a novel posttranslational modification that leads to anti-inflammatory activity of antibodies of immunoglobulin G, isotype 4 (IgG4). IgG4 antibodies are dynamic molecules that exchange Fab arms by swapping a heavy chain and attached light chain (half-molecule) with a heavy-light chain pair from another molecule, which results in bispecific antibodies. Mutagenesis studies revealed that the third constant domain is critical for this activity. The impact of IgG4 Fab arm exchange was confirmed in vivo in a rhesus monkey model with experimental autoimmune myasthenia gravis. IgG4 Fab arm exchange is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 antibodies.

  10. Survival and digestibility of orally-administered immunoglobulin preparations containing IgG through the gastrointestinal tract in humans.

    PubMed

    Jasion, Victoria S; Burnett, Bruce P

    2015-03-07

    Oral immunoglobulin (Ig) preparations are prime examples of medicinal nutrition from natural sources. Plasma products containing Ig have been used for decades in animal feed for intestinal disorders to mitigate the damaging effects of early weaning. These preparations reduce overall mortality and increase feed utilization in various animal species leading to improved growth. Oral administration of Ig preparations from human serum as well as bovine colostrum and serum have been tested and proven to be safe as well as effective in human clinical trials for a variety of enteric microbial infections and other conditions which cause diarrhea. In infants, children, and adults, the amount of intact IgG recovered in stool ranges from trace amounts up to 25% of the original amount ingested. It is generally understood that IgG can only bind to antigens within the GI tract if the Fab structure is intact and has not been completely denatured through acidic pH or digestive proteolytic enzymes. This is a comprehensive review of human studies regarding the survivability of orally-administered Ig preparations, with a focus on IgG. This review also highlights various biochemical studies on IgG which potentially explain which structural elements are responsible for increased stability against digestion.

  11. 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

  12. Cadmium and the reticuloendothelial system (RES). A specific defect in blood clearance of soluble aggregates of IgG by the liver in mice given cadmium.

    PubMed Central

    Knutson, D W; Vredevoe, D L; Aoki, K R; Hays, E J; Levy, L

    1980-01-01

    The ability of the reticuloendothelial system (RES) to bind and catabolize soluble stable heat aggregates of 125I-IgG (A-IgG) was studied in mice given oral cadmium. Cadmium caused a delay in the circulation clearance of A-IgG in intact animals. The defect was due to impaired liver uptake of A-IgG and correlated with increased liver cadmium. Subsequent catabolism of bound A-IgG by liver slices was not affected. The defect was specific in that clearance of aggregated human serum albumin and colloidal carbon was normal in cadmium mice; this suggests that cadmium may affect either Fc or complement receptors of Kupffer cells in liver. PMID:7419241

  13. IgG from patients with systemic sclerosis bind to DNA antitopoisomerase 1 in normal human fibroblasts extracts

    PubMed Central

    Tamby, Mathieu C; Servettaz, Amélie; Tamas, Nicolas; Reinbolt, Joseph; Caux, Frédéric; Meyer, Olivier; Allanore, Yannick; Kahan, André; Guillevin, Loïc; Mouthon, Luc

    2008-01-01

    By using a semi-quantitative immunoblotting technique, we have analyzed serum immunoglobulin G (IgG) reactivities of patients with limited cutaneous systemic sclerosis and anticentromere antibodies, patients with diffuse systemic sclerosis and antitopoisomerase 1 antibodies, patients with diffuse systemic sclerosis without antitopoisomerase 1 or anticentromere antibodies and age- and gender-matched healthy controls with normal human skin fibroblasts and HEp-2 cells antigens. Serum IgG reactivities of patients with diffuse systemic sclerosis and antitopoisomerase 1 antibodies differed significantly from those of healthy controls or systemic sclerosis patients in other groups for reactivity with fibroblast proteins. IgG from patients with antitopoisomerase 1 antibodies bound to a 90 kDa fibroblast band and to a 100 kDa protein band in a HEp-2 cell protein extract. These two bands were further identified as DNA topoisomerase 1. Our results indicate that IgG from patients with diffuse systemic sclerosis bind DNA topoisomerase 1 in normal human fibroblasts extracts. PMID:19707389

  14. Influence of IgG Subclass on Human Antimannan Antibody-Mediated Resistance to Hematogenously Disseminated Candidiasis in Mice.

    PubMed

    Nishiya, Casey T; Boxx, Gayle M; Robison, Kerry; Itatani, Carol; Kozel, Thomas R; Zhang, Mason X

    2015-11-16

    Candida albicans is a yeast-like pathogen and can cause life-threatening systemic candidiasis. Its cell surface is enriched with mannan that is resistant to complement activation. Previously, we developed the recombinant human IgG1 antimannan antibody M1g1. M1g1 was found to promote complement activation and phagocytosis and protect mice from systemic candidiasis. Here, we evaluate the influence of IgG subclass on antimannan antibody-mediated protection. Three IgG subclass variants of M1g1 were constructed: M1g2, M1g3, and M1g4. The IgG subclass identity for each variant was confirmed with DNA sequence and subclass-specific antibodies. These variants contain identical M1 Fabs and exhibited similar binding affinities for C. albicans yeast and purified mannan. Yeast cells and hyphae recovered from the kidney of antibody-treated mice with systemic candidiasis showed uniform binding of each variant, indicating constitutive expression of the M1 epitope and antibody opsonization in the kidney. All variants promoted deposition of both murine and human C3 onto the yeast cell surface, with M1g4 showing delayed activation, as determined by flow cytometry and immunofluorescence microscopy. M1g4-mediated complement activation was found to be associated with its M1 Fab that activates the alternative pathway in an Fc-independent manner. Treatment with each subclass variant extended the survival of mice with systemic candidiasis (P < 0.001). However, treatment with M1g1, M1g3, or M1g4, but not with M1g2, also reduced the kidney fungal burden (P < 0.001). Thus, the role of human antimannan antibody in host resistance to systemic candidiasis is influenced by its IgG subclass.

  15. 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.

  16. Activation of human platelets by antibodies to thymocytes and beta 2-microglobulin. I. Qualitative and quantitative aspects of the platelet aggregation induced by HATG and SA beta 2mG.

    PubMed Central

    Csákó, G; Suba, E A; Wistar, R

    1980-01-01

    The platelet effect of antibody preparations known to have immunosuppressive action was investigated by a turbidimetric method in vitro. Both horse anti-human thymocyte globulin (HATG) and sheep anti-human beta 2-microglobulin IgG (SA beta 2mG) caused the platelets to aggregate in all human platelet-rich plasmas (PRP) tested. The aggregation was usually irreversible and characterized by a sigmoid curve. The action is specific for the antibodies in HATG and SA beta 2mG because control preparations (horse normal IgG and IgG fractions of sheep normal, anti-dog IgG and anti-human IgA sera) were ineffective; further, heating (30 min at 56 degrees C) and BaSO4 or Al(OH)3 adsorption of HATG and SA beta 2mG did not alter their aggregating capability. When HATG and SA beta 2mG were added together to PRP, they induced aggregation in a simple additive manner. High antibody doses tended to decrease the extent of aggregation. The effect of platelet count on aggregation varied with both the dose level ('low' or 'high') and type (HATG or SA beta 2mG) of the inducer antibody. Using fixed submaximal doses, four main aggregation patterns could be recognized among 60 PRP: (i) high responses to both HATG and SA beta 2mG; (ii) high to HATG, low to SA beta 2mG; (iii) low to HATG, high to SA beta 2mG; and (iv) low to both. The results provide guidelines for quantitative aggregation studies with platelet antibodies and suggest that HATG and SA beta 2mG act through distinct platelet membrane components, the receptor for the latter being the best characterized protein of the mammalian cell membrane. PMID:6156042

  17. 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

  18. 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

  19. 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

    2015-12-07

    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.

  20. Deficiency of complement component C3 is associated with accelerated removal of soluble 123I-labelled aggregates of IgG from the circulation.

    PubMed Central

    Halma, C; Daha, M R; Camps, J A; Evers-Schouten, J H; Pauwels, E K; Van, E s

    1992-01-01

    Complement and erythrocyte complement receptors CR1 (CD35) play an important role in the clearance of immune complexes. We studied the elimination of soluble 123I-labelled aggregates of human immunoglobulin G (123I-AIgG), used as a model for immune complexes, in two patients with a congenital and two patients with an acquired deficiency of complement component C3, and compared these with 10 healthy controls. The first disappearance halflife of 123I-AIgG was shorter (3.3 +/- 0.4 versus 7.0 +/- 0.4 min in the controls, P = 0.005) and maximal hepatic uptake of aggregates was increased in the C3 deficient patients (maximal liver/background ratio 3.6 +/- 0.4 versus 2.7 +/- 0.2 in controls, P = 0.04). Apparently, in the absence of C3, removal of circulating immune complexes by the liver is accelerated, probably through Fc receptor-dependent mechanisms. PMID:1458675

  1. 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

  2. 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.

  3. 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

  4. Clearance of human IgG1-sensitised red blood cells in vivo in humans relates to the in vitro properties of antibodies from alternative cell lines.

    PubMed

    Armour, Kathryn L; Smith, Cheryl S; Ip, Natasha C Y; Ellison, Cara J; Kirton, Christopher M; Wilkes, Anthony M; Williamson, Lorna M; Clark, Michael R

    2014-01-01

    We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance.

  5. Cardamom extract as inhibitor of human platelet aggregation.

    PubMed

    Suneetha, W Jessie; Krishnakantha, T P

    2005-05-01

    The inhibitory activity of cardamom extract was studied on human platelets. Platelet aggregation and lipid peroxidation were evaluated with platelet rich plasma (PRP) and platelet membranes, respectively, obtained from blood of healthy volunteers. Human platelets were subjected to stimulation with a variety of agonists including ADP (2.5 mM), epinephrine (2.5 mM), collagen (10 mM), calcium ionophore A 23187 (6 microM) and ristocetin (1.25 microg/mL). The IC50 were 0.49, 0.21, 0.55 and 0.59 mg with ADP, epinephrine, collagen and calcium ionophore A 23187, respectively, and no inhibition with ristocetin. The inhibitory effect was dose dependent with concentrations varying between 0.14 and 0.70 mg and time dependent at IC50. Lipid peroxidation induced by iron--ascorbic acid system in platelet membranes was analysed with malondialdehyde (MDA) as an index. An increase in concentration of cardamom has decreased the MDA formation significantly. Hence, it may be said that aqueous extract of cardamom may have component(s), which protect platelets from aggregation and lipid peroxidation.

  6. 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.

  7. Abnormal clearance of soluble aggregates of human immunoglobulin G in patients with systemic lupus erythematosus.

    PubMed

    Lobatto, S; Daha, M R; Breedveld, F C; Pauwels, E K; Evers-Schouten, J H; Voetman, A A; Cats, A; Van Es, L A

    1988-04-01

    In the present study, we tested mononuclear phagocyte system function in nine healthy controls and 15 SLE patients with complement activating 123I-labelled aggregates of human IgG (AIgG). Clearance half-time of AIgG was 26 +/- 8 min in controls, compared to 58 +/- 27 min in patients (P less than 0.005). Binding of AIgG to erythrocytes was significantly lower in patients, 9.3 +/- 8.1 vs 24 +/- 20% (P less than 0.05). The increase of C3a-levels in plasma was significantly lower in patients than in controls (P less than 0.05 at 3 and 8 min), suggesting less complement activation. Liver and spleen uptake of 123I-AIgG was measured with a gamma camera and expressed as liver/spleen uptake ratios. In patients, the liver/spleen uptake ratios were significantly higher than in controls at 15 min, 3.8 +/- 2.0 vs 2.31 +/- 0.7 (P less than 0.05), due to less splenic uptake of AIgG. Correlations between clearance half-time or liver/spleen uptake ratios and immune complex levels or disease activity were not found. This study indicates that clearance of soluble AIgG is abnormal in patients with SLE, due to decreased splenic uptake of AIgG.

  8. First Attempt To Validate Human IgG Antibody Response to Nterm-34kDa Salivary Peptide as Biomarker for Evaluating Exposure to Aedes aegypti Bites

    PubMed Central

    Elanga Ndille, Emmanuel; Doucoure, Souleymane; Damien, Georgia; Mouchet, François; Drame, Papa Makhtar; Cornelie, Sylvie; Noukpo, Herbert; Yamadjako, Sandra; Djenontin, Armel; Moiroux, Nicolas; Misse, Dorothee; Akogbeto, Martin; Corbel, Vincent; Henry, Marie-Claire; Chandre, Fabrice; Baldet, Thierry; Remoue, Franck

    2012-01-01

    Background Much effort is being devoted for developing new indicators to evaluate the human exposure to Aedes mosquito bites and the risk of arbovirus transmission. Human antibody (Ab) responses to mosquito salivary components could represent a promising tool for evaluating the human-vector contact. Methodology/Principal findings To develop a specific biomarker of human exposure to Aedes aegypti bites, we measured IgG Ab response to Ae. aegypti Nterm-34 kDa salivary peptide in exposed children in 7 villages of Southern Benin (West Africa). Results showed that specific IgG response presented high inter-individual heterogeneity between villages. IgG response was associated with rainfall and IgG level increased from dry (low exposure) to rainy (high exposure) seasons. These findings indicate that IgG Ab to Nterm-34 kDa salivary peptide may represent a reliable biomarker to detect variation in human exposure to Ae. aegypti bites. Conclusion/Significance This preliminary study highlights the potential use of Ab response to this salivary peptide for evaluating human exposure to Ae. aegypti. This biomarker could represent a new promising tool for assessing the risk of arbovirus transmission and for evaluating the efficacy of vector control interventions. PMID:23166852

  9. Aggregation of biopharmaceuticals in human plasma and human serum: implications for drug research and development.

    PubMed

    Arvinte, Tudor; Palais, Caroline; Green-Trexler, Erin; Gregory, Sonia; Mach, Henryk; Narasimhan, Chakravarthy; Shameem, Mohammed

    2013-01-01

    Analytical methods based on light microscopy, 90° light-scattering and surface plasmon resonance (SPR) allowed the characterization of aggregation that can occur when antibodies are mixed with human plasma. Light microscopy showed that aggregates formed when human plasma was mixed with 5% dextrose solutions of Herceptin(®) (trastuzumab) or Avastin(®) (bevacizumab) but not Remicade(®) (infliximab). The aggregates in the plasma-Herceptin(®)-5% dextrose solution were globular, size range 0.5-9 μm, with a mean diameter of 4 μm. The aggregates in the plasma-Avastin(®)-5% dextrose samples had a mean size of 2 μm. No aggregation was observed when 0.9% NaCl solutions of Herceptin(®), Avastin(®) and Remicade(®) were mixed with human plasma. 90° light-scattering measurements showed that aggregates were still present 2.5 h after mixing Herceptin(®) or Avastin(®) with 5% dextrose-plasma solution. A SPR method was utilized to qualitatively describe the extent of interactions of surface-bound antibodies with undiluted human serum. Increased binding was observed in the case of Erbitux(®) (cetuximab), whereas no binding was measured for Humira(®) (adalimumab). The binding of sera components to 13 monoclonal antibodies was measured and correlated with known serum binding properties of the antibodies. The data presented in this paper provide analytical methods to study the intrinsic and buffer-dependent aggregation tendencies of therapeutic proteins when mixed with human plasma and serum.

  10. Successful post-exposure prophylaxis of Ebola infected non-human primates using Ebola glycoprotein-specific equine IgG

    PubMed Central

    Pyankov, Oleg V.; Setoh, Yin Xiang; Bodnev, Sergey A.; Edmonds, Judith H.; Pyankova, Olga G.; Pyankov, Stepan A.; Pali, Gabor; Belford, Shane; Lu, Louis; La, Mylinh; Lovrecz, George; Volchkova, Valentina A.; Chappell, Keith J.; Watterson, Daniel; Marsh, Glenn; Young, Paul R.; Agafonov, Alexander A.; Farmer, Jillann F.; Volchkov, Victor E.; Suhrbier, Andreas; Khromykh, Alexander A.

    2017-01-01

    Herein we describe production of purified equine IgG obtained from horses immunized with plasmid DNA followed by boosting with Kunjin replicon virus-like particles both encoding a modified Ebola glycoprotein. Administration of the equine IgG over 5 days to cynomolgus macaques infected 24 hours previously with a lethal dose of Ebola virus suppressed viral loads by more than 5 logs and protected animals from mortality. Animals generated their own Ebola glycoprotein-specific IgG responses 9–15 days after infection, with circulating virus undetectable by day 15–17. Such equine IgG may find utility as a post-exposure prophylactic for Ebola infection and provides a low cost, scalable alternative to monoclonal antibodies, with extensive human safety data and WHO-standardized international manufacturing capability available in both high and low income countries. PMID:28155869

  11. Effect of IDA and TREN chelating agents and buffer systems on the purification of human IgG with immobilized nickel affinity membranes.

    PubMed

    Ribeiro, Mariana Borsoi; Vijayalakshmi, Mookambesvaran; Todorova-Balvay, Daniele; Bueno, Sonia Maria Alves

    2008-01-01

    The purification of IgG from human plasma was studied by comparing two affinity membranes complexed with Ni(II), prepared by coupling iminodiacetic acid (IDA) and Tris(2-aminoethyl)amine (TREN) to poly(ethylenevinyl alcohol), PEVA, hollow fiber membranes. The Ni(II)-TREN-PEVA hollow fiber membrane had lower capacity for human IgG than the complex Ni(II)-IDA-PEVA, but with similar selectivity. The IgG in peak fractions eluted from the Ni(II)-IDA-PEVA with a stepwise concentration gradient of Tris-HCl pH 7.0 (100-700 mM) reached a purity of 98% (based on IgG, IgM, IgA, albumin, and transferrin nephelometric analysis). Adsorption IgG data at different temperatures (4-37 degrees C) were analyzed using Langmuir model resulting in a calculated maximum capacity at 25 degrees C of 204.6 mg of IgG/g of dry membrane. Decrease in Kd with increasing temperature (1.7x10(-5) to 5.3x10(-6) M) indicated an increase in affinity with increased temperature. The positive value of enthalpy change (26.2 kJ/mol) indicated that the adsorption of IgG in affinity membrane is endothermic. Therefore, lower temperature induces adsorption as verified experimentally.

  12. Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp.

    PubMed

    Sjögren, Jonathan; Andersson, Linda; Mejàre, Malin; Olsson, Fredrik

    2017-01-01

    Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG. For all human IgG subclasses, the IdeS enzyme from Streptococcus pyogenes has been applied to generate F(ab')2 fragments that subsequently can be reduced under mild conditions to generate a homogenous pool of Fab' fragments. The enzyme Kgp from Porphyromonas gingivalis has been applied to generate intact Fab fragments from human IgG1 and the Fab fragments can be purified using a CH1-specific affinity resin. The SpeB protease, also from S. pyogenes, is able to digest mouse IgGs and has been applied to digest antibodies and Fab fragments can be purified on light chain affinity resins. In this chapter, we describe methodologies that can be used to obtain Fab fragments from human and mouse IgG using bacterial proteases.

  13. Immunogenic and antigenic epitopes of immunoglobulins I. Cross-reactivity of murine monoclonal antibodies to human IgG with the immunoglobulins of certain animal species.

    PubMed Central

    Jefferis, R; Lowe, J; Ling, N R; Porter, P; Senior, S

    1982-01-01

    Antibody-producing hybridoma clones have been isolated following immunization of mice with human IgG. Twenty-five monoclonal antibodies (nine anti-C gamma 3, fourteen anti-C gamma 2, one anit-kappa and one anti-lambda) were selected for study of their cross-reactivity with the IgG of fifteen mammalian species and chicken immunoglobulin. Each antibody exhibited a unique reaction profile suggesting that human IgG expresses a very large repertoire of immunogenic epitopes. Whilst some antibodies showed a very restricted cross-reactivity profile for others a very wide reactivity profile was observed-including two clones producing autoantibodies. Antibodies demonstrating cross-reactivity between human Fc gamma and 7S chicken immunoglobulin allow its definitive assignment as a homologue of human IgG. Four clones demonstrated specificity for bovine IgG subclass gamma 1 and gamma 2 and the degree of reactivity allows their application to qualitative and quantitative assay systems. These studies suggest new perspectives for the characterization of immunoglobulins and the standardization of anti-immunoglobulin reagents. PMID:6173313

  14. 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.

  15. Crystallization and preliminary X-ray diffraction studies of an RNA aptamer in complex with the human IgG Fc fragment

    PubMed Central

    Sugiyama, Shigeru; Nomura, Yusuke; Sakamoto, Taiichi; Kitatani, Tomoya; Kobayashi, Asako; Miyakawa, Shin; Takahashi, Yoshinori; Adachi, Hiroaki; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Nakamura, Yoshikazu; Matsumura, Hiroyoshi

    2008-01-01

    Aptamers, which are folded DNA or RNA molecules, bind to target molecules with high affinity and specificity. An RNA aptamer specific for the Fc fragment of human immunoglobulin G (IgG) has recently been identified and it has been demonstrated that an optimized 24-nucleotide RNA aptamer binds to the Fc fragment of human IgG and not to other species. In order to clarify the structural basis of the high specificity of the RNA aptamer, it was crystallized in complex with the Fc fragment of human IgG1. Preliminary X-ray diffraction studies revealed that the crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 83.7, b = 107.2, c = 79.0 Å. A data set has been collected to 2.2 Å resolution. PMID:18931441

  16. Novel blocking human IgG directed against the pentapeptide repeat motifs of Neisseria meningitidis Lip/H.8 and Laz lipoproteins.

    PubMed

    Ray, Tathagat Dutta; Lewis, Lisa A; Gulati, Sunita; Rice, Peter A; Ram, Sanjay

    2011-04-15

    Ab-initiated, complement-dependent killing contributes to host defenses against invasive meningococcal disease. Sera from nonimmunized individuals vary widely in their bactericidal activity against group B meningococci. We show that IgG isolated from select individuals can block killing of group B meningococci by human sera that are otherwise bactericidal. This IgG also reduced the bactericidal efficacy of Abs directed against the group B meningococcal protein vaccine candidates factor H-binding protein currently undergoing clinical trials and Neisserial surface protein A. Immunoblots revealed that the blocking IgG was directed against a meningococcal Ag called H.8. Killing of meningococci in reactions containing bactericidal mAbs and human blocking Abs was restored when binding of blocking Ab to meningococci was inhibited using either synthetic peptides corresponding to H.8 or a nonblocking mAb against H.8. Furthermore, genetic deletion of H.8 from target organisms abrogated blocking. The Fc region of the blocking IgG was required for blocking because F(ab')(2) fragments were ineffective. Blocking required IgG glycosylation because deglycosylation with peptide:N-glycanase eliminated blocking. C4b deposition mediated by an anti-factor H-binding protein mAb was reduced by intact blocking IgG, but not by peptide:N-glycanase-treated blocking IgG, suggesting that blocking resulted from inhibition of classical pathway of complement. In conclusion, we have identified H.8 as a meningococcal target for novel blocking Abs in human serum. Such blocking Abs may reduce the efficacy of select antigroup B meningococcal protein vaccines. We also propose that outer membrane vesicle-containing meningococcal vaccines may be more efficacious if purged of subversive immunogens such as H.8.

  17. Human IgA and IgG F(ab')2 that bind to staphylococcal protein A belong to the VHIII subgroup.

    PubMed

    Sasso, E H; Silverman, G J; Mannik, M

    1991-09-15

    Staphylococcal protein A (SPA) is a bacterial membrane protein that possesses, in addition to its Fc gamma-binding activity, a distinct specificity for the Fab region of some IgM, IgA, IgG, and IgE. The Fab site that binds to SPA has been localized to the V region of the Ig H chain. In a previous study of human monoclonal and polyclonal IgM, we demonstrated that binding to SPA was highly restricted to molecules of the VHIII subgroup, and that nearly all VHIII IgM were able to bind SPA. The present study examines the VH composition of SPA-binding and SPA-nonbinding fractions of purified human polyclonal IgA, and IgG F(ab')2 fragments. We found that 22% of the IgA and 15% of the IgG F(ab')2 bound to SPA-agarose. Analysis with VH subgroup-specific antisera indicated that the SPA-binding fraction of IgA was dominated by the VHIII subgroup, and the SPA-binding fraction of IgG F(ab')2 contained only VHIII molecules. Furthermore, substantial portions of the total VHIII protein in IgA and in IgG F(ab')2 bound to SPA. We conclude that Fab binding to SPA is both restricted to and highly prevalent among human VHIII molecules, regardless of Ig class. These results suggest that protein A is an Ig superantigen.

  18. A human monoclonal IgG1 potently neutralizing the pro-inflammatory cytokine GM-CSF.

    PubMed

    Krinner, Eva-Maria; Raum, Tobias; Petsch, Silke; Bruckmaier, Sandra; Schuster, Ioana; Petersen, Laetitia; Cierpka, Ronny; Abebe, Derege; Mølhøj, Michael; Wolf, Andreas; Sørensen, Poul; Locher, Mathias; Baeuerle, Patrick A; Hepp, Julia

    2007-02-01

    The pro-inflammatory cytokine GM-CSF is aberrantly produced in many autoimmune and chronic inflammatory human diseases. GM-CSF neutralization by antibodies has been shown to have a profound therapeutic effect in animal models of rheumatoid arthritis, inflammatory lung diseases, psoriasis and multiple sclerosis. Moreover, the absence of GM-CSF in null mutant mice ameliorates or prevents certain of these diseases. Here we describe the biophysical and biological properties of a human anti-GM-CSF IgG1 antibody designated MT203, which was derived by phage display guided selection. MT203 bound with picomolar affinity to an epitope on human and macaque GM-CSF involved in high-affinity receptor interaction. As a consequence, the antibody potently prevented both GM-CSF-induced proliferation of TF-1 cells with a sub-nanomolar IC50 value and the production of the chemokine IL-8 by U937 cells. MT203 neutralized equally well recombinant (r) human (h) GM-CSF from Escherichia coli and yeast, and also normally glycosylated GM-CSF secreted by human lung epithelial cells in response to IL-1beta stimulation. Furthermore, MT203 significantly reduced both survival and activation of peripheral human eosinophils as may be required for effective treatment of inflammatory lung diseases. The antibody did not show a detectable loss of neutralizing activity after 5 days in human serum at 37 degrees C. Based on its favorable properties, MT203 has been selected for development as a novel anti-inflammatory human monoclonal antibody with therapeutic potential in a multitude of human autoimmune and inflammatory diseases.

  19. Study of chronic hemolytic anaemia patients in Rio de Janeiro: prevalence of anti-human parvovirus B19 IgG antibodies and the development aplastic crises.

    PubMed

    Sant'Anna, Anadayr L M; Garcia, Rita de Cássia N Cubel; Marzoche, Mônica; da Rocha, Heloisa Helena A Gallo; Paula, Maria Tereza M; Lobo, Clarisse C; Nascimento, Jussara P

    2002-01-01

    The prevalence of anti-human parvovirus B19 IgG antibodies was determined in sera from 165 chronic hemolytic anemia patients, receiving medical care at Instituto Estadual de Hematologia (IEHE), Rio de Janeiro, during the year of 1994. This sample represents around 10% of the chronic hemolytic anemia patients attending at IEHE. Most of these patients (140) have sickle cell disease. Anti-B19 IgG antibodies were detected in 32.1% of patients. No statistically significant difference (p > 0.05) was seen between IgG antibody prevalence in male (27.8%) and female (35.5%) patients. Anti-B19 IgG antibodies were more frequent in older (37.6%) than younger (28.2%) than 20 years old patients, although this difference had no statistical significance (p > 0.05). Anti-B19 IgG antibody prevalence showed that 67.9% of patients enrolled in the study were susceptible to B19 acute infection. With the aim to detect acute B19 infection, patients follow up continued until February 1996. During this period four patients presented transient aplastic crisis due to human parvovirus B19 as confirmed by the detection of specific IgM antibodies. All four patients were younger than 20 years old, and 3 were younger than 10 years old. Three of them were sickle cell disease patients. Three of the four acute B19 infection occurred during 1994 springtime.

  20. Detection of Human IgG on Poly(pyrrole-3-carboxylic acid) Thin Film by Electrochemical-Surface Plasmon Resonance Spectroscopy

    NASA Astrophysics Data System (ADS)

    Janmanee, Rapiphun; Baba, Akira; Phanichphant, Sukon; Sriwichai, Saengrawee; Shinbo, Kazunari; Kato, Keizo; Kaneko, Futao

    2011-01-01

    An electrochemically controlled surface plasmon resonance (SPR) immunosensor for the detection of human immunoglobulin G (IgG) has been developed using poly(pyrrole-3-carboxylic acid) (PP3C) film. In this work, a pyrrole-3-carboxylic acid monomer was used for electropolymerization of a PP3C film on a gold-coated high-refractive-index glass slide. In situ electrochemical (EC)-SPR spectroscopy was performed to study the kinetic property and electroactivity property of the PP3C film. Moreover, ultraviolet-visible (UV-vis) spectroscopy was performed to characterize the PP3C film. Finally, the immunosensor-based PP3C film was constructed. The carboxylic acid surface of the PP3C film was activated for the immobilization of anti-human IgG. The immunosensor electrode was used for probing the binding reaction of anti-human IgG/human IgG with several concentrations of human IgG at different constant applied potentials. The probe immobilization and immunosensing process were in situ monitored by EC-SPR technique. The sensitivity of the sensor was improved by controlling the morphology of the PP3C film by applying the potential.

  1. 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

  2. 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

    2016-08-03

    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.

  3. Immunoglobulin (Ig)G purified from human sera mirrors intravenous Ig human leucocyte antigen (HLA) reactivity and recognizes one's own HLA types, but may be masked by Fab complementarity-determining region peptide in the native sera.

    PubMed

    Ravindranath, M H; Terasaki, P I; Maehara, C Y; Jucaud, V; Kawakita, S; Pham, T; Yamashita, W

    2015-02-01

    Intravenous immunoglobulin (IVIg) reacted with a wide array of human leucocyte antigen (HLA) alleles, in contrast to normal sera, due possibly to the purification of IgG from the pooled plasma. The reactivity of IgG purified from normal sera was compared with that of native sera to determine whether any serum factors mask the HLA reactivity of anti-HLA IgG and whether IgG purified from sera can recognize the HLA types of the corresponding donors. The purified IgG, unlike native sera, mirrored IVIg reactivity to a wide array of HLA-I/-II alleles, indicating that anti-HLA IgG may be masked in normal sera - either by peptides derived from soluble HLA or by those from antibodies. A < 3 kDa peptide from the complementarity-determining region (CDR) of the Fab region of IgG (but not the HLA peptides) masked HLA recognition by the purified IgG. Most importantly, some of the anti-HLA IgG purified from normal sera - and serum IgG from a few donors - indeed recognized the HLA types of the corresponding donors, confirming the presence of auto-HLA antibodies. Comparison of HLA types with the profile of HLA antibodies showed auto-HLA IgG to the donors' HLA antigens in this order of frequency: DPA (80%), DQA (71%), DRB345 (67%), DQB (57%), Cw (50%), DBP (43%), DRB1 (21%), A (14%) and B (7%). The auto-HLA antibodies, when unmasked in vivo, may perform immunoregulatory functions similar to those of therapeutic preparations of IVIg.

  4. IgG antinuclear antibodies with cross-reactive rheumatoid factor activity.

    PubMed

    Darwin, B S; Grudier, J P; Klatt, C L; Pisetsky, D S

    1986-12-15

    To investigate whether IgG antinuclear antibodies have cross-reactive rheumatoid factor activity, monoclonal IgG antibodies to DNA and Sm from autoimmune MRL-lpr/lpr mice were assayed by ELISA for binding to IgG antigens. Of the nine anti-DNA and anti-Sm monoclonals tested, six showed significant binding to affinity-purified rabbit IgG (RIgG) and human IgG (HIgG). To confirm that cross-reactivities were due to a single antibody, immunoabsorption of a representative polyspecific monoclonal termed C11 (anti-DNA, anti-Sm) on either Sepharose-DNA or Sepharose-RIgG resulted in marked loss of activity to the three antigens DNA, Sm and RIgG compared with immunoabsorption on Sepharose-bovine serum albumin. The monomolecular nature of the cross-reacting antibody was also suggested by inhibition analysis of C11; DNA inhibited C11 binding to RIgG 64%, whereas Sm inhibited binding to RIgG 33%. Aggregated RIgG and HIgG, however, did not inhibit binding of C11 to DNA, Sm, or solid-phase RIgG, probably reflecting the low affinity of this antibody for fluid phase Ig. Together, these findings suggest that antinuclear autoantibodies of the IgG, as well as the IgM, class have polyspecific IgG binding activity and suggest that IgG antinuclear antibodies may emerge from rheumatoid factor responses.

  5. CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display.

    PubMed

    Miescher, S; Zahn-Zabal, M; De Jesus, M; Moudry, R; Fisch, I; Vogel, M; Kobr, M; Imboden, M A; Kragten, E; Bichler, J; Mermod, N; Stadler, B M; Amstutz, H; Wurm, F

    2000-10-01

    Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones. LD-6-3 and LD-6-33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth-adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.

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

    PubMed

    Horrocks, Mathew H; Lee, Steven F; Gandhi, Sonia; Magdalinou, Nadia K; Chen, Serene W; Devine, Michael J; Tosatto, Laura; Kjaergaard, Magnus; Beckwith, Joseph S; Zetterberg, Henrik; Iljina, Marija; Cremades, Nunilo; Dobson, Christopher M; Wood, Nicholas W; Klenerman, David

    2016-03-16

    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.

  7. 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

  8. Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells.

    PubMed

    Nogales-Gadea, Gisela; Saxena, Abhishek; Hoffmann, Carolin; Hounjet, Judith; Coenen, Daniëlle; Molenaar, Peter; Losen, Mario; Martinez-Martinez, Pilar

    2015-06-05

    Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

  9. Human platelet aggregation inhibitors from thyme (Thymus vulgaris L.).

    PubMed

    Okazaki, Kenji; Kawazoe, Kazuyoshi; Takaishi, Yoshihisa

    2002-06-01

    Two antiaggregant compounds, thymol (compound 1) and 3,4,3',4'-tetrahydroxy-5,5'-diisopropyl-2,2'-dimethylbiphenyl (compound 2) were isolated from the leaves of thyme (Thymus vulgaris L.). The structures were determined by (1)H-, (13)C-NMR and mass spectra (MS) studies. These compounds inhibited platelet aggregation induced by collagen, ADP, arachidonic acid (AA) and thrombin except that compound 2 did not inhibit platelet aggregation induced by thrombin.

  10. 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.

  11. Detection of IgM and IgG antibodies against Brucella by dot-ELISA in humans.

    PubMed

    Barbuddhe, S B; Yadava, V K; Singh, D K

    1994-03-01

    Dot-enzyme linked immunosorbent assay (dot-ELISA) with autoclaved extract of B. abortus S99 was used for the detection of Brucella antibodies in human sera. Results were compared with those of STAT, RBPT and CFT. Out of 80 sera 8(10 per cent), 15(18.7 per cent), 20(25 per cent) and 13(16.25 per cent) were found positive by RBPT, STAT, IgM dot-ELISA and IgG dot-ELISA respectively. Out of 74 sera samples tested with CFT 9(12.16 per cent) proved positive. The relative sensitivity and relative specificity in comparison with CFT was found to be 33.33 per cent, 96.92 percent for RBPT, 33.33 per cent and 84.61 per cent for STAT and 88.88 per cent and 76.92 per cent for dot-ELISA, respectively. The dot-ELISA was found to be a more sensitive, economical and a rapid test for screening of human brucellosis under field conditions.

  12. 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.

  13. 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

  14. 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

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

    PubMed Central

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

    1987-01-01

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

  16. 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.

  17. Editor’s Highlight: Subvisible Aggregates of Immunogenic Proteins Promote a Th1-Type Response

    PubMed Central

    Ratanji, Kirsty D.; Dearman, Rebecca J.; Kimber, Ian; Thorpe, Robin; Wadhwa, Meenu; Derrick, Jeremy P.

    2016-01-01

    Protein aggregation is associated with enhanced immunogenicity of biotherapeutics. As a result, regulatory guidelines recommend screening for aggregation during bioprocessing. However, the mechanisms underlying the enhanced immunogenicity of aggregates are poorly understood. In the investigations described herein, the immunogenicity in mice of a humanized single chain variable antibody fragment (scFv) purified after expression in Escherichia coli has been examined. Reproducible scFv aggregates were obtained within the subvisible particle size range (mean diameter 2 µm) using thermal and mechanical stresses. Intraperitoneal immunization of BALB/c strain mice with 1 mg/ml of aggregated or monomeric scFv induced similar IgG and IgG1 antibody responses. In contrast, aggregate preparations stimulated significantly higher levels of anti-scFv IgG2a antibody than did the monomer. In comparative studies, aggregates of ovalbumin (OVA) within the subvisible particle size range were prepared by stir stress, and their immunogenicity compared with that of monomeric OVA in mice. Aggregated and monomeric OVA induced similar anti-OVA IgG and IgG1 antibody responses, whereas IgG2a antibody levels were significantly higher in aggregate-immunized mice. Furthermore, cytokine profiles in supernatants taken from splenocyte-dendritic cell co-cultures were consistent with aggregated preparations inducing a T helper (Th) 1-type response. Aggregated proteins within the subvisible range were therefore shown to induce a preferential Th1 type response, whereas monomeric proteins elicited a selective Th2 response. These data indicate that protein aggregation can impact on both the vigor and quality of immune responses. PMID:27370416

  18. Multistep Aggregation Pathway of Human Interleukin-1 Receptor Antagonist: Kinetic, Structural, and Morphological Characterization

    PubMed Central

    Krishnan, Sampathkumar; Raibekas, Andrei A.

    2009-01-01

    Abstract The complex, multistep aggregation kinetic and structural behavior of human recombinant interleukin-1 receptor antagonist (IL-1ra) was revealed and characterized by spectral probes and techniques. At a certain range of protein concentration (12–27 mg/mL) and temperature (44–48°C), two sequential aggregation kinetic transitions emerge, where the second transition is preceded by a lag phase and is associated with the main portion of the aggregated protein. Each kinetic transition is linked to a different type of aggregate population, referred to as type I and type II. The aggregate populations, isolated at a series of time points and analyzed by Fourier-transform infrared spectroscopy, show consecutive protein structural changes, from intramolecular (type I) to intermolecular (type II) β-sheet formation. The early type I protein spectral change resembles that seen for IL-1ra in the crystalline state. Moreover, Fourier-transform infrared data demonstrate that type I protein assembly alone can undergo a structural rearrangement and, consequently, convert to the type II aggregate. The aggregated protein structural changes are accompanied by the aggregate morphological changes, leading to a well-defined population of interacting spheres, as detected by scanning electron microscopy. A nucleation-driven IL-1ra aggregation pathway is proposed, and assumes two major activation energy barriers, where the second barrier is associated with the type I → type II aggregate structural rearrangement that, in turn, serves as a pseudonucleus triggering the second kinetic event. PMID:19134476

  19. High-yield production of a human monoclonal IgG by rhizosecretion in hydroponic tobacco cultures.

    PubMed

    Madeira, Luisa M; Szeto, Tim H; Henquet, Maurice; Raven, Nicole; Runions, John; Huddleston, Jon; Garrard, Ian; Drake, Pascal M W; Ma, Julian K-C

    2016-02-01

    Rhizosecretion of recombinant pharmaceuticals from in vitro hydroponic transgenic plant cultures is a simple, low cost, reproducible and controllable production method. Here, we demonstrate the application and adaptation of this manufacturing platform to a human antivitronectin IgG1 monoclonal antibody (mAb) called M12. The rationale for specific growth medium additives was established by phenotypic analysis of root structure and by LC-ESI-MS/MS profiling of the total protein content profile of the hydroponic medium. Through a combination of optimization approaches, mAb yields in hydroponic medium reached 46 μg/mL in 1 week, the highest figure reported for a recombinant mAb in a plant secretion-based system to date. The rhizosecretome was determined to contain 104 proteins, with the mAb heavy and light chains the most abundant. This enabled evaluation of a simple, scalable extraction and purification protocol and demonstration that only minimal processing was necessary prior to protein A affinity chromatography. MALDI-TOF MS revealed that purified mAb contained predominantly complex-type plant N-glycans, in three major glycoforms. The binding of M12 purified from hydroponic medium to vitronectin was comparable to its Chinese hamster ovary (CHO)-derived counterpart. This study demonstrates that in vitro hydroponic cultivation coupled with recombinant protein rhizosecretion can be a practical, low-cost production platform for monoclonal antibodies.

  20. Monitoring the Systemic Human Memory B Cell Compartment of Melanoma Patients for Anti-Tumor IgG Antibodies

    PubMed Central

    Gilbert, Amy E.; Karagiannis, Panagiotis; Dodev, Tihomir; Koers, Alexander; Lacy, Katie; Josephs, Debra H.; Takhar, Pooja; Geh, Jenny L. C.; Healy, Ciaran; Harries, Mark; Acland, Katharine M.; Rudman, Sarah M.; Beavil, Rebecca L.; Blower, Philip J.; Beavil, Andrew J.; Gould, Hannah J.; Spicer, James; Nestle, Frank O.; Karagiannis, Sophia N.

    2011-01-01

    Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001). Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001). Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer. PMID:21559411

  1. Monitoring the systemic human memory B cell compartment of melanoma patients for anti-tumor IgG antibodies.

    PubMed

    Gilbert, Amy E; Karagiannis, Panagiotis; Dodev, Tihomir; Koers, Alexander; Lacy, Katie; Josephs, Debra H; Takhar, Pooja; Geh, Jenny L C; Healy, Ciaran; Harries, Mark; Acland, Katharine M; Rudman, Sarah M; Beavil, Rebecca L; Blower, Philip J; Beavil, Andrew J; Gould, Hannah J; Spicer, James; Nestle, Frank O; Karagiannis, Sophia N

    2011-04-29

    Melanoma, a potentially lethal skin cancer, is widely thought to be immunogenic in nature. While there has been much focus on T cell-mediated immune responses, limited knowledge exists on the role of mature B cells. We describe an approach, including a cell-based ELISA, to evaluate mature IgG antibody responses to melanoma from human peripheral blood B cells. We observed a significant increase in antibody responses from melanoma patients (n = 10) to primary and metastatic melanoma cells compared to healthy volunteers (n = 10) (P<0.0001). Interestingly, we detected a significant reduction in antibody responses to melanoma with advancing disease stage in our patient cohort (n = 21) (P<0.0001). Overall, 28% of melanoma patient-derived B cell cultures (n = 1,800) compared to 2% of cultures from healthy controls (n = 600) produced antibodies that recognized melanoma cells. Lastly, a patient-derived melanoma-specific monoclonal antibody was selected for further study. This antibody effectively killed melanoma cells in vitro via antibody-mediated cellular cytotoxicity. These data demonstrate the presence of a mature systemic B cell response in melanoma patients, which is reduced with disease progression, adding to previous reports of tumor-reactive antibodies in patient sera, and suggesting the merit of future work to elucidate the clinical relevance of activating humoral immune responses to cancer.

  2. A quantitative study of the distribution of IgG sub-classes in a group of normal human sera.

    PubMed

    Shakib, F; Stanworth, D R; Drew, R; Catty, D

    1975-01-01

    The radial immunodiffusion method of Mancini has been applied to quantitative study of IgG subclasses in a normal population. The method was assessed in terms of both reproducibility and antiserum consumption. The distribution of IgG subclasses in a group of normal individuals was studied, and compared with their incidence in a series of monoclonal proteins investigated by previous workers and particularly with other quantitative studies on groups of normal individual's sera.

  3. Type IV collagen aggregates promote keratinocyte proliferation and formation of epidermal layer in human skin equivalents.

    PubMed

    Matsuura-Hachiya, Yuko; Arai, Koji Y; Muraguchi, Taichi; Sasaki, Tasuku; Nishiyama, Toshio

    2017-03-07

    Type IV collagen isolated from lens capsule without enzymatic treatment is known to form a gel under physiological condition and influences cellular activities. In case of human keratinocytes, the suppression of proliferation on reconstituted type IV collagen gels was reported in monolayer culture. In this study, we examined effects of type IV collagen isolated from porcine lens capsule on epidermal formation in human skin equivalents. Type IV collagen aggregates were prepared under the culture condition and the aggregates suppressed keratinocyte proliferation in monolayer culture as well as the culture on the gels. In human skin equivalents type IV collagen aggregates were reconstituted on the surface of contracted collagen gels containing human dermal fibroblasts and the keratinocytes were then cultured on the aggregates for 14 days. Interestingly, in human skin equivalents with type IV collagen aggregates, the BrdU-positive keratinocytes were increased and the thickness of the epidermal layer was around twice than that of control culture. Epidermal differentiation markers were expressed in the upper layer of the epidermis and the defined deposition of human basement membrane components were increased at the dermal-epidermal junction. These results indicate that the type IV collagen aggregates stimulate the proliferation of basal keratinocytes and improve the stratification of epidermal layers in human skin equivalents. This article is protected by copyright. All rights reserved.

  4. Mucosal pemphigus vulgaris anti-Dsg3 IgG are pathogenic to the oral mucosa of humanized Dsg3 mice

    PubMed Central

    Culton, Donna A.; McCray, Suzanne K.; Park, Moonhee; Roberts, James C.; Li, Ning; Zedek, Daniel C.; Anhalt, Grant J.; Cowley, Dale; Liu, Zhi; Diaz, Luis A.

    2015-01-01

    There are two major clinical subsets of pemphigus vulgaris (PV), mucosal PV (mPV) and mucocutaneous PV (mcPV). The mPV subset exhibits anti-human desmoglein (Dsg) 3 autoantibodies that fail to recognize murine Dsg3; thus, passive transfer experiments of mPV IgG into WT mice have been unsuccessful at inducing disease. We therefore generated a fully humanized Dsg3 (hDSG3) murine model utilizing a human Dsg3 transgenic animal crossed to the murine Dsg3 knockout line. Expression of hDsg3 in the mucosa rescues the murine Dsg3 knockout phenotype. Well characterized mPV sera bind mucosal epithelia from the hDsg3 mice, but not mucosal tissues from WT mice by as detected by indirect immunofluorescence. The majority of mPV sera preferentially recognize hDsg3 compared to mDsg3 by immunoprecipitation as well. Passive transfer of mPV IgG into adult hDsg3 mice, but not WT mice, induces suprabasilar acantholysis in mucosal tissues, thus confirming pathogenicity of mPV anti-hDsg3 IgG in vivo. Human anti-hDsg3 antibodies are detected in perilesional mucosa as well as in sera of recipient mice by immunofluorescence. These findings suggest that the Dsg3 epitopes targeted by pathogenic mPV IgG are human specific. This hDsg3 mouse model will be invaluable in studying the clinical transition from mPV to mcPV. PMID:25695683

  5. Human IgG Antibody Response to Aedes Nterm-34kDa Salivary Peptide, an Epidemiological Tool to Assess Vector Control in Chikungunya and Dengue Transmission Area

    PubMed Central

    Elanga Ndille, Emmanuel; Doucoure, Souleymane; Poinsignon, Anne; Mouchet, François; Cornelie, Sylvie; D’Ortenzio, Eric; DeHecq, Jean Sébastien; Remoue, Franck

    2016-01-01

    Background Arboviral diseases are an important public health concerns. Vector control remains the sole strategy to fight against these diseases. Because of the important limits of methods currently used to assess human exposure to Aedes mosquito bites, much effort is being devoted to develop new indicators. Recent studies have reported that human antibody (Ab) responses to Aedes aegypti Nterm-34kDa salivary peptide represent a promising biomarker tool to evaluate the human-Aedes contact. The present study aims investigate whether such biomarker could be used for assessing the efficacy of vector control against Aedes. Methodology/Principal findings Specific human IgG response to the Nterm-34kDa peptide was assessed from 102 individuals living in urban area of Saint-Denis at La Reunion Island, Indian Ocean, before and after the implementation of vector control against Aedes mosquitoes. IgG response decreased after 2 weeks (P < 0.0001), and remained low for 4 weeks post-intervention (P = 0.0002). The specific IgG decrease was associated with the decline of Aedes mosquito density, as estimated by entomological parameters and closely correlated to vector control implementation and was not associated with the use of individual protection, daily commuting outside of the house, sex and age. Our findings indicate a probable short-term decrease of human exposure to Aedes bites just after vector control implementation. Conclusion/Significance Results provided in the present study indicate that IgG Ab response to Aedes aegypti Nterm-34kDa salivary peptide could be a relevant short-time indicator for evaluating the efficacy of vector control interventions against Aedes species. PMID:27906987

  6. 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

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

    PubMed

    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.

  8. Soluble Amyloid-beta Aggregates from Human Alzheimer’s Disease Brains

    PubMed Central

    Esparza, Thomas J.; Wildburger, Norelle C.; Jiang, Hao; Gangolli, Mihika; Cairns, Nigel J.; Bateman, Randall J.; Brody, David L.

    2016-01-01

    Soluble amyloid-beta (Aβ) aggregates likely contribute substantially to the dementia that characterizes Alzheimer’s disease. However, despite intensive study of in vitro preparations and animal models, little is known about the characteristics of soluble Aβ aggregates in the human Alzheimer’s disease brain. Here we present a new method for extracting soluble Aβ aggregates from human brains, separating them from insoluble aggregates and Aβ monomers using differential ultracentrifugation, and purifying them >6000 fold by dual antibody immunoprecipitation. The method resulted in <40% loss of starting material, no detectible ex vivo aggregation of monomeric Aβ, and no apparent ex vivo alterations in soluble aggregate sizes. By immunoelectron microscopy, soluble Aβ aggregates typically appear as clusters of 10–20 nanometer diameter ovoid structures with 2-3 amino-terminal Aβ antibody binding sites, distinct from previously characterized structures. This approach may facilitate investigation into the characteristics of native soluble Aβ aggregates, and deepen our understanding of Alzheimer’s dementia. PMID:27917876

  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. Unraveling the aggregation propensity of human insulin C-peptide.

    PubMed

    Tsiolaki, Paraskevi L; Louros, Nikolaos N; Zompra, Aikaterini A; Hamodrakas, Stavros J; Iconomidou, Vassiliki A

    2017-03-01

    Over the last 20 years, proinsulin C-peptide emerged as an important player in various biological events. Much time and effort has been spent in exploring all functional features of C-peptide and recording its implications in Diabetes mellitus. Only a few studies, though, have addressed C-peptide oligomerization and link this procedure with Diabetes. The aim of our work was to examine the aggregation propensity of C-peptide, utilizing Transmission Electron Microscopy, Congo Red staining, ATR-FTIR, and X-ray fiber diffraction at a 10 mg ml(-1) concentration. Our experimental work clearly shows that C-peptide self-assembles into amyloid-like fibrils and therefore, the aggregation propensity of C-peptide is a characteristic novel feature that should be related to physiological and also pathological conditions. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 108: 1-8, 2017.

  11. 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 Central

    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

    ABSTRACT 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

  12. Mucosal pemphigus vulgaris anti-Dsg3 IgG is pathogenic to the oral mucosa of humanized Dsg3 mice.

    PubMed

    Culton, Donna A; McCray, Suzanne K; Park, Moonhee; Roberts, James C; Li, Ning; Zedek, Daniel C; Anhalt, Grant J; Cowley, Dale O; Liu, Zhi; Diaz, Luis A

    2015-06-01

    There are two major clinical subsets of pemphigus vulgaris (PV)-mucosal PV (mPV) and mucocutaneous PV (mcPV). The mPV subset exhibits anti-human desmoglein (Dsg) 3 autoantibodies that fail to recognize murine Dsg3 (mDsg3); thus, passive transfer experiments of mPV IgG into wild-type (WT) mice have been unsuccessful at inducing disease. We therefore generated a fully humanized Dsg3 (hDSG3) murine model utilizing a hDsg3 transgenic animal crossed to the mDsg3 knockout line. Expression of hDsg3 in the mucosa rescues the mDsg3 knockout phenotype. Well-characterized mPV sera bind mucosal epithelia from the hDsg3 mice, but not mucosal tissues from WT mice, as detected by indirect immunofluorescence (IF). The majority of mPV sera preferentially recognize hDsg3 compared with mDsg3 by immunoprecipitation as well. Passive transfer of mPV IgG into adult hDsg3 mice, but not WT mice, induces suprabasilar acantholysis in mucosal tissues, thus confirming the pathogenicity of mPV anti-hDsg3 IgG in vivo. Human anti-hDsg3 antibodies are detected in perilesional mucosa as well as in sera of recipient mice by IF. These findings suggest that the Dsg3 epitopes targeted by pathogenic mPV IgG are human specific. This hDsg3 mouse model will be invaluable in studying the clinical transition from mPV to mcPV.

  13. 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.

  14. Dynamic and equilibrium performance of sensors based on short peptide ligands for affinity adsorption of human IgG using surface plasmon resonance.

    PubMed

    Islam, Nafisa; Shen, Fei; Gurgel, Patrick V; Rojas, Orlando J; Carbonell, Ruben G

    2014-08-15

    This paper characterizes the potential of novel hexameric peptide ligands for on-line IgG detection in bioprocesses. Surface Plasmon Resonance (SPR) was used to study the binding of human IgG to the hexameric peptide ligand HWRGWV, which was covalently grafted to alkanethiol self-assembled monolayers (SAM) on gold surfaces. Peptide coupling on SAMs was verified, followed by covalent grafting of peptides with a removable Fmoc or acetylated N-termini via their C-termini to produce active peptide SPR sensors that were tested for IgG binding. The dynamics and extent of peptide-IgG binding were compared with results from a conventional system using protein A attached on a gold surface via disulfide monolayers. IgG binding to protein A on disulfide monolayers yielded equilibrium dissociation constants of 1.4×10(-7)M. The corresponding dissociation constant value for the acetylated version of the peptide (Ac-HWRGWV) supported on alkanethiol SAM was 5.8×10(-7)M and that for HWRGWV on the alkanethiol SAM (after de-protection of Fmoc-HWRGWVA) was 1.2×10(-6)M. Maximum IgG binding capacities, Qm of 6.7, 3.8, and 4.1mgm(-2) were determined for the protein A and the two forms of HWRGWV-based biosensors, respectively. Real-time data for the kinetics of adsorption were used to determine the apparent rate constants for adsorption and desorption. The results were analyzed to understand the mechanism of IgG binding to the protein and peptide ligands. It was found that the peptide-IgG binding was reaction controlled, however the protein A-IgG binding mechanism was partially mass transfer (diffusion) controlled. The adsorption rate constants, ka, for the protein A ligand increased with decreasing concentration of analyte and the peptide ligand ka values was constant at different IgG concentrations and flow rates.

  15. Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

    PubMed

    Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

    2015-05-01

    Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding.

  16. Novel feline autoimmune blistering disease resembling bullous pemphigoid in humans: IgG autoantibodies target the NC16A ectodomain of type XVII collagen (BP180/BPAG2).

    PubMed

    Olivry, T; Chan, L S; Xu, L; Chace, P; Dunston, S M; Fahey, M; Marinkovich, M P

    1999-07-01

    In humans and dogs, bullous pemphigoid (BP) is an autoimmune blistering disease associated with the production of basement membrane autoantibodies that target the 180-kd type XVII collagen (BP180, BPAG2) and/or the 230-kd plakin epidermal isoform BPAG1e (BP230). In two adult cats, an acquired dermatosis and stomatitis was diagnosed as BP subsequent to the fulfillment of the following criteria: 1) presence of cutaneous vesicles, erosions, and ulcers; 2) histologic demonstration of subepidermal vesiculation with inflammatory cells, including eosinophils; 3) in vivo deposition of IgG autoantibodies at the epidermal basement membrane zone; and 4) serum IgG autoantibodies targeting a 180-kd epidermal protein identified as type XVII collagen. In both cats, the antigenic epitopes targeted by IgG autoantibodies were shown to be situated in the NC16A ectodomain of type XVII collagen, a situation similar to that of humans and dogs with BP. Feline BP therefore can be considered a clinical, histopathologic, and immunologic homologue of BP in humans and dogs.

  17. 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.

  18. Production and Characterization of a Set of Mouse-Human Chimeric Immunoglobulin G (IgG) Subclass and IgA Monoclonal Antibodies with Identical Variable Regions Specific for Pseudomonas aeruginosa Serogroup O6 Lipopolysaccharide

    PubMed Central

    Preston, Michael J.; Gerçeker, A. Alev; Reff, Mitchell E.; Pier, Gerald B.

    1998-01-01

    The heavy- and light-chain variable regions from a murine monoclonal antibody that recognize Pseudomonas aeruginosa serogroup O6 lipopolysaccharide (LPS) were used to generate a series of chimeric mouse-human monoclonal antibodies with identical variable regions. The murine variable-region gene segments were cloned into an immunoglobulin (Ig) cDNA expression vector that contained the human kappa light-chain and IgG1 constant regions. The IgG1 heavy-chain constant region was then replaced with the human IgG2, IgG3, IgG4, or IgA1 heavy-chain constant region. The five different expression vectors were transfected into Chinese hamster ovary cells for antibody production. The chimeric antibodies exhibited immunoreactivity and affinity similar to that of the parental murine IgG antibody toward whole cells of a serogroup O6 strain. In vitro complement deposition assays demonstrated that the chimeric IgG4 and IgA antibodies did not mediate the deposition of complement component C3 onto the surface of either purified LPS or whole bacteria. The chimeric IgG1 and IgG3 antibodies were similar in their ability to deposit C3 onto the surface of both bacteria and LPS, while IgG2 antibody was more effective at depositing C3 onto the surface of bacteria than onto purified LPS. The pattern of opsonophagocytic activity of the chimeric monoclonal antibodies was similar to that of complement deposition onto bacterial cells in that the chimeric IgG1 and IgG3 had the highest opsonic activity. Although IgG2 deposited more C3 onto the bacterial surface than did IgG4 or IgA, all three of these isotypes had low opsonic activity against the serogroup O6 target strain. This series of related antibodies will help reveal functional differences in efficacy among protective antibodies to P. aeruginosa and will be critical for defining the optimal formulation of either a vaccine for active immunization or a polyclonal intravenous IgG or monoclonal antibody cocktail for passive immunotherapy. PMID

  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. 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.

  2. 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

  3. Effects of tomato extract on human platelet aggregation in vitro.

    PubMed

    Dutta-Roy, A K; Crosbie, L; Gordon, M J

    2001-06-01

    Among all fruits tested in vitro for their anti-platelet property, tomato had the highest activity followed by grapefruit, melon, and strawberry, whereas pear and apple had little or no activity. Tomato extract (20-50 microl of 100% juice) inhibited both ADP- and collagen-induced aggregation by up to 70% but could not inhibit arachidonic acid-induced platelet aggregation and concomitant thromboxane synthesis under similar experimental conditions. The anti-platelet components (MW <1000 Da) in tomatoes are water soluble, heat stable and are concentrated in the yellow fluid around the seeds. The active fractions were separated using gel filtration and HPLC. The aqueous fraction (110 000 xg supernatant) of tomatoes containing anti-platelet activity was subjected to gel filtration column chromatography (Biogel P2 column). The activity was fractionated into two peaks, peak-3 and peak-4 (major peak). Subsequently, peak-4 was further purified by HPLC using a reversed-phase column. NMR and mass spectroscopy studies indicated that peak F2 (obtained from peak 4) contained adenosine and cytidine. Deamination of peak F2 with adenosine deaminase almost completely abolished its anti-platelet activity, confirming the presence of adenosine in this fraction. In comparison, deamination of peak-4 resulted in only partial loss of inhibitory activity while the activity of peak-3 remained unaffected. These results indicate that tomatoes contain anti-platelet compounds in addition to adenosine. Unlike aspirin, the tomato-derived compounds inhibit thrombin-induced platelet aggregation. All these data indicate that tomato contains very potent anti-platelet components, and consuming tomatoes might be beneficial both as a preventive and therapeutic regime for cardiovascular disease.

  4. 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.

  5. 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

  6. Quantifying local characteristics of velocity, aggregation and hematocrit of human erythrocytes in a microchannel flow.

    PubMed

    Kaliviotis, Efstathios; Dusting, Jonathan; Sherwood, Joseph M; Balabani, Stavroula

    2015-09-25

    The effect of erythrocyte aggregation on blood viscosity and microcirculatory flow is a poorly understood area of haemodynamics, especially with relevance to serious pathological conditions. Advances in microfluidics have made it possible to study the details of blood flow in the microscale, however, important issues such as the relationship between the local microstructure and local flow characteristics have not been investigated extensively. In the present study an experimental system involving simple brightfield microscopy has been successfully developed for simultaneous, time-resolved quantification of velocity fields and local aggregation of human red blood cells (RBC) in microchannels. RBCs were suspended in Dextran and phosphate buffer saline solutions for the control of aggregation. Local aggregation characteristics were investigated at bulk and local levels using statistical and edge-detection image processing techniques. A special case of aggregating flow in a microchannel, in which hematocrit gradients were present, was studied as a function of flowrate and time. The level of aggregation was found to strongly correlate with local variations in velocity in both the bulk flow and wall regions. The edge detection based analysis showed that near the side wall large aggregates are associated with regions corresponding to high local velocities and low local shear. On the contrary, in the bulk flow region large aggregates occurred in regions of low velocity and high erythrocyte concentration suggesting a combined effect of hematocrit and velocity distributions on local aggregation characteristics. The results of this study showed that using multiple methods for aggregation quantification, albeit empirical, could help towards a robust characterisation of the structural properties of the fluid.

  7. 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

  8. 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

  9. Expression of all six human Torque teno virus (TTV) proteins in bacteria and in insect cells, and analysis of their IgG responses.

    PubMed

    Kakkola, Laura; Bondén, Heidi; Hedman, Lea; Kivi, Niina; Moisala, Susanna; Julin, Jaakko; Ylä-Liedenpohja, Jussi; Miettinen, Simo; Kantola, Kalle; Hedman, Klaus; Söderlund-Venermo, Maria

    2008-12-20

    Torque teno virus (TTV) is a non-enveloped human virus with a circular ( approximately 3800 nt) ssDNA genome. TTV transcription results in three viral mRNAs and six proteins, the function or antigenicity of which are unknown. The six open reading frames of TTV genotype 6 were expressed in bacteria and insect cells. Expression of the ORF1/1-encoded protein was inefficient, while expression of the others was successful, with ORF1 and ORF1/2 as arginine-rich region depleted. All six recombinant TTV proteins were antigenic. Of healthy adults, 11/25 (44%) showed strong IgG reactivity with one or more proteins. Four subjects, two of whom were genotype-6-DNA positive, were followed. One of the latter showed concurrently a strong IgG response against the ORF1 protein. The other showed appearance of IgG against the ORF2 protein concomitantly with resolution of the genotype-6 viremia. The genotype-6 sequences remained unaltered for years, suggesting that some mechanisms other than amino acid substitutions play a role in TTV immune evasion.

  10. Curcumin promotes fibril formation in F isomer of human serum albumin via amorphous aggregation.

    PubMed

    Mothi, Nivin; Muthu, Shivani A; Kale, Avinash; Ahmad, Basir

    2015-12-01

    We here describe the amyloid fibrils promoting behavior of curcumin, which ability to inhibit amyloid fibrillization of several globular proteins is well documented. Transmission electron microscopy (TEM), 90° light scattering (RLS), thioflavine T (ThT) and Congo red (CR) binding studies demonstrated that both F (pH3.4) and E (pH1.8) isomers of human serum albumin (HSA) in the absence and presence of curcumin initially converted into amorphous aggregates. Interestingly, only the sample containing F isomer preincubated with curcumin formed fibrils on incubation for longer period. We also found that curcumin strongly bind to the F isomer, alter its secondary, tertiary structures and thermal stability. We conclude that the conversion of intermediate states into amorphous aggregate to fibrils is dictated by its conformation. This study provides unique insights into ligand-controlled HSA aggregation pathway and should provide a useful model system to study both amorphous and the fibrillar aggregation of multidomain proteins.

  11. An Extended Minimal Physiologically Based Pharmacokinetic Model: Evaluation of Type II Diabetes Mellitus and Diabetic Nephropathy on Human IgG Pharmacokinetics in Rats.

    PubMed

    Chadha, Gurkishan S; Morris, Marilyn E

    2015-11-01

    Although many studies have evaluated the effects of type 2 diabetes mellitus (T2DM) on the pharmacokinetics (PK) of low molecular weight molecules, there is limited information regarding effects on monoclonal antibodies. Our previous studies have reported significant increases in total (2-4 fold) and renal (100-300 fold) clearance of human IgG, an antibody isotype, in Zucker diabetic fatty (ZDF) rats. Pioglitazone treatment incompletely reversed the disease-related PK changes. The objective of this study was to construct a mechanistic model for simultaneous fitting plasma and urine data, to yield physiologically relevant PK parameters. We propose an extended minimal physiologically based PK (mPBPK) model specifically for IgG by classifying organs as either leaky or tight vascular tissues, and adding a kidney compartment. The model incorporates convection as the primary mechanism of IgG movement from plasma into tissues, interstitial fluid (ISF) in extravascular distribution space, and glomerular filtration rate (GFR), sieving coefficient and fraction reabsorbed in the kidney. The model captured the plasma and urine PK profiles well, and simulated concentrations in ISF. The model estimated a 2-4 fold increase in nonrenal clearance from plasma and 30-120 fold increase in renal clearance with T2DM, consistent with the experimental findings, and these differences in renal clearance were related to changes in GFR, sieving coefficient, and proximal tubular reabsorption. In conclusion, the mPBPK model offers a more relevant approach for analyzing plasma and urine IgG concentration-time data than conventional models and provides insight regarding alterations in distributional and elimination parameters occurring with T2DM.

  12. Salivary production of IgA and IgG to human herpes virus 8 latent and lytic antigens by patients in whom Kaposi's sarcoma has regressed.

    PubMed

    Mbopi-Keou, Francois-Xavier; Legoff, Jerome; Piketty, Christophe; Hocini, Hakim; Malkin, Jean-Elie; Inoue, Naoki; Scully, Crispian M; Porter, Stephen R; Teo, Chong-Gee; Belec, Laurent

    2004-01-23

    IgG and IgA antibodies with specificities to a latent and a lytic antigen of human herpes virus 8 (HHV-8) were detectable in the saliva and serum of eight patients whose Kaposi's sarcoma had regressed, seven of whom were HIV-1 infected. The measurement of antibody-specific activity and secretion rate, and the detection of secretory IgA all indicate anti-HHV-8 antibody activity in saliva. The specific humoral responses possibly influence mucosal replication of HHV-8, and in turn, that of HIV.

  13. Simplification of aggregate culture of human mesenchymal stem cells as a chondrogenic screening assay.

    PubMed

    Welter, Jean F; Solchaga, Luis A; Penick, Kitsie J

    2007-06-01

    Aggregate culture provides a three-dimensional (3-D) environment for differentiating or differentiated cells; it is particularly useful to study in vitro chondrogenesis and cartilage biology. We have recently ported this method from a conical tube-based format to a 96-well plate format for the study of mesenchymal stem cell (MSC) chondrogenesis. The microplate format has greatly reduced the workload and materials cost, while maintaining reproducible chondrogenic differentiation. A long-term goal is to fully automate aggregate culture--this requires critically identifying all the indispensable steps of the protocol. Robotic laboratory equipment for manipulating microplate assays are commercially available; however centrifugation steps are difficult to implement automatically. We, therefore, tested whether the centrifugation step can be eliminated, thus significantly streamlining the assay workflow. By comparing aggregates prepared from human bone marrow-derived MSCs (hMSCs) that were formed either through centrifugation or through free sedimentation, we found that both methods produce aggregates with similar formation kinetics, and that there was no perceptible difference in the timing of the appearance of markers of chondrogenesis. Thus, it appears safe to eliminate the centrifugation step from the aggregate culture protocol. This results in significant time and effort savings and paves the way for future full automation of the aggregate assay.

  14. 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...

  15. 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 ...

  16. Structured illumination microscopy of autofluorescent aggregations in human tissue.

    PubMed

    Best, Gerrit; Amberger, Roman; Baddeley, David; Ach, Thomas; Dithmar, Stefan; Heintzmann, Rainer; Cremer, Christoph

    2011-06-01

    Sections from human eye tissue were analyzed with Structured Illumination Microscopy (SIM) using a specially designed microscope setup. In this microscope the structured illumination was generated with a Twyman-Green Interferometer. This SIM technique allowed us to acquire light-optical images of autofluorophore distributions in the tissue with previously unmatched optical resolution. In this work the unique setup of the microscope made possible the application of SIM with three different excitation wavelengths (488, 568 and 647 nm), thus enabling us to gather spectral information about the autofluorescence signal.

  17. Release of leukotrienes C4 and B4 (LTC4, LTB4) and prostaglandin E2 (PGE2) from human monocytes (M phi) induced with aggregated immunoglobulins (Ig) of different classes

    SciTech Connect

    Ferreri, N.R.; Howland, W.C.; Spiegelberg, H.L.

    1986-03-01

    Purified human peripheral blood monocytes were stimulated with aggregated human myeloma proteins or the calcium ionophore A23187. Release of LTC4, LTB4 and PGE2 into the supernate was determined by radioimmunoassay and high performance liquid chromatography. The ionophore induced release of 10 +/- 5 ng LTC4 and 25 +/- 8 ng LTB4/10/sup 6/ M phi. Aggregated IgG, IgA and IgE but not IgM or monomeric Ig induced release of LTC4 and LTB4 that was approximately 10-20% of that induced by ionophore. Similarly, IgG, IgA and IgE but not IgM induced release of PGE2 (range 0.015-0.22 ng/10/sup 6/ M phi). Absence of calcium or preincubation with nordihydroguaiaretic acid (10/sup -6/ M) inhibited Ig-induced LTC4 and LTB4 release and indomethacin (10/sup -6/ M) inhibited PGE2 release. Phagocytosis of the Ig aggregates was not required since release was not inhibited by cytochalasin B. Release of PGE2 and LTC4/LTB4 induced by all classes except IgM correlated with the presence or absence of M phi Fc receptors (FcR) for each class as determined by rosette assay. The data indicate that IgG, IgA and IgE immune complexes can induce M phi arachidonic acid metabolism via interaction with FcR despite inhibition of phagocytosis. Such a mechanism may contribute to inflammatory reactions characterized by mononuclear cell infiltrates.

  18. Technetium-99m-human polyclonal IgG radiolabeled via the hydrazino nicotinamide derivative for imaging focal sites of infection in rats

    SciTech Connect

    Abrams, M.J.; Juweid, M.; tenKate, C.I.; Schwartz, D.A.; Hauser, M.M.; Gaul, F.E.; Fuccello, A.J.; Rubin, R.H.; Strauss, H.W.; Fischman, A.J. )

    1990-12-01

    The biologic behavior of human polyclonal immunoglobulin (IgG) radiolabeled with technetium-99m ({sup 99m}Tc) by a novel method, via a nicotinyl hydrazine derivative, was evaluated in rats. Technetium-99m- and indium-111-IgG were co-administered to normal rats and biodistribution was determined at 2, 6, and 16 hr. The inflammation imaging properties of the two reagents were compared in rats with deep-thigh infection due to Escherichia coli. Blood clearance of both antibody preparations was well described by a bi-exponential function: ({sup 99m}Tc-IgG: t1/2 = 3.82 +/- 0.89 and 57.52 +/- 1.70 hr. {sup 111}In-IgG: 3.93 +/- 0.117 and 40.71 +/- 1.26 hr). Biodistributions in the solid organs were similar, however, small but statistically significant differences were detected: {sup 99m}Tc-IgG greater than {sup 111}In-IgG in lung, liver, and spleen; {sup 99m}Tc-IgG less than {sup 111}In-IgG in kidney and skeletal muscle (p less than 0.01). At all three imaging times, target-to-background ratio and percent residual activity for the two compounds were remarkably similar. These studies establish that human polyclonal IgG labeled with {sup 99m}Tc via a nicotinyl hydrazine modified intermediate is equivalent to {sup 111}In-IgG for imaging focal sites of infection in experimental animals.

  19. Formation of bobierrite (magnesium phosphate) crystal aggregates by bacteria from human urine and renal calculi.

    PubMed

    del Moral, A; Rivadeneyra, M A; Roldán, E; Perez-García, I; Ramos-Cormenzana, A; García-Cervigón, A

    1989-01-01

    The formation of extracellular crystal aggregates of bobierrite [Mg3(PO4)2.8H2O] by bacteria isolated from renal calculi and urine of urolithiasic patients is found with the use of B-17 and B-43 media. The crystal aggregates were observed in the colonies as deposits of brown-yellow coloration, and were identified by X-ray powder diffraction, chemical analysis and scanning electron microscopy. The production of bobierrite by bacteria from human urine and renal calculi implies further versatility of crystal formation by microorganisms, and it may be interesting to investigate the possible relationships between calculi precipitation and urinary infection.

  20. Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Huang, Richard Y.-C.; Iacob, Roxana E.; Krystek, Stanley R.; Jin, Mi; Wei, Hui; Tao, Li; Das, Tapan K.; Tymiak, Adrienne A.; Engen, John R.; Chen, Guodong

    2016-08-01

    Aggregation of protein therapeutics has long been a concern across different stages of manufacturing processes in the biopharmaceutical industry. It is often indicative of aberrant protein therapeutic higher-order structure. In this study, the aggregation propensity of a human Fc-fusion protein therapeutic was characterized. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was applied to examine the conformational dynamics of dimers collected from a bioreactor. HDX-MS data combined with spatial aggregation propensity calculations revealed a potential aggregation interface in the Fc domain. This study provides a general strategy for the characterization of the aggregation propensity of Fc-fusion proteins at the molecular level.

  1. IgG rheumatoid factors against the four human Fc-gamma subclasses in early rheumatoid arthritis (the Swedish TIRA project).

    PubMed

    Kanmert, D; Kastbom, A; Almroth, G; Skogh, T; Enander, K; Wetterö, J

    2012-01-01

    Rheumatoid factor (RF), i.e. a family of autoantibodies against the Fc part of IgG, is an important seromarker of rheumatoid arthritis (RA). Traditional particle agglutination without disclosing the antibody isotype remains the predominating diagnostic method in clinical routine. Although IgG-RF attracts pathogenic interest, its detection remains technically challenging. The present study aimed at developing a set of tests identifying IgG-RFs directed against the four IgG subclasses. IgG-RF against either subclass of human IgG-Fc were analysed with four novel enzyme-linked immunosorbent assays (ELISAs) utilizing four recombinant human Fc-gamma fragments (hIgG1-4) as sources of antigen. Sera from 40 patients with recent onset RA (20 seropositive and 20 seronegative by IgM-RF and IgA-RF-isotype-specific ELISA) were analysed. Sera from 20 healthy blood donors served as reference. Among the IgM-/IgA-RF-positive RA-sera, IgG-RF was found directed against hIgG1 and hIgG2, but not against hIgG3 or hIgG4. Significant correlations were seen between IgG-RF against hIgG2-Fc and IgM-RF (r = 0.666) levels. Further prospective studies are warranted to elucidate any correlation to disease course and outcome.

  2. Evaluation of the Human IgG Antibody Response to Aedes albopictus Saliva as a New Specific Biomarker of Exposure to Vector Bites

    PubMed Central

    Doucoure, Souleymane; Mouchet, François; Cornelie, Sylvie; DeHecq, Jean Sébastien; Rutee, Abdul Hamid; Roca, Yelin; Walter, Annie; Hervé, Jean Pierre; Misse, Dorothée; Favier, François; Gasque, Philippe; Remoue, Franck

    2012-01-01

    Background The spread of Aedes albopictus, a vector for re-emergent arbovirus diseases like chikungunya and dengue, points up the need for better control strategies and new tools to evaluate transmission risk. Human antibody (Ab) responses to mosquito salivary proteins could represent a reliable biomarker for evaluating human-vector contact and the efficacy of control programs. Methodology/Principal Findings We used ELISA tests to evaluate specific immunoglobulin G (IgG) responses to salivary gland extracts (SGE) in adults exposed to Aedes albopictus in Reunion Island. The percentage of immune responders (88%) and levels of anti-SGE IgG Abs were high in exposed individuals. At an individual level, our results indicate heterogeneity of the exposure to Aedes albopictus bites. In addition, low-level immune cross-reactivity between Aedes albopictus and Aedes aegypti SGEs was observed, mainly in the highest responders. Conclusion/Significance Ab responses to saliva could be used as an immuno-epidemiological tool for evaluating exposure to Aedes albopictus bites. Combined with entomological and epidemiological methods, a “salivary” biomarker of exposure to Aedes albopictus could enhance surveillance of its spread and the risk of arbovirus transmission, and could be used as a direct tool for the evaluation of Aedes albopictus control strategies. PMID:22363823

  3. Ellipsometric studies on deposition of complement from human serum to IgG and IgA immobilized on hydrophobic silicon

    NASA Astrophysics Data System (ADS)

    Tengvall, Pentti; Askendal, Agneta; Lundstroem, Ingemar

    1999-01-01

    Human immunoglobulin G (IgG) and colostrum immunoglobulin A (IgA) were absorbed to hydrophobic silicon and the deposition of complement (C) was studied by ellipsometry- antibody techniques after incubation at 37 degree(s)C in human serum for up to 1 hour. In parallel experiments soluble iC3b, C4d and Bb were detected by ELISA-techniques. IgG coated surfaces rapidly activated the classical pathway and caused deposition of anti-C3c, antiproperdin, and during short serum incubation times also of anti-C1q and anti-IgG. The IgA-coated surfaces activated the alternative pathway and displayed a lag phase in the complement protein deposition. Anti-IgG, Clq, -C4, -factor H and -factor B were not deposited into IgA-surfaces at any time. Ellipsometry and antibody techniques offer a convenient and rapid means to display activation of the complement system by solid light-reflecting surfaces and facilitate a time-resolved determination of the activation pathway(s).

  4. 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.

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

    PubMed Central

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

    2014-01-01

    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

  6. Recombinant human IgG antibodies recognizing distinct extracellular domains of EGF receptor exhibit different degrees of growth inhibitory effects on human A431 cancer cells.

    PubMed

    Chang, Chialun; Takayanagi, Atsushi; Yoshida, Tetsuhiko; Shimizu, Nobuyoshi

    2013-05-01

    Recently, we isolated 4 distinct kinds of single chain antibody against human EGF receptor (EGFR) after screening the Keio phage display scFv library by using two methods of target-guided proximity labeling. In the current study, these monovalent scFv antibodies were converted to bivalent IgGs of humanized forms (hIgGs) by recombinant technology using the specially designed expression vectors followed by protein production in CHO cells. The resulting recombinant hIgGs were examined for their binding specificity using several different transformed human BJ cell lines that express deletion mutants of EGFR, each lacking one of 4 distinct extracellular domains (L1, L2, C1 and C2). Immuno-fluorescent microscopy and immuno-precipitation assay on these cells indicated that 4 distinct kinds of hIgGs bind to one of 3 different domains (L1, C1 and C2). Then, these hIgGs were further examined for biological effects on human A431 cancer cells, which overexpress EGFR. The results indicated that hIgG38 binding to L1 and hIgG45 binding to C2 substantially suppressed the EGF-induced phosphorylation of EGFR, resulting in the growth inhibition of A431 cancer cells. On the contrary, hIgG40 binding to C1 and hIgG42 binding to another site (epitope) of C2 exhibited no such inhibitory effects. Thus, the newly produced four recombinant hIgG antibodies recognize 4 different sites (epitopes) in 3 different extracellular domains of EGFR and exhibit different biological effects on cancer cells. These characteristics are somewhat different from the currently utilized therapeutic anti-EGFR antibodies. Hence, these hIgG antibodies will be invaluable as a research tool for the detailed molecular analysis of the EGFR-mediated signal transduction mechanism and more importantly a possible application as new therapeutic agents to treat certain types of cancers.

  7. Structural insights into complexes of glucose-regulated Protein94 (Grp94) with human immunoglobulin G. relevance for Grp94-IgG complexes that form in vivo in pathological conditions.

    PubMed

    Pagetta, Andrea; Tramentozzi, Elisa; Tibaldi, Elena; Cendron, Laura; Zanotti, Giuseppe; Brunati, Anna Maria; Vitadello, Maurizio; Gorza, Luisa; Finotti, Paola

    2014-01-01

    While the mechanism by which Grp94 displays its chaperone function with client peptides in the cell has been elucidated extensively, much less is known about the nature and properties of how Grp94 can engage binding to proteins once it is exposed on the cell surface or liberated in the extra-cellular milieu, as occurs in pathological conditions. In this work, we wanted to investigate the molecular aspects and structural characteristics of complexes that Grp94 forms with human IgG, posing the attention on the influence that glycosylation of Grp94 might have on the binding capacity to IgG, and on the identification of sites involved in the binding. To this aim, we employed both native, fully glycosylated and partially glycosylated Grp94, and recombinant, non-glycosylated Grp94, as well as IgG subunits, in different experimental conditions, including the physiological setting of human plasma. Regardless of the species and type, Grp94 engages a similar, highly specific and stable binding with IgG that involves sites located in the N-terminal domain of Grp94 and the hinge region of whole IgG. Grp94 does not form stable complex with Fab, F(ab)2 or Fc. Glycosylation turns out to be an obstacle to the Grp94 binding to IgG, although this negative effect can be counteracted by ATP and spontaneously also disappears in time in a physiological setting of incubation. ATP does not affect at all the binding capacity of non-glycosylated Grp94. However, complexes that native, partially glycosylated Grp94 forms with IgG in the presence of ATP show strikingly different characteristics with respect to those formed in absence of ATP. Results have relevance for the mechanism regulating the formation of stable Grp94-IgG complexes in vivo, in the pathological conditions associated with the extra-cellular location of Grp94.

  8. A Multistage Pathway for Human Prion Protein Aggregation in Vitro: From Multimeric Seeds to β-Oligomers and Nonfibrillar Structures

    PubMed Central

    Cho, Kang R.; Yu, Shuiliang; Yin, Shaoman; Plomp, Marco; Qiu, S. Roger; Lakshminarayanan, Rajamani; Moradian-Oldak, Janet; Sy, Man-Sun

    2015-01-01

    Aberrant protein aggregation causes numerous neurological diseases including Creutzfeldt—Jakob disease (CJD), but the aggregation mechanisms remain poorly understood. Here, we report AFM results on the formation pathways of β-oligomers and nonfibrillar aggregates from wild-type full-length recombinant human prion protein (WT) and an insertion mutant (10OR) with five additional octapeptide repeats linked to familial CJD. Upon partial denaturing, seeds consisting of 3–4 monomers quickly appeared. Oligomers of ∼11–12 monomers then formed through direct interaction of seeds, rather than by subsequent monomer attachment. All larger aggregates formed through association of these β-oligomers. Although both WT and 10OR exhibited identical aggregation mechanisms, the latter oligomerized faster due to lower solubility and, hence, thermodynamic stability. This novel aggregation pathway has implications for prion diseases as well as others caused by protein aggregation. PMID:21534611

  9. The heterogeneity of the non-aggregating proteoglycans of the human intervertebral disc.

    PubMed Central

    DiFabio, J L; Pearce, R H; Caterson, B; Hughes, H

    1987-01-01

    Non-aggregating proteoglycans of differing average hydrodynamic volumes were prepared from nuclei pulposi and anuli fibrosi of three human lumbar spines and characterized by biochemical and immunochemical analyses. The hexose-to-hexuronate and protein-to-hexuronate ratios increased with decreasing hydrodynamic volume. Analysis by composite agarose/polyacrylamide-gel electrophoresis has demonstrated two aggregating subpopulations [McDevitt, Jahnke & Green (1982) Trans. Annu. Meet. Orthop. Res. Soc. 7, 50]. In the present study, electrophoresis of the non-aggregating pools has shown three additional subpopulations, here named bands III, IV and V. The two smallest proteoglycan pools from each tissue contained two and three components respectively. These components were isolated by preparative electrophoresis and analysed. Band III was a proteoglycan richer in keratan sulphate than in chondroitin sulphate; band IV was a proteoglycan richer in chondroitin sulphate than in keratan sulphate; band V contained only chondroitin sulphate. Unsaturated disaccharides prepared from the chondroitin sulphate of all bands were predominantly 6-sulphated, with only 5-15% 4-sulphated. The molecular masses of the chondroitin sulphate and keratan sulphate did not differ between the bands. The amino acid composition of band III differed from that of band IV. Thus three distinct subpopulations of non-aggregating proteoglycan were demonstrated in the human intervertebral disc. PMID:3117036

  10. Development of antihuman IgG antibodies and hematologic deficits but not clinical abnormalities in C57BL/6 mice after repeated administration of human intravenous immunoglobulin.

    PubMed

    Loeffler, David A; Smith, Lynnae M; Klaver, Andrea C; Brzezinski, Heather A; Morrison, Essie I; Coffey, Mary P; Steficek, Barbara A; Cook, Susan S

    2012-02-01

    Intravenous immunoglobulin (IvIg) preparations consist of purified human immunoglobulins collected from large numbers of healthy persons and are used to treat autoimmune, immunodeficiency, and inflammatory disorders. Studying the effects of IvIg effects in experimental animal models might clarify its mechanisms of action in these disorders, but whether 'serum sickness' or other abnormalities occur after repeated IvIg administration to immunocompetent animals is unknown. In the current study, male C57BL/6 mice (8 to 10 wk old; n = 27) received IvIg (1 g/kg IP) weekly for 6 wk. They were observed for clinical abnormalities, and body weight, temperature, renal function, hematologic parameters, and serum antihuman IgG antibodies were measured before and during treatment. Postmortem evaluations were performed on kidney, spleen, liver, and heart. No clinical or histologic abnormalities were noted despite a transient increase in BUN. Mean antibody levels to human IgG on days 21 and 43 after IvIg administration were increased by 23-fold compared with pretreatment levels. 88% and 89% of the mice were antibody responders on those days. Unexpectedly, hemoglobin, hematocrit, and RBC, WBC, lymphocyte, and platelet counts decreased after IvIg administration. These findings suggest that although it does not produce serum sickness, repeated IvIg administration to immunocompetent mice induces a strong humoral immune response and hematologic deficits of unknown etiology. These factors could cause the effects of IvIg preparations in mouse models of human disease to differ from their effects in the human disorders.

  11. 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

  12. Inhibitory effects of yuzu and its components on human platelet aggregation.

    PubMed

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

    2015-03-01

    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.

  13. 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

  14. Acetaminophen and meloxicam inhibit platelet aggregation and coagulation in blood samples from humans.

    PubMed

    Martini, Angela K; Rodriguez, Cassandra M; Cap, Andrew P; Martini, Wenjun Z; Dubick, Michael A

    2014-12-01

    Acetaminophen (Ace) and meloxicam (Mel) are the two types of analgesic and antipyretic medications. This study investigated the dose responses of acetaminophen and meloxicam on platelet aggregation and coagulation function in human blood samples. Blood samples were collected from six healthy humans and processed to make platelet-adjusted (100 × 10 cells/μl) blood samples. Acetaminophen (Tylenol, Q-PAP, 100 mg/ml) was added at the doses of 0 μg/ml (control), 214 μg/ml (the standard dose, 1 ×), 4 ×, 8 ×, 10 ×, 12 ×, 16 ×, and 20 ×. Similarly, meloxicam (Metacam, 5 mg/ml) was added at doses of 0 μg/ml (control), 2.85 μg/ml (the standard dose, 1 ×), 4 ×, 8 ×, 10 ×, 12 ×, 16 ×, and 20 ×. Fifteen minutes after the addition of acetaminophen and/or meloxicam, platelet aggregation was stimulated with collagen (2 μg/ml) or arachidonic acid (0.5 mmol/l) and assessed using a Chrono-Log 700 aggregometer. Coagulation function was assessed by prothrombin time (PT), activated partial thromboplastin time (aPTT), and using Rotem thrombelastogram. A robust inhibition by acetaminophen and/or meloxicam was observed in arachidonic acid-stimulated platelet aggregation starting at 1 × dose. Collagen-stimulated platelet aggregation was inhibited by ACE starting at 1 × (78 ± 10% of control), and by meloxicam starting at 4 × (72 ± 5% of control, both P < 0.05). The inhibitions by acetaminophen and meloxicam combined were similar to those by acetaminophen or meloxicam. aPTT was prolonged by meloxicam starting at 4 ×. No changes were observed in PT or any of Rotem measurements by acetaminophen and/or meloxicam. Acetaminophen and meloxicam compromised platelet aggregation and aPTT. Further effort is warranted to characterize the effects of acetaminophen and meloxicam on bleeding in vivo.

  15. LC-MS analysis of polyclonal human anti-Neu5Gc Xeno-autoantibodies IgG subclass and partial sequence using multi-step IVIG affinity purification and multi-enzymatic digestion

    PubMed Central

    Lu, Qiaozhen; Padler-Karavani, Vered; Yu, Hai; Chen, Xi; Wu, Shiaw-Lin; Varki, Ajit; Hancock, William S.

    2014-01-01

    Human polyclonal IgG antibodies directly against the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) are potential biomarkers and mechanistic contributors to cancer and other diseases associated with chronic inflammation. Using a sialoglycan microarray, we screened the binding pattern of such antibodies (anti-Neu5Gc IgG) in several samples of clinically-approved human IVIG (IgG). These results were used to select an appropriate sample for a multi-step affinity purification of the xeno-autoantibody fraction. The sample was then analyzed via our multi-enzyme digestion procedure followed by nanoLC coupled to LTQ-FTMS. We used characteristic and unique peptide sequences to determine the IgG subclass distribution and thus provided direct evidence that all four IgG subclasses can be generated during a xeno-autoantibody immune response to carbohydrate Neu5Gc-antigens. Furthermore, we obtained a significant amount of sequence coverage of both the constant and variable regions. The approach described here, therefore, provides a way to characterize these clinically significant antibodies, helping to understand their origins and significance. PMID:22390546

  16. [Role of protein kinases of human red cell membrane in deformability and aggregation changes].

    PubMed

    Murav'ev, A V; Maĭmistova, A A; Tikhomirova, I A; Bulaeva, S V; Mikhaĭlov, P V; Murav'ev, A A

    2012-01-01

    The proteomic analysis has showed that red cell membrane contains several kinases and phosphatases. Therefore the aim of this study was to investigate the role of protein kinases of human red cell membrane in deformability and aggregation changes. Exposure of red blood cells (RBCs) to some chemical compounds led to change in the RBC microrheological properties. When forskolin (10 microM), an adenylyl cyclase (AC) and a protein kinase A (PKA) stimulator was added to RBC suspension, the RBC deformability (RBCD) was increased by 20% (p < 0.05). Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP (by 26%; p < 0.01). Red cell aggregation (RBCA) was significantly decreased under these conditions (p < 0.01). Markedly less changes of deformability was found after RBC incubation with protein kinase stimulator C (PKC)--phorbol 12-myristate 13-acetate (PMA). This drug reduced red cell aggregation only slightly. It was inhibited red cell tyrosine phosphotase activity by N-vanadat and was obtained a significant RBCD rise and RBCA lowering. The similar effect was found when cells were incubated with cisplatin as a tyrosine protein kinase (TPK) activator. It is important to note that a selective TPK inhibitor--lavendustin eliminated the above mention effects. On the whole the total data clearly show that the red cell aggregation and deformation changes were connected with an activation of the different intracellular signaling pathways.

  17. Manganese exposure induces α-synuclein aggregation in the frontal cortex of non-human primates.

    PubMed

    Verina, Tatyana; Schneider, Jay S; Guilarte, Tomás R

    2013-03-13

    Aggregation of α-synuclein (α-syn) in the brain is a defining pathological feature of neurodegenerative disorders classified as synucleinopathies. They include Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Occupational and environmental exposure to manganese (Mn) is associated with a neurological syndrome consisting of psychiatric symptoms, cognitive impairment and parkinsonism. In this study, we examined α-syn immunoreactivity in the frontal cortex of Cynomolgus macaques as part of a multidisciplinary assessment of the neurological effects produced by exposure to moderate levels of Mn. We found increased α-syn-positive cells in the gray matter of Mn-exposed animals, typically observed in pyramidal and medium-sized neurons in deep cortical layers. Some of these neurons displayed loss of Nissl staining with α-syn-positive spherical aggregates. In the white matter we also observed α-syn-positive glial cells and in some cases α-syn-positive neurites. These findings suggest that Mn exposure promotes α-syn aggregation in neuronal and glial cells that may ultimately lead to degeneration in the frontal cortex gray and white matter. To our knowledge, this is the first report of Mn-induced neuronal and glial cell α-syn accumulation and aggregation in the frontal cortex of non-human primates.

  18. Influence of Aluminium and EGCG on Fibrillation and Aggregation of Human Islet Amyloid Polypeptide

    PubMed Central

    Xu, Zhi-Xue; Zhang, Qiang; Ma, Gong-Li; Chen, Cong-Heng; He, Yan-Ming; Xu, Li-Hui; Zhang, Yuan; Zhou, Guang-Rong; Li, Zhen-Hua

    2016-01-01

    The abnormal fibrillation of human islet amyloid polypeptide (hIAPP) has been implicated in the development of type II diabetes. Aluminum is known to trigger the structural transformation of many amyloid proteins and induce the formation of toxic aggregate species. The (−)-epigallocatechin gallate (EGCG) is considered capable of binding both metal ions and amyloid proteins with inhibitory effect on the fibrillation of amyloid proteins. However, the effect of Al(III)/EGCG complex on hIAPP fibrillation is unclear. In the present work, we sought to view insight into the structures and properties of Al(III) and EGCG complex by using spectroscopic experiments and quantum chemical calculations and also investigated the influence of Al(III) and EGCG on hIAPP fibrillation and aggregation as well as their combined interference on this process. Our studies demonstrated that Al(III) could promote fibrillation and aggregation of hIAPP, while EGCG could inhibit the fibrillation of hIAPP and lead to the formation of hIAPP amorphous aggregates instead of the ordered fibrils. Furthermore, we proved that the Al(III)/EGCG complex in molar ratio of 1 : 1 as Al(EGCG)(H2O)2 could inhibit the hIAPP fibrillation more effectively than EGCG alone. The results provide the invaluable reference for the new drug development to treat type II diabetes. PMID:28074190

  19. 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

  20. Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.

    PubMed

    Irani, Vashti; Guy, Andrew J; Andrew, Dean; Beeson, James G; Ramsland, Paul A; Richards, Jack S

    2015-10-01

    Monoclonal antibodies are being developed as therapeutics to complement drugs and vaccines or to fill the gap where no drugs or vaccines exist. These therapeutic antibodies (ThAb) may be especially important for infectious diseases in which there is antibiotic resistance, toxin-mediated pathogenesis, or for emerging pathogens. The unique structure of antibodies determines the specific nature of the effector function, so when developing ThAb, the desired effector functions need to be considered and integrated into the design and development processes to ensure maximum efficacy and safety. Antibody subclass is a critical consideration, but it is noteworthy that almost all ThAb that are licenced or currently in development utilise an IgG1 backbone. This review outlines the major structural properties that vary across subclasses, how these properties affect functional immunity, and discusses the various approaches used to study subclass responses to infectious diseases. We also review the factors associated with the selection of antibody subclasses when designing ThAb and highlight circumstances where different subclass properties might be beneficial when applied to particular infectious diseases. These approaches are critical to the future design of ThAb and to the study of naturally-acquired and vaccine-induced immunity.

  1. Influence of methionine oxidation on the aggregation of recombinant human growth hormone.

    PubMed

    Mulinacci, Filippo; Poirier, Emilie; Capelle, Martinus A H; Gurny, Robert; Arvinte, Tudor

    2013-09-01

    Oxidation of methionine (Met) residues is one of the major chemical degradations of therapeutic proteins. This chemical degradation can occur at various stages during production and storage of a biotherapeutic drug. During the oxidation process, the side chain of methionine residue undergoes a chemical modification, with the thioether group substituted by a sulfoxide group. In previous papers, we showed that oxidation of the two most accessible methionine residues of recombinant human growth hormone (r-hGH), Met¹⁴ and Met¹²⁵, has no influence on the conformation of the protein [1]. However, the oxidized r-hGH is less thermally stable than the native protein [2]. In the current work, the consequences of the oxidation of these two methionine residues on the aggregation of r-hGH were investigated. The aggregation properties and kinetics of the native and oxidized r-hGH were measured in different buffers with both spectroscopic and chromatographic methods. Stabilities of oxidized and non-oxidized r-hGH were studied after storage at 37°C and freeze/thawing cycles. Methionine oxidation influenced the aggregation properties of r-hGH. In accelerated stability studies at 37°C, oxidized hormone aggregated more and faster than non-oxidized hormone. In freezing/thawing stability studies, it was found that oxidized r-hGH was less stable than its non-oxidized counterpart. In case of hGH, we have shown that chemical degradations such as oxidation can affect its physical stability and can induce aggregation.

  2. Inhibitory effect of corcin on aggregation of 1N/4R human tau protein in vitro

    PubMed Central

    Karakani, Ali Mohammadi; Riazi, Gholamhossein; Mahmood Ghaffari, Seyed; Ahmadian, Shahin; Mokhtari, Farzad; Jalili Firuzi, Mahshad; Zahra Bathaie, Seyedeh

    2015-01-01

    Objective(s): Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. One of the hallmarks of AD is an abnormal accumulation of fibril forms of tau protein which is known as a microtubule associated protein. In this regard, inhibition of tau aggregation has been documented to be a potent therapeutic approach in AD and tauopathies. Unfortunately, the available synthetic drugs have modest beneficial efficacy with several side effects. Therefore, pipeline drugs from natural sources with anti-aggregation properties can be useful in the prevention and treatment of AD. Among medicinal plants, saffron (Crocus sativus, L.), as a traditional herbal medicine has different pharmacological properties and can be used as treatment for several nervous system impairment including depression and dementia. Crocin as a major constituent of saffron is the glycosylated form of crocetin. Materials and Methods: In this study, we investigated the inhibitory effect of crocin on aggregation of recombinant human tau protein 1N/4R isoform using biochemical methods and cell culture. Results: Results revealed that tau protein under the fibrillation condition and in the presence of crocin had enough stability with low tendency for aggregation. Crocin inhibited tau aggregation with IC50 of 100 µg/ml. Furthermore, transmission electron microscopy images confirmed that crocin could suppress the formation of tau protein filaments. Conclusion: Inhibitory effect of crocin could be related to its interference with nucleation phase that led to increases in monomer species of tau protein. Based on our results, crocin is recommended as a proper candidate to be used in AD treatment. PMID:26124935

  3. Further insights into the anti-aggregating activity of NMDA in human platelets

    PubMed Central

    Franconi, Flavia; Miceli, Mauro; Alberti, Luisa; Seghieri, Giuseppe; De Montis, M Graziella; Tagliamonte, Alessandro

    1998-01-01

    In the present study the effect of N-methyl-D-aspartate (NMDA) on thromboxane B2 synthesis and on [Ca2+]i was studied in human platelets.NMDA (10−7 M) completely inhibited the synthesis of thromboxane B2 from exogenous arachidonic acid (AA), while it did not interfere with the aggregating effect of the thromboxane A2 receptor agonist U-46619.NMDA (0.1 μM–10 μM) dose-dependently increased intracellular calcium in washed platelets pre-loaded with fura 2 AM, and this effect was not additive with that of AA.NMDA shifted the dose-response curve of AA to the right. At the highest AA concentrations platelet aggregation was not inhibited.The antiaggregating effect of NMDA was not antagonized by NG-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase (NOS) inhibitor.Finally, NMDA (0.01 nM–100 nM) associated with either aspirin or indomethacin significantly potentiated the antiaggregating activity of both cyclo-oxygenase inhibitors.It was concluded that NMDA is a potent inhibitor of platelet aggregation and thromboxane B2 synthesis in human platelet rich plasma (PRP). PMID:9630340

  4. 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.

  5. Effects of an angelica extract on human erythrocyte aggregation, deformation and osmotic fragility.

    PubMed

    Wang, X; Wei, L; Ouyang, J P; Muller, S; Gentils, M; Cauchois, G; Stoltz, J F

    2001-01-01

    In Chinese traditional medicine, angelica is widely used for its known clinical effects of ameliorating blood microcirculation. But the mechanism of these beneficial effects still remains unclear. In this work the rheological behaviour of human erythrocytes treated by angelica was studied in vitro. Normal RBCs incubated with an angelica extract at different concentrations (5, 10 or 20 mg/ml) for 60 min at 37 degrees C and then their aggregation, deformation and osmotic fragility were measured with different recently developed optical techniques, namely Erythroaggregometer (Regulest, Florange, France), LORCA (Mechatronics, Amsterdam) and Fragilimeter (Regulest, Florange, France). Experimental results show that angelica (20 mg/ml) significantly decreased normal RBCs' aggregation speed (p<0.01) and could inhibit the hyperaggregability caused by dextran 500. However, the strength of normal RBCs aggregates were not influenced by angelica. When a calcium ionophore A23187 (1.9 microM) was used to harden cell membrane, angelica (20 mg/ml) could significantly (p<0.01) protect erythrocytes against the loss of their deformability even it had no effects on normal RBCs deformation. Finally angelica (5 and 10 mg/ml) decreased significantly (p<0.01) normal RBCs osmotic fragility. In conclusion angelica plays a rheologically active role on human erythrocytes, and this study suggests a possible mechanism for angelica's positive effects against certain cardiovascular diseases.

  6. The FcγR of humans and non-human primates and their interaction with IgG: implications for induction of inflammation, resistance to infection and the use of therapeutic monoclonal antibodies.

    PubMed

    Hogarth, P Mark; Anania, Jessica C; Wines, Bruce D

    2014-01-01

    Considerable effort has focused on the roles of the individual members of the FcγR receptor (FcγR) family in inflammatory diseases and humoral immunity. Recent work has revealed major roles in infection and in particular HIV pathogenesis and immunity. In addition, FcγR functions underpin the action of many of the successful therapeutic monoclonal antibodies. This emphasises the need for a greater understanding of FcγR function in humans and in the NHP which provides a key model for human immunity and preclinical testing of antibodies. We discuss recent key aspects of the human FcγR receptor biology and structure to define differences and similarities in activity between the human and macaque Fc receptors. These differences and similarities nuance the interpretation of infection and vaccine studies in the macaque. Indeed passive IgG antibody protection in lentivirus infection models in the macaque provided early evidence for the role of Fc receptors in anti-HIV immunity that have subsequently gained support from human vaccine trials. None-the-less the diverse functions and cellular contexts of FcγR receptor expression ensure there is much still to understand of the protective and deleterious effects of FcγRs in HIV infection. Careful comparative studies of human and non-human primate FcγRs will facilitate our appreciation of what attributes of HIV specific IgG antibodies, either acquired naturally or via vaccination, are most important for protection.

  7. Human CD100, a novel leukocyte semaphorin that promotes B-cell aggregation and differentiation.

    PubMed Central

    Hall, K T; Boumsell, L; Schultze, J L; Boussiotis, V A; Dorfman, D M; Cardoso, A A; Bensussan, A; Nadler, L M; Freeman, G J

    1996-01-01

    Herein we describe the molecular characterization of the human leukocyte activation antigen CD100 and identify it as the first semaphorin, to our knowledge, in the immune system. Semaphorins have recently been described as neuronal chemorepellants that direct pioneering neurons during nervous system development. In this study we demonstrate that CD100 induces B cells to aggregate and improves their viability in vitro. We show that CD100 modifies CD40-CD40L B-cell signaling by augmenting B-cell aggregation and survival and down-regulating CD23 expression. Thus, these results suggest that semaphorins as exemplified by CD100 also play a functional role in the immune system. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8876214

  8. Determination of the molecular size distribution of immunoglobulin G (IgG) in intravenous IgG-albumin formulations by high-performance liquid chromatography.

    PubMed

    Lee, J K; Deluccia, F J; Kelly, E L; Davidson, C; Borger, F R

    1988-07-01

    Recent reports have expressed concern about the safety of intravenous human immunoglobulin G (IgG) preparations. Evidence seems to indicate that aggregates in these formulations are responsible for several adverse reactions in some patients, including anaphylaxis and dyspnea. Therefore, monitoring the molecular size distribution of IgG in these products is essential for ensuring their safety. This paper describes a sensitive and precise two-stage high-performance liquid chromatographic method for determining the molecular size distribution profile of IgG in an intravenous formulation stabilized with albumin. In the first step, all molecular forms of IgG are separated from all molecular forms of albumin by anion-exchange chromatography. In the second stage, a portion of the collected IgG fraction is re-chromatographed by size-exclusion chromatography and separated into its aggregate, dimer, and monomer components. Although some minor losses of aggregate do occur in the procedure, the overall molecular size distribution is not significantly affected. The method described is both time-efficient and accurate, and represents an improvement over existing methods.

  9. 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.

  10. TRU-016, a humanized anti-CD37 IgG fusion protein for the potential treatment of B-cell malignancies.

    PubMed

    Robak, Tadeusz; Robak, Pawel; Smolewski, Piotr

    2009-12-01

    TRU-016, under development by Trubion Pharmaceuticals Inc and Facet Biotech Corp, is an intravenously administered anti-CD37 IgG fusion protein for the potential treatment of B-cell malignancies, including chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL), as well as for autoimmune and inflammatory diseases. TRU-016 was created by humanizing SMIP-016, a mouse/human chimeric protein that demonstrated antitumor activity against lymphoid malignancies in preclinical studies, including in human B-cell tumor mouse xenograft models. In addition, TRU-016 demonstrated synergistic or additive activity in NHL cells in combination with rituximab, rapamycin, doxorubicin and bendamustine. In a phase I/II clinical trial in refractory or relapsed patients with CLL or small lymphocytic lymphoma, TRU-016 was well tolerated, with clinical benefit and a reduced absolute lymphocyte count observed in all cohorts dosed at > 0.1 mg/kg. TRU-016 is a promising therapeutic agent for patients with B-cell lymphoid malignancies, especially patients refractory to standard treatment.

  11. A new human IgG avidity test, using mixtures of recombinant antigens (rROP1, rSAG2, rGRA6), for the diagnosis of difficult-to-identify phases of toxoplasmosis.

    PubMed

    Drapała, Dorota; Holec-Gąsior, Lucyna; Kur, Józef; Ferra, Bartłomiej; Hiszczyńska-Sawicka, Elżbieta; Lautenbach, Dariusz

    2014-07-01

    The preliminary diagnostic utility of two mixtures of Toxoplasma gondii recombinant antigens (rROP1+rSAG2 and rROP1+rGRA6) in IgG ELISA and IgG avidity test has been evaluated. A total of 173 serum samples from patients with toxoplasmosis and seronegative people were examined. The sensitivity of IgG ELISA for rROP1+rSAG2 and rROP1+rGRA6 was 91.1% and 76.7%, respectively, while the reactivity for sera from patients where acute toxoplasmosis was suspected was higher, at 100% and 95.4%, respectively, than for people with chronic infection, at 88.2% and 70.6%. In this study a different trend in avidity maturation of IgG antibodies for two mixtures of proteins in comparison with native antigen was observed. The results suggest that a new IgG avidity test using the mixtures of recombinant antigens may be useful for the diagnosis of difficult-to-identify phases of toxoplasmosis. For this reason, selected mixtures after the additional tests on groups of sera with well-defined dates of infection could be used as a better alternative to the native antigens of the parasite in the serodiagnosis of human T. gondii infection.

  12. Aggregation of human recombinant monoclonal antibodies influences the capacity of dendritic cells to stimulate adaptive T-cell responses in vitro.

    PubMed

    Rombach-Riegraf, Verena; Karle, Anette C; Wolf, Babette; Sordé, Laetitia; Koepke, Stephan; Gottlieb, Sascha; Krieg, Jennifer; Djidja, Marie-Claude; Baban, Aida; Spindeldreher, Sebastian; Koulov, Atanas V; Kiessling, Andrea

    2014-01-01

    Subvisible proteinaceous particles which are present in all therapeutic protein formulations are in the focus of intense discussions between health authorities, academics and biopharmaceutical companies in the context of concerns that such particles could promote unwanted immunogenicity via anti-drug antibody formation. In order to provide further understanding of the subject, this study closely examines the specific biological effects proteinaceous particles may exert on dendritic cells (DCs) as the most efficient antigen-presenting cell population crucial for the initiation of the adaptive immune response. Two different model IgG antibodies were subjected to three different types of exaggerated physical stress to generate subvisible particles in far greater concentrations than the ones typical for the currently marketed biotherapeutical antibodies. The aggregated samples were used in in vitro biological assays in order to interrogate the early DC-driven events that initiate CD4 T-cell dependent humoral adaptive immune responses--peptide presentation capacity and co-stimulatory activity of DCs. Most importantly, antigen presentation was addressed with a unique approach called MHC-associated Peptide Proteomics (MAPPs), which allows for identifying the sequences of HLA-DR associated peptides directly from human dendritic cells. The experiments demonstrated that highly aggregated solutions of two model mAbs generated under controlled conditions can induce activation of human monocyte-derived DCs as indicated by upregulation of typical maturation markers including co-stimulatory molecules necessary for CD4 T-cell activation. Additional data suggest that highly aggregated proteins could induce in vitro T-cell responses. Intriguingly, strong aggregation-mediated changes in the pattern and quantity of antigen-derived HLA-DR associated peptides presented on DCs were observed, indicating a change in protein processing and presentation. Increasing the amounts of subvisible

  13. 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

  14. Polymorphisms and Interspecies Differences of the Activating and Inhibitory FcγRII of Macaca nemestrina Influence the Binding of Human IgG Subclasses1

    PubMed Central

    Trist, Halina M.; Tan, Peck Szee; Wines, Bruce D.; Ramsland, Paul A.; Orlowski, Eva; Stubbs, Janine; Gardiner, Elizabeth E.; Pietersz, Geoffrey A.; Kent, Stephen J.; Stratov, Ivan; Burton, Dennis R.; Hogarth, P. Mark

    2014-01-01

    Little is known of the impact of Fc receptor polymorphism in macaques on the binding of human IgG (huIgG) and nothing is known of this interaction in the pigtail macaque (M. nemestrina) which is used in preclinical evaluation of vaccines and therapeutic antibodies. We defined the sequence and huIgG binding characteristics of the M. nemestrina activating FcγRIIa (mnFcγRIIa) and inhibitory FcγRIIb (mnFcγRIIb) and predicted their structures using the huIgGFc:huFcγRIIa crystal structure. Large differences were observed in the binding of huIgG by mnFcγRIIa and mnFcγRIIb compared to their human FcR counterparts. MnFcγRIIa has markedly impaired binding of huIgG1 and huIgG2 immune complexes compared to huFcγRIIa (His131). In contrast, mnFcγRIIb has enhanced binding of huIgG1 and broader specificity as, unlike huFcγRIIb, it avidly binds IgG2. Mutagenesis and molecular modelling of mnFcγRIIa showed that Pro159 and Tyr160 impair the critical FG loop interaction with huIgG. The enhanced binding of huIgG1 and huIgG2 by mnFcγRIIb was shown to be dependent on His131 and Met132. Significantly, both His131 and Met132 are conserved across FcγRIIb of rhesus and cynomolgus macaques. We identified functionally significant polymorphism of mnFcγRIIa, wherein Pro at position 131, also an important polymorphic site in huFcγRIIa, almost abolished binding of huIgG2 and huIgG1 and reduced binding of huIgG3 compared to mnFcγRIIa His131. These marked interspecies differences in IgG-binding between human and macaque FcRs and polymorphisms within species have implications for pre-clinical evaluation of antibodies and vaccines in macaques. PMID:24342805

  15. Polymorphisms and interspecies differences of the activating and inhibitory FcγRII of Macaca nemestrina influence the binding of human IgG subclasses.

    PubMed

    Trist, Halina M; Tan, Peck Szee; Wines, Bruce D; Ramsland, Paul A; Orlowski, Eva; Stubbs, Janine; Gardiner, Elizabeth E; Pietersz, Geoffrey A; Kent, Stephen J; Stratov, Ivan; Burton, Dennis R; Hogarth, P Mark

    2014-01-15

    Little is known of the impact of Fc receptor (FcR) polymorphism in macaques on the binding of human (hu)IgG, and nothing is known of this interaction in the pig-tailed macaque (Macaca nemestrina), which is used in preclinical evaluation of vaccines and therapeutic Abs. We defined the sequence and huIgG binding characteristics of the M. nemestrina activating FcγRIIa (mnFcγRIIa) and inhibitory FcγRIIb (mnFcγRIIb) and predicted their structures using the huIgGFc/huFcγRIIa crystal structure. Large differences were observed in the binding of huIgG by mnFcγRIIa and mnFcγRIIb compared with their human FcR counterparts. MnFcγRIIa has markedly impaired binding of huIgG1 and huIgG2 immune complexes compared with huFcγRIIa (His(131)). In contrast, mnFcγRIIb has enhanced binding of huIgG1 and broader specificity, as, unlike huFcγRIIb, it avidly binds IgG2. Mutagenesis and molecular modeling of mnFcγRIIa showed that Pro(159) and Tyr(160) impair the critical FG loop interaction with huIgG. The enhanced binding of huIgG1 and huIgG2 by mnFcγRIIb was shown to be dependent on His(131) and Met(132). Significantly, both His(131) and Met(132) are conserved across FcγRIIb of rhesus and cynomolgus macaques. We identified functionally significant polymorphism of mnFcγRIIa wherein proline at position 131, also an important polymorphic site in huFcγRIIa, almost abolished binding of huIgG2 and huIgG1 and reduced binding of huIgG3 compared with mnFcγRIIa His(131). These marked interspecies differences in IgG binding between human and macaque FcRs and polymorphisms within species have implications for preclinical evaluation of Abs and vaccines in macaques.

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

    PubMed

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

    1989-07-01

    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.

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

    PubMed

    Lo, Agnes S; Mao, Xuming; Mukherjee, Eric M; Ellebrecht, Christoph T; Yu, Xiaocong; Posner, Marshall R; Payne, Aimee S; Cavacini, Lisa A

    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.

  18. 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

  19. In vitro and in vivo properties of a fully human IgG1 monoclonal antibody that combats multidrug resistant Pseudomonas aeruginosa.

    PubMed

    Adawi, Azmi; Bisignano, Carlo; Genovese, Tiziana; Filocamo, Angela; Khouri-Assi, Camellia; Neville, Anat; Feuerstein, Giora Z; Cuzzocrea, Salvatore; Neville, Lewis F

    2012-09-01

    The development of an anti-bacterial drug in the form of a monoclonal antibody (mAb) targeting an exposed virulence factor, represents an innovative therapeutic strategy. Consequently, a fully human IgG1 mAb (LST-007) targeting Pseudomonas aeruginosa (PA) flagellin type b was recombinantly expressed and characterized in vitro and in an infection model driven by a multidrug resistant (MDR) PA strain. LST-007 demonstrated a highly specific binding towards whole PA bacteria harboring flagellin type b and its recombinant counterpart, with a K(D) of 7.4x10(-10) M. In bioactivity assays, LST-007 or titers of Cmax sera derived from pharmacokinetic studies, markedly attenuated PA motility in an equipotent manner. In vivo, parenteral LST-007 (20 mg/kg) given as a single or double-dosing paradigm post-infection, afforded survival (up to 75% at Day 7) in a lethal model of pneumonia driven by the intratracheal (i.t.) instillation of an LD(80) of the MDR PA isolate. This protective effect was markedly superior to that of imipenem (30% survival at Day 7) and totally devoid with an irrelevant, human isotype mAb. These data lay credence that LST-007 may be a valuable adjunct to the limited list of anti-bacterials that can tackle MDR PA strains, thereby warranting its continued development for eventual clinical evaluation.

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

    PubMed

    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.

  1. Chondrogenic differentiation of human mesenchymal stem cell aggregates via controlled release of TGF-β1 from incorporated polymer microspheres

    PubMed Central

    Solorio, Loran D.; Fu, Andrew S.; Hernández-Irizarry, Roberto; Alsberg, Eben

    2013-01-01

    Aggregate culture is a useful method for inducing chondrogenic differentiation of human mesenchymal stem cells (hMSC) in a three-dimensional in vitro culture environment. Conventional aggregate culture, however, typically requires repeated growth factor supplementation during media changes, which is both expensive and time-intensive. In addition, homogenous cell differentiation is limited by the diffusion of chondrogenic growth factor from the culture medium into the aggregate and peripheral cell consumption of the growth factor. We have engineered a technology to incorporate growth factor-loaded polymer microspheres within hMSC aggregates themselves. Here, we report on the system’s capacity to induce chondrogenesis via sustained delivery of transforming growth factor-b1 (TGF-β1). Cartilage formation after 3 weeks in the absence of externally supplied growth factor approached that of aggregates cultured by conventional methods. Chondrogenesis in the central region of the aggregates is enabled at TGF-β1 levels much lower than those required by conventional culture using exogenously supplied TGF-β1, which is likely a result of the system’s ability to overcome limitations of growth factor diffusion from cell culture media surrounding the exterior of the aggregates. Importantly, the inclusion of growth factor-releasing polymer microspheres in hMSC aggregates could enable in vivo chondrogenesis for cartilage tissue engineering applications without extensive in vitro culture. PMID:19322820

  2. Aggregate human health risk assessment from dust of daily life in the urban environment of Beijing.

    PubMed

    Xu, L Y; Shu, X

    2014-04-01

    Because of the high emissions of polycyclic aromatic hydrocarbons (PAHs) into the environment by the increasing number of vehicles in Beijing and the absorption of these PAHs onto particulates, the performance of a preliminary health risk assessment of the aggregate exposure to PAHs of urban citizens in daily life is very important. Urban dust can be used to examine the aggregation of atmospheric particulates from local pollution sources over a long time period and the direct exposure of the urban human population. The environment's correlative with clothing, dining, residing, and traveling in urban daily life was assessed using exposure-receptor-oriented analysis. The multipathway exposure model was used to simulate the lifetime exposure of a female citizen to PAHs in dust. All of the PAH concentrations in dust for each behavior and its correlative environment in Beijing were acceptable because all of the carcinogenic risks of PAHs in the dust were approximately 1.0 × 10(-6). The dominant induced carcinogenic risks in the dust were Benzo(a)pyrene and Dibenzo(a,h)anthracene. The main carcinogenic risk routes for humans were dermal contact and oral intake, which contributed on average 99.78% of the risk. Indoor risk is especially important, as the decoration and height within the building were important impact factors for carcinogenic risk induced by indoor PAHs. For people living in an urban area, a healthy lifestyle includes less decoration per room, living on a low floor, wearing a respirator, and reducing exposed skin area when traveling.

  3. A new Caenorhabditis elegans model of human huntingtin 513 aggregation and toxicity in body wall muscles

    PubMed Central

    Lee, Amy L.; Ung, Hailey M.; Sands, L. Paul

    2017-01-01

    Expanded polyglutamine repeats in different proteins are the known determinants of at least nine progressive neurodegenerative disorders whose symptoms include cognitive and motor impairment that worsen as patients age. One such disorder is Huntington’s Disease (HD) that is caused by a polyglutamine expansion in the human huntingtin protein (htt). The polyglutamine expansion destabilizes htt leading to protein misfolding, which in turn triggers neurodegeneration and the disruption of energy metabolism in muscle cells. However, the molecular mechanisms that underlie htt proteotoxicity have been somewhat elusive, and the muscle phenotypes have not been well studied. To generate tools to elucidate the basis for muscle dysfunction, we engineered Caenorhabditis elegans to express a disease-associated 513 amino acid fragment of human htt in body wall muscle cells. We show that this htt fragment aggregates in C. elegans in a polyglutamine length-dependent manner and is toxic. Toxicity manifests as motor impairment and a shortened lifespan. Compared to previous models, the data suggest that the protein context in which a polyglutamine tract is embedded alters aggregation propensity and toxicity, likely by affecting interactions with the muscle cell environment. PMID:28282438

  4. 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.

  5. Flow cytometry-based algorithm to analyze the anti-fixed Toxoplasma gondii tachyzoites IgM and IgG reactivity and diagnose human acute toxoplasmosis.

    PubMed

    Silva-dos-Santos, Priscila Pinto; Barros, Geisa Baptista; Mineo, José Roberto; de Oliveira Silva, Deise Aparecida; Menegaz, Mauro Hygino Weinert; Serufo, José Carlos; Dietze, Reynaldo; Martins-Filho, Olindo de Assis; Lemos, Elenice Moreira

    2012-04-30

    In the present study we evaluated the performance of a flow cytometry-based algorithm as a new serological approach to detect antibodies to T. gondii and specific IgG avidity to diagnose acute toxoplasmosis. The results showed that using FC-AFTA-IgM assay, all serum samples from patients with acute toxoplasmosis demonstrated seropositivity, whereas 90% of patients with chronic infection and 100% of non-infected individuals presented negative results. Thus, only 10% of patients with chronic toxoplasmosis showed residual IgM, in contrast with other methodologies used to diagnosis acute toxoplasmosis. On the order hand, FC-AFTA-IgG assay as well as FC-AFTA-IgG subclasses is unlikely to discriminate acute from chronic toxoplasmosis. We have also evaluated the performance of FC-AFTA-IgG avidity as a tool to exclude chronic toxoplasmosis in patients with positive FC-AFTA-IgM. Our data showed an excellent performance of FC-AFTA-IgG avidity employing the cut-off of 60% for Avidity Index (AI) with sensitivity and specificity of 100%. All serum samples from patients presenting acute toxoplasmosis showed low avidity index (AI≤60%), whereas all chronic patients showed high avidity index (AI>60%). The outstanding performance indexes of this novel flow cytometry-based algorithm support its use as a non-conventional alternative serological approach to diagnose human acute toxoplasmosis.

  6. 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.

  7. Detection of parasite-specific IgG and IgA in paired serum and saliva samples for diagnosis of human strongyloidiasis in northern Paraná state, Brazil.

    PubMed

    Bosqui, Larissa R; Gonçalves, Ana Lúcia R; Gonçalves-Pires, Maria do Rosário F; Custodio, Luiz Antonio; de Menezes, Maria Cláudia N D; Murad, Valter A; de Paula, Fabiana M; Pavanelli, Wander R; Conchon-Costa, Ivete; Costa-Cruz, Julia Maria; Costa, Idessania N

    2015-10-01

    Human strongyloidiasis is an infection caused by the helminth Strongyloides stercoralis that can be fatal, especially in immunosuppressed patients. The aim of this study is to evaluate parasite-specific IgG and IgA levels using S. venezuelensis third-stage (L3) infective larvae alkaline extract as a heterologous antigen by ELISA in paired serum and saliva samples with improved sensitivity and specificity. Individuals from northern Paraná state, Brazil were divided into three groups: 30 patients copropositive for S. stercoralis (Group I); 30 clinically healthy individuals (Group II); and 30 patients copropositive for other parasites (Group III). The area under ROC curve (AUC), an overall index of diagnostic accuracy, and Kappa index were calculated. Data were analyzed using analysis of variance (ANOVA) followed by a Kruskal-Wallis test. Probability (p) values of <0.05 were regarded as significant. In Group I, IgG was detected in 96.7% serum and in 6.7% saliva samples. IgG was not detected in Group II. In Group III, cross-reactivity was observed for serum IgG in 26.7% and in 6.7% for saliva samples. In Group I, IgA was detected in 76.7% serum and 56.7% saliva samples. In Group II, 3.3% were positive for IgA in serum, whereas IgA was not detected in any saliva samples. Group III showed 6.7% serum and 26.7% saliva-positive samples. The sensitivity values for detection of IgG and IgA in serum samples were 96.7% and 76.7%, respectively. In saliva samples, the sensitivity values for detection of IgG and IgA were 6.7% and 56.7%, respectively. The specificity value was 100% for the detection of IgG in serum and for detection of IgG and IgA in saliva, and 96.7% for detection of IgA in serum samples. The proper choice of immunological diagnosis to supplement parasitological methods is essential to estimate the true prevalence of the parasite, and will permit analysis of population immune response profiles, particularly in northern Paraná state, where there are no previous

  8. Inhibitory Mechanism of EGCG on Fibrillation and Aggregation of Amidated Human Islet Amyloid Polypeptide.

    PubMed

    Xu, Zhixue; Ma, Gongli; Zhang, Qiang; Chen, Congheng; He, Yanming; Xu, Lihui; Zhou, Guangrong; Li, Zhenhua; Yang, Hongjie; Zhou, Ping

    2017-03-15

    The abnormal fibrillation of human islet amyloid polypeptide (hIAPP) is associated with development of T2DM. EGCG can bind amyloid proteins to inhibit the fibrillation of these proteins. Here, we sought to investigate the effect of EGCG on amidated hIAPP (hIAPP-NH2) fibrillation and aggregation by using spectroscopic and microscopic techniques, and also sought to view insight into the interaction of EGCG and hIAPP22-27 by using spectroscopic experiments and quantum chemical calculations. ThT fluorescence, real-time NMR and TEM studies demonstrated that EGCG could inhibit the formation of hIAPP-NH2 fibrils, while promote the formation of hIAPP-NH2 amorphous aggregates. Phenylalanine intrinsic fluorescence and NMR studies of EGCG/hIAPP22-27 complex revealed three important binding sites including A-ring of EGCG, residue Phe23 and residue Ile26. DFT calculation identified the dominant binding structures of EGCG/Phe23 and EGCG/Ile26 complexes, named Structure I and Structure II, respectively. Our study demonstrates the inhibitory mechanism of EGCG on fibrillation and aggregation of hIAPP-NH2 in which EGCG interacts with hIAPP-NH2 through hydrogen bonding and π-π interaction between A-ring and residue Phe23 as well as hydrophobic interactions between A-ring and residue Ile26, thus can inhibit the inter-peptide interaction between hIAPP-NH2 monomers and finally inhibit fibrillation of hIAPP-NH2. This study offers an intuitive explanation at molecular level.

  9. Characterization of Two Human Skeletal Calsequestrin Mutants Implicated in Malignant Hyperthermia and Vacuolar Aggregate Myopathy*

    PubMed Central

    Lewis, Kevin M.; Ronish, Leslie A.; Ríos, Eduardo; Kang, ChulHee

    2015-01-01

    Calsequestrin 1 is the principal Ca2+ storage protein of the sarcoplasmic reticulum of skeletal muscle. Its inheritable D244G mutation causes a myopathy with vacuolar aggregates, whereas its M87T “variant” is weakly associated with malignant hyperthermia. We characterized the consequences of these mutations with studies of the human proteins in vitro. Equilibrium dialysis and turbidity measurements showed that D244G and, to a lesser extent, M87T partially lose Ca2+ binding exhibited by wild type calsequestrin 1 at high Ca2+ concentrations. D244G aggregates abruptly and abnormally, a property that fully explains the protein inclusions that characterize its phenotype. D244G crystallized in low Ca2+ concentrations lacks two Ca2+ ions normally present in wild type that weakens the hydrophobic core of Domain II. D244G crystallized in high Ca2+ concentrations regains its missing ions and Domain II order but shows a novel dimeric interaction. The M87T mutation causes a major shift of the α-helix bearing the mutated residue, significantly weakening the back-to-back interface essential for tetramerization. D244G exhibited the more severe structural and biophysical property changes, which matches the different pathophysiological impacts of these mutations. PMID:26416891

  10. Optimization of a human IgG B-cell ELISpot assay for the analysis of vaccine-induced B-cell responses.

    PubMed

    Jahnmatz, Maja; Kesa, Gun; Netterlid, Eva; Buisman, Anne-Marie; Thorstensson, Rigmor; Ahlborg, Niklas

    2013-05-31

    B-cell responses after infection or vaccination are often measured as serum titers of antigen-specific antibodies. Since this does not address the aspect of memory B-cell activity, it may not give a complete picture of the B-cell response. Analysis of memory B cells by ELISpot is therefore an important complement to conventional serology. B-cell ELISpot was developed more than 25 years ago and many assay protocols/reagents would benefit from optimization. We therefore aimed at developing an optimized B-cell ELISpot for the analysis of vaccine-induced human IgG-secreting memory B cells. A protocol was developed based on new monoclonal antibodies to human IgG and biotin-avidin amplification to increase the sensitivity. After comparison of various compounds commonly used to in vitro-activate memory B cells for ELISpot analysis, the TLR agonist R848 plus interleukin (IL)-2 was selected as the most efficient activator combination. The new protocol was subsequently compared to an established protocol, previously used in vaccine studies, based on polyclonal antibodies without biotin avidin amplification and activation of memory B-cells using a mix of antigen, CpG, IL-2 and IL-10. The new protocol displayed significantly better detection sensitivity, shortened the incubation time needed for the activation of memory B cells and reduced the amount of antigen required for the assay. The functionality of the new protocol was confirmed by analyzing specific memory B cells to five different antigens, induced in a limited number of subjects vaccinated against tetanus, diphtheria and pertussis. The limited number of subjects did not allow for a direct comparison with other vaccine studies. Optimization of the B-cell ELISpot will facilitate an improved analysis of IgG-secreting B cells in vaccine studies.

  11. 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.

  12. Copper and Zinc Ions Specifically Promote Nonamyloid Aggregation of the Highly Stable Human γ-D Crystallin.

    PubMed

    Quintanar, Liliana; Domínguez-Calva, José A; Serebryany, Eugene; Rivillas-Acevedo, Lina; Haase-Pettingell, Cameron; Amero, Carlos; King, Jonathan A

    2016-01-15

    Cataract is the leading cause of blindness in the world. It results from aggregation of eye lens proteins into high-molecular-weight complexes, causing light scattering and lens opacity. Copper and zinc concentrations in cataractous lens are increased significantly relative to a healthy lens, and a variety of experimental and epidemiological studies implicate metals as potential etiological agents for cataract. The natively monomeric, β-sheet rich human γD (HγD) crystallin is one of the more abundant proteins in the core of the lens. It is also one of the most thermodynamically stable proteins in the human body. Surprisingly, we found that both Cu(II) and Zn(II) ions induced rapid, nonamyloid aggregation of HγD, forming high-molecular-weight light-scattering aggregates. Unlike Zn(II), Cu(II) also substantially decreased the thermal stability of HγD and promoted the formation of disulfide-bridged dimers, suggesting distinct aggregation mechanisms. In both cases, however, metal-induced aggregation depended strongly on temperature and was suppressed by the human lens chaperone αB-crystallin (HαB), implicating partially folded intermediates in the aggregation process. Consistently, distinct site-specific interactions of Cu(II) and Zn(II) ions with the protein and conformational changes in specific hinge regions were identified by nuclear magnetic resonance. This study provides insights into the mechanisms of metal-induced aggregation of one of the more stable proteins in the human body, and it reveals a novel and unexplored bioinorganic facet of cataract disease.

  13. Human IgE, IgG and IgA antibody responses to T101, a murine monoclonal antibody against human lymphocytes: implications for pathogenesis, risk and avoidance of adverse immunologic reactions.

    PubMed

    Dykewicz, M S; Cranberg, J A; Patterson, R; Rosen, S T; Shaughnessy, M A; Zimmer, A M

    1990-01-01

    To study immune responses that may play a role in immediate-type allergic reactions (ITAR) and serum-sickness-like reactions that have been reported with administration of monoclonal antibodies (MAb), we measured by enzyme-linked immunosorbent assay serum levels of IgE, IgG, and IgA human antimurine antibody (HAMA) to T101, a murine IgG2a antibody that has been used in the treatment of cutaneous T cell lymphoma (CTCL) and chronic lymphocytic leukemia (CLL). None of 8 patients (4 with CLL, 4 CTCL) pretreated with T101 had elevated titers of any HAMA class tested as compared to normal control sera. All CTCL patients who had elevated total serum IgE levels, but normal total serum levels of other antibody classes, had significant rises in IgE, IgG, and IgA HAMA to T101 after intravenous MAb infusion. However, the CLL patients who were hypogammaglobulinemic failed to develop significant rises in HAMA. Three patients (2 CLL, 1 CTCL) had ITAR (e.g., urticaria, angioedema, bronchospasm) associated with infusion of T101; prophylactic medication regimens based upon the control of radiographic contrast media reactions were used with apparent benefit in subsequent infusions in these patients. All 8 patients had negative immediate intradermal skin tests (1 microgram/ml) to T101 prior to its infusion. Our data confirm that (1) non-IgE-mediated mechanisms cause ITAR from this MAb, possibly by a mechanism inherent in the action of this MAb against lymphocytes, and (2) that isotypic antibody responses to MAb vary with the type of malignancy being treated.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Evaluation of genetic damage in human peripheral lymphocytes exposed to mineral trioxide aggregate and Portland cements.

    PubMed

    Braz, M G; Camargo, E A; Salvadori, D M F; Marques, M E A; Ribeiro, D A

    2006-03-01

    summary Mineral trioxide aggregate (MTA) and Portland cement are being used in dentistry as root-end-filling material for periapical surgery and for the sealing of communications between the root canal system and the surrounding tissues. However, genotoxicity tests for complete risk assessment of these compounds have not been conducted up to now. In the present study, the genotoxic effects of MTA and Portland cements were evaluated in peripheral lymphocytes from 10 volunteers by the alkaline single cell gel (comet) assay. The results pointed out that the single cell gel (comet) assay failed to detect the presence of DNA damage after a treatment of peripheral lymphocytes by MTA and Portland cements for concentrations up to 1000 mug mL(-1). In summary, our results indicate that exposure to MTA or Portland cements may not be a factor that increases the level of DNA lesions in human peripheral lymphocytes as detected by single cell gel (comet) assay.

  15. Aggregates of human erythrocyte membrane sialoglycoproteins in the presence of deoxycholate and dodecyl sulfate.

    PubMed

    Liljas, L

    1978-02-15

    Gel electrophoresis in the presence of deoxycholate of human erythrocyte membranes solubilized with deoxycholate resolves four glycoprotein zones. Electrophoresis in dodecyl sulfate in a second dimension reveals several components, three of which migrate in the region of PAS-2. One of the zones in deoxycholate gel electrophoresis contains component PAS-3, and this glycoprotein seems to exist as a monomer in deoxycholate, but aggregates partially upon addition of dodecyl sulfate. The major sialoglycoprotein migrates as a diffuse zone in dodecyl sulfate. The major sialoglycoprotein migrates as a diffuse zone in deoxycholate gel electrophoresis, indicating association and dissociation during the electrophoresis. The use of deoxycholate followed by dodecyl sulfate in two-dimentional electrophoresis gave high resolution of membrane proteins and can be used for detection of complexes in one of the detergents.

  16. 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.

  17. IgG4 can induce an M2-like phenotype in human monocyte-derived macrophages through FcγRI.

    PubMed

    Swisher, Jennifer F A; Haddad, Devin A; McGrath, Anna G; Boekhoudt, Gunther H; Feldman, Gerald M

    2014-01-01

    Antibodies evoke cellular responses through the binding of their Fc region to Fc receptors, most of which contain immunoreceptor tyrosine-based activation motif domains and are thus considered "activating." However, there is a growing appreciation of these receptors for their ability to deliver an inhibitory signal as well. We previously described one such phenomenon whereby interferon (IFN)γ signaling is inhibited by immune complex signaling through FcγRI. To understand the implications of this in the context of therapeutic antibodies, we assessed individual IgG subclasses to determine their ability to deliver this anti-inflammatory signal in monocyte-derived macrophages. Like IgG1, we found that IgG4 is fully capable of inhibiting IFNγ-mediated events. In addition, F(ab')2 fragments that interfere with FcγRI signaling reversed this effect. For mAbs developed with either an IgG1 or an IgG4 constant region for indications where inflammation is undesirable, further examination of a potential Fc-dependent contribution to their mechanism of action is warranted.

  18. IgG subclass co-expression brings harmony to the quartet model of murine IgG function.

    PubMed

    Collins, Andrew M

    2016-11-01

    A model of murine IgG function is presented in which the co-expression of the IgG subclasses is a central feature, class switching occurs before the commencement of somatic hypermutation, and there is little switching between subclasses. It is named the quartet model to emphasize the harmony that comes from the simultaneous presence of the four subclasses. In this model, IgG3 and IgG2b antibodies are particularly important early in the response, when T-cell help may be limiting. IgG3 initiates inflammation through complement fixation, whereas IgG2b provides early FcγR-mediated effector functions. As T-cell help strengthens, IgG2a antibodies increase the power of the response, whereas IgG1 production helps limit the inflammatory drive and limits immunopathology. The model highlights the fact that murine IgG subclasses function quite differently to human IgG subclasses. This allows them to serve the special immunological needs of a species that is vulnerable because of its small size.

  19. 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

  20. 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.

  1. A Human Anti-Insulin IgG Autoantibody Apparently Arises Through Clonal Selection from an Insulin-Specific “Germ-Line” Natural Antibody Template

    PubMed Central

    Ichiyoshi, Yuji; Zhou, Min; Casali, Paolo

    2015-01-01

    We analyzed the structural correlates underlying the insulin-dependent selection of the specific anti-insulin IgG1 κ mAb13-producing cell clone, derived from a patient with insulin-dependent diabetes mellitus treated with recombinant human insulin. First, we cloned the germ-line genes that putatively gave rise to the expressed VH and Vκ segments and used them to generate the full (unmutated) “germ-line revertant” of the “wild-type” (somatically mutated) mAb13, using recombinant PCR methods and an in vitro human Cγ1 and Cκ expression system. The full “germ-line revertant” bound insulin specifically and in a dose-saturable fashion, but with a relative avidity (Avrel) more than three-fold lower than that of its wild-type counterpart (Avrel, 1.69 × 10−8 vs 4.91 × 10−9 g/μl). Second, we established, by reassorting wild-type and germ-line revertant forms of the mAb13 VH and Vκ segments, that the increased Avrel for insulin of mAb13 when compared with its full “germ-line revertant” counterpart was entirely dependent on the mutations in the VH not those in the Vκ chain. Third, we determined, by site-directed mutagenesis experiments, that of the three mutations in the mAb13 VH segment (Ser→Gly, Ser→Thr, and Ser→Arg at positions 31, 56, and 58, respectively), only Arg58 was crucial in increasing the mAb13 Avrel (from 1.44 × 10−8 to 5.14 × 10−9 g/μl) and affinity (Kd, from 189 to 59 nM) for insulin. The affinity enhancement mediated by the VH segment Arg58 residue reflected about a threefold decrease in dissociation rate constant (Koff, from 4.92 × 10−3 to 1.54 × 10−3 s−1)but not an increase in association rate constant (Kon, from 2.60 × 104 to 2.61 × 104 M−1 s−1), and it contrasted with the complete loss of insulin binding resulting from the substitution of the VH segment Asn52 by Lys. The present findings suggest that human insulin, a self Ag, has the potential to recruit a natural autoantibody-producing cell precursor

  2. Family Aggregation of Human T-Lymphotropic Virus 1-Associated Diseases: A Systematic Review

    PubMed Central

    Alvarez, Carolina; Gotuzzo, Eduardo; Vandamme, Anne-Mieke; Verdonck, Kristien

    2016-01-01

    Human T-lymphotropic virus 1 (HTLV-1) is a retrovirus that produces a persistent infection. Two transmission routes (from mother to child and via sexual intercourse) favor familial clustering of HTLV-1. It is yet unknown why most HTLV-1 carriers remain asymptomatic while about 10% of them develop complications. HTLV-1 associated diseases were originally described as sporadic entities, but familial presentations have been reported. To explore what is known about family aggregation of HTLV-1-associated diseases we undertook a systematic review. We aimed at answering whether, when, and where family aggregation of HTLV-1-associated diseases was reported, which relatives were affected and which hypotheses were proposed to explain aggregation. We searched MEDLINE, abstract books of HTLV conferences and reference lists of selected papers. Search terms used referred to HTLV-1 infection, and HTLV-1-associated diseases, and family studies. HTLV-1-associated diseases considered are adult T-cell leukemia/lymphoma (ATLL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), HTLV-1-associated uveitis, and infective dermatitis. Seventy-four records reported HTLV-1-associated diseases in more than one member of the same family and were included. Most reports came from HTLV-1-endemic countries, mainly Japan (n = 30) and Brazil (n = 10). These reports described a total of 270 families in which more than one relative had HTLV-1-associated diseases. In most families, different family members suffered from the same disease (n = 223). The diseases most frequently reported were ATLL (115 families) and HAM/TSP (102 families). Most families (n = 144) included two to four affected individuals. The proportion of ATLL patients with family history of ATLL ranged from 2 to 26%. The proportion of HAM/TSP patients with family history of HAM/TSP ranged from 1 to 48%. The predominant cluster types for ATLL were clusters of siblings and parent-child pairs and for HAM/TSP, an affected

  3. Coupled human erythrocyte velocity field and aggregation measurements at physiological haematocrit levels.

    PubMed

    Dusting, Jonathan; Kaliviotis, Efstathios; Balabani, Stavroula; Yianneskis, Michael

    2009-07-22

    Simultaneous measurement of erythrocyte (RBC) velocity fields and aggregation properties has been successfully performed using an optical shearing microscope and Particle Image Velocimetry (PIV). Blood at 45% haematocrit was sheared at rates of 5.4< or =gamma < or = 252 s(-1) and imaged using a high speed camera. The images were then processed to yield aggregation indices and flow velocities. Negligible levels of aggregation were observed for gamma > or = 54.0 s(-1), while high levels of aggregation and network formation occurred for gamma < or = 11.7 s(-1). The results illustrate that the velocity measurements are dependent on the extent of RBC aggregation. High levels of network formation cause the velocities at gamma > or = 5.4 s(-1) to deviate markedly from the expected solid body rotation profile. The effect of aggregation level on the PIV accuracy was assessed by monitoring the two-dimensional (2D) correlation coefficients. Lower levels of aggregation result in poorer image correlation, from which it can be inferred that PIV accuracy is reduced. Moreover, aggregation is time-dependent, and consequently PIV accuracy may decrease during recording as the cells break up. It is therefore recommended that aggregation and its effects are taken into account in future when undertaking blood flow studies using PIV. The simplicity of the technique, which requires no lasers, filters, or special pretreatments, demonstrates the potential wide-spread applicability of the data acquisition system for accurate blood flow PIV and aggregation measurement.

  4. 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.

  5. Exploring the 'aggregation-prone' core of human Cystatin C: A structural study.

    PubMed

    Tsiolaki, Paraskevi L; Louros, Nikolaos N; Hamodrakas, Stavros J; Iconomidou, Vassiliki A

    2015-09-01

    Amyloidogenic proteins like human Cystatin C (hCC) have been shown to form dimers and oligomers by exchange of subdomains of the monomeric proteins. Normally, the hCC monomer, a low molecular type 2 Cystatin, consists of 120 amino acid residues and functions as an inhibitor of cysteine proteases. The oligomerization of hCC is involved in the pathophysiology of a rare form of amyloidosis namely Icelandic hereditary cerebral amyloid angiopathy, in which an L68Q mutant is deposited as amyloid in brain arteries of young adults. In order to find the shortest stretch responsible to drive the fibril formation of hCC, we have previously demonstrated that the LQVVR peptide forms amyloid fibrils, in vitro (Tsiolaki et al., 2015). Predictions by AMYLPRED, an amyloidogenic determinant prediction algorithm developed in our lab, led us to synthesize and experimentally study two additional predicted peptides derived from hCC. Along with our previous findings, in this work, we reveal that these peptides self-assemble, in a similar way, into amyloid-like fibrils in vitro, as electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy and Congo red staining studies have shown. Further to our experimental results, all three peptides seem to have a fundamental contribution in forming the "aggregation-prone" core of human Cystatin C.

  6. [Effect of dauricine on rat and human platelet aggregation and metabolism of arachidonic acid in washed rat platelets].

    PubMed

    Tong, L; Yue, T L

    1989-01-01

    Dauricine (Dau), an isoquinoline alkaloid extracted from the roots of Menispermum dauricum D. C. and used as an antiarrhythmic agent in China recently, was shown to inhibit rat platelet aggregation induced by arachidonic acid (AA) and ADP, as well as human platelet aggregation induced by AA, ADP and adrenaline (Adr) in vitro in a dose-dependent manner. The concentration of Dau required for 50% inhibition (IC50) of rat platelet aggregation induced by AA and ADP was 26 and 37 mumol/L, respectively. For human platelet aggregation induced by AA, ADP and Adr the IC50 of Dau was found to be 39, 55 and 43 mumol/L, respectively. Dau inhibited the cyclooxygenase pathway metabolites of AA (TXB2 and HHT) in washed intact rat platelets. The production of TXB2 and HHT was reduced by 26% and 19%, respectively, when the Dau concentration was 50 mumol/L and by 46 and 45%, respectively, when the concentration of Dau was 100 mumol/L. The formation of 12-HETE was also inhibited at 100 mumol/L of Dau. The inhibitory effect of Dau on AA metabolism may be one of the mechanisms related to its inhibition of platelet aggregation.

  7. 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.

  8. DETECTION OF HUMAN ANTI-ZIKA VIRUS IgG BY ELISA USING AN ANTIGEN FROM in vitro INFECTED VERO CELLS: PRELIMINARY RESULTS

    PubMed Central

    SUMITA, Laura Masami; RODRIGUES, Jaqueline Polizeli; FERREIRA, Noely Evangelista; FELIX, Alvina Clara; SOUZA, Nathalia Caroline Santiago; MACHADO, Clarisse Martins; JÚNIOR, Heitor Franco de ANDRADE

    2016-01-01

    SUMMARY Zika virus (ZKV) infection is a huge public health problem in Brazil because of the increased incidence of microcephaly in neonates from infected mothers. Detection of specific IgG antibodies in maternal serum samples constitutes an important approach for diagnosing ZKV infection and evaluating its relationship with neonatal microcephaly. However, as there is no serological test produced in Brazil to detect IgM and IgG antibodies against ZKV, we sought to examine specific IgG in serum samples from patients or suspected mothers to detect previous infection and to test for specificity with regard to flaviviral infections occurring in the same area. Brazilian Zika virus native antigens were obtained from infected Vero cell layers or free virions in the culture medium and then used in ELISA. We tested sera from eight ZKV RNA-diagnosed infected patients (ZKVR), seven neonates with microcephaly and their mothers after delivery (MM), 140 dengue virus IgM-positive (DM) and IgG (DG)-positive patients, and 100 yellow fever (YF)-vaccinated patients. According to the ELISA, ZKVR samples were mostly positive (7/8), and all the MM serum samples were positive for ZKV IgG (7/7). In contrast, cross-reactions for dengue or yellow fever-vaccinated patients were observed, including DM (48/95), DG (10/45) or YF (3/100) serum samples; however, these cross-reactions exhibited low antigen avidity so that 6 M urea largely removed this cross-reactivity, with only a few cross-reacting samples remaining (8/140). ELISA based on extracted virions was much more specific, with all ZKVR (8/8) and MM sera being positive for ZKV IgG (7/7) and only borderline cross-reactivity found for DM (6/95), DG (3/45) or YF (4/100)-vaccinated serum samples. This technique (ELISA) can identify specific IgG in ZKV-infected patients and may be helpful in diagnosing congenital infetions after maternal RNA virus clearance or in epidemiological studies. PMID:27982355

  9. DETECTION OF HUMAN ANTI-ZIKA VIRUS IgG BY ELISA USING AN ANTIGEN FROM in vitro INFECTED VERO CELLS: PRELIMINARY RESULTS.

    PubMed

    Sumita, Laura Masami; Rodrigues, Jaqueline Polizeli; Ferreira, Noely Evangelista; Felix, Alvina Clara; Souza, Nathalia Caroline Santiago; Machado, Clarisse Martins; Júnior, Heitor Franco de Andrade

    2016-12-08

    Zika virus (ZKV) infection is a huge public health problem in Brazil because of the increased incidence of microcephaly in neonates from infected mothers. Detection of specific IgG antibodies in maternal serum samples constitutes an important approach for diagnosing ZKV infection and evaluating its relationship with neonatal microcephaly. However, as there is no serological test produced in Brazil to detect IgM and IgG antibodies against ZKV, we sought to examine specific IgG in serum samples from patients or suspected mothers to detect previous infection and to test for specificity with regard to flaviviral infections occurring in the same area. Brazilian Zika virus native antigens were obtained from infected Vero cell layers or free virions in the culture medium and then used in ELISA. We tested sera from eight ZKV RNA-diagnosed infected patients (ZKVR), seven neonates with microcephaly and their mothers after delivery (MM), 140 dengue virus IgM-positive (DM) and IgG (DG)-positive patients, and 100 yellow fever (YF)-vaccinated patients. According to the ELISA, ZKVR samples were mostly positive (7/8), and all the MM serum samples were positive for ZKV IgG (7/7). In contrast, cross-reactions for dengue or yellow fever-vaccinated patients were observed, including DM (48/95), DG (10/45) or YF (3/100) serum samples; however, these cross-reactions exhibited low antigen avidity so that 6 M urea largely removed this cross-reactivity, with only a few cross-reacting samples remaining (8/140). ELISA based on extracted virions was much more specific, with all ZKVR (8/8) and MM sera being positive for ZKV IgG (7/7) and only borderline cross-reactivity found for DM (6/95), DG (3/45) or YF (4/100)-vaccinated serum samples. This technique (ELISA) can identify specific IgG in ZKV-infected patients and may be helpful in diagnosing congenital infetions after maternal RNA virus clearance or in epidemiological studies.

  10. Clonal analysis of a human antibody response. Quantitation of precursors of antibody-producing cells and generation and characterization of monoclonal IgM, IgG, and IgA to rabies virus

    PubMed Central

    1990-01-01

    We quantitated and characterized the changes in the human B cell repertoire, at the clonal level, before and after immunization with rabies virus. Moreover, we generated 10 monoclonal cell lines producing IgM, IgG, and IgA antibodies to the virus. We found that in healthy subjects, not previously exposed to the virus, nearly 2% of the circulating B lymphocytes were committed to the production of antibodies that bound the virus. These B cells expressed the surface CD5 molecule. The antibodies they produced were polyreactive IgM that displayed a relatively low affinity for the virus components (Kd, 1.0- 2.4 x 10(-6) g/microliters). After immunization, different anti-virus (IgG and IgA) antibody-producing cells consistently appeared in the circulation and increased from less than 0.005% to greater than 10% of the total B cells committed to the production of IgG and IgA, respectively. Most of such B cells do not express CD5 and produce monoreactive antibodies of high affinity for rabies virus (Kd, 6.5 x 10(-9) to 1.2 x 10(-10) g/microliters). One of these IgG mAbs efficiently neutralized rabies virus in vitro and in vivo, as detailed elsewhere (Dietzschold, B., P. Casali, Y. Ueki, M. Gore, C. E. Rupprecht, A. L. Notkins, and H. Koprowski, manuscript submitted for publication). Hybridization experiments using probes specific for the different human V gene segment families revealed that cell precursors producing low affinity IgM binding to rabies virus utilized a restricted number of VH gene segments (i.e., only members of the VHIIIb subfamily), whereas cell precursors producing high affinity IgG and IgA to rabies virus utilized an assortment of different VH gene segments (i.e., members of the VHI, VHIII, VHIV, and VHVI families and VHIIIb subfamily). In conclusion, our studies show that EBV transformation in conjunction with limiting dilution technology and somatic cell hybridization techniques are useful methods for quantitating, at the B cell clonal level, the human

  11. IgG4 Characteristics and Functions in Cancer Immunity.

    PubMed

    Crescioli, Silvia; Correa, Isabel; Karagiannis, Panagiotis; Davies, Anna M; Sutton, Brian J; Nestle, Frank O; Karagiannis, Sophia N

    2016-01-01

    IgG4 is the least abundant subclass of IgG in normal human serum, but elevated IgG4 levels are triggered in response to a chronic antigenic stimulus and inflammation. Since the immune system is exposed to tumor-associated antigens over a relatively long period of time, and tumors notoriously promote inflammation, it is unsurprising that IgG4 has been implicated in certain tumor types. Despite differing from other IgG subclasses by only a few amino acids, IgG4 possesses unique structural characteristics that may be responsible for its poor effector function potency and immunomodulatory properties. We describe the unique attributes of IgG4 that may be responsible for these regulatory functions, particularly in the cancer context. We discuss the inflammatory conditions in tumors that support IgG4, the emerging and proposed mechanisms by which IgG4 may contribute to tumor-associated escape from immune surveillance and implications for cancer immunotherapy.

  12. IgGs containing light chains of the λ- and κ- type and of all subclasses (IgG1-IgG4) from the sera of patients with systemic lupus erythematosus hydrolyze myelin basic protein.

    PubMed

    Bezuglova, Anna M; Konenkova, Ludmila P; Buneva, Valentina N; Nevinsky, Georgy A

    2012-12-01

    Human myelin basic protein (hMBP)-hydrolyzing activity was recently shown to be an intrinsic property of antibodies from systemic lupus erythematosus (SLE) patients. Here, we present the first evidence demonstrating a significant diversity of different fractions of polyclonal IgGs (pIgGs) from SLE patients in their affinity for hMBP and in the ability of pIgGs to hydrolyze hMBP at different optimal pH values (5.3-9.5); the pH profiles of IgG1, IgG2, IgG3 and IgG4 were unique. IgGs containing the λ-type of light chains demonstrated higher relative activities (RAs) in the hydrolysis of hMBP and its oligopeptides (OPs) than κ-IgGs. IgGs of all four subclasses were catalytically active; their RAs in the hydrolysis of hMBP increased in the following order: IgG4 < IgG2 < IgG3 < IgG1. Metal-dependent proteolytic activity of λ-IgG, IgG1, IgG2 and IgG3 was higher than their serine protease-like activity, while these activities of κ-IgG were comparable. Phenylmethylsulfonylfluoride had almost no effect on the activity of IgG4, while EDTA significantly suppressed its activity. The RAs of λ-IgG in the hydrolysis of four OPs corresponding to different cleavage sites of hMBP were remarkably higher than those for κ-IgGs. IgG1-IgG4 demonstrated different RAs and patterns of hydrolysis of these four OPs. Although combination of Ca²⁺ plus Mg²⁺ was the best in the activation of IgG1 and IgG2, IgG3 and IgG4 demonstrated the highest activity in the presence of Ca²⁺ plus Co²⁺. The ratio of the RAs of λ-IgG, κ-IgG and IgG1-IgG4 preparations in all analyzed cases was individual for each preparation.

  13. Rational design of aggregation-resistant bioactive peptides: reengineering human calcitonin.

    PubMed

    Fowler, Susan B; Poon, Stephen; Muff, Roman; Chiti, Fabrizio; Dobson, Christopher M; Zurdo, Jesús

    2005-07-19

    A high propensity to aggregate into intractable deposits is a common problem limiting the production and use of many peptides and proteins in a wide range of biotechnological and pharmaceutical applications. Many therapeutic polypeptides are frequently abandoned at an early stage in their development because of problems with stability and aggregation. It has been shown recently that parameters describing the physicochemical properties of polypeptides can be used as predictors of protein aggregation. Here we demonstrate that these and similar tools can be applied to the rational redesign of bioactive molecules with a significantly reduced aggregation propensity without loss of physiological activity. This strategy has been exemplified by designing variants of the hormone calcitonin that show a significantly reduced aggregation propensity, yet maintain, or even increase, their potency when compared to the current therapeutic forms. The results suggest that this approach could be used successfully to enhance the solubility and efficacy of a wide range of other peptide and protein therapeutics.

  14. Diminished clearance of soluble aggregates of human immunoglobulin G in patients with rheumatoid arthritis.

    PubMed

    Lobatto, S; Daha, M R; Westedt, M L; Pauwels, E K; Evers-Schouten, J H; Voetman, A A; Cats, A; van Es, L A

    1989-01-01

    Investigation of the capacity of the mononuclear phagocyte system to remove immune complexes from the circulation was performed by the administration of 125I-labelled aggregates of human immunoglobulin G (AIgG) to patients with seropositive rheumatoid arthritis and healthy volunteers. It was found that the rate at which AIgG disappeared from the circulation was significantly prolonged in patients with RA, t1/2 61 +/- 49 min, versus 26 +/- 8 min in healthy volunteers (p less than 0.01). We were not able to establish a correlation between the t1/2 of AIgG and immune complex levels in the circulation, or between t1/2 and articular disease activity (Ritchie index). The sites of removal of AIgG from the circulation were analysed by determining radioactivity levels detectable over liver, spleen and heart. No correlation was found between t1/2 and liver/spleen uptake ratios. We have demonstrated that the removal of AIgG from the circulation of patients with RA is abnormal, though the biological significance of this finding remains to be determined.

  15. Cytotoxicity of two available mineral trioxide aggregate cements and a new formulation on human gingival fibroblasts

    PubMed Central

    Torshabi, Maryam; Amid, Reza; Kadkhodazadeh, Mahdi; Shahrbabaki, Sara Eslami; Tabatabaei, Fahimeh S.

    2016-01-01

    Aim: The purpose of this study was to investigate the cytotoxicity of nanohybrid mineral trioxide aggregate (MTA) in comparison with calcium-enriched mixture (CEM) cement and MTA-Angelus, using human gingival fibroblasts (HGFs). Materials and Methods: Nine disc-shaped specimens of each material (in 2 set stat: A, set for 24 h; B, set for 30 min; and C, fresh stat) were prepared. HGFs were exposed to tested materials’ extracts or control media. Cytotoxicity testing was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay in two time intervals. Statistical Analysis: Results were evaluated by one-way ANOVA and t-test. Statistical significance was set at P < 0.05. Results: CEM cement demonstrated favorable cell viability values when completely set (24 h set MTA = 24 h set CEM) at both time intervals. Interestingly, 24 h after incubation, CEM in Groups B and C demonstrated higher cell viability values than MTA (P < 0.05). However, after 72 h of incubation, these groups of CEM and MTA showed equal cell viability. All samples of nanohybrid MTA had slight cytotoxic effects after 24 h of incubation, and moderate cytotoxic effects after 72 h of incubation. Conclusion: Set CEM and set MTA-Angelus exerted similar, favorable effects on cell viability. However, within the limitations of this in vitro study, the results suggest that nanohybrid MTA could not be recommended as a material of choice for cervical root resorption. PMID:27994312

  16. Biocompatibility of Mineral Trioxide Aggregate with TiO2 Nanoparticles on Human Gingival Fibroblasts

    PubMed Central

    Samiei, Mohammad; Aghazadeh, Marzieh; Divband, Baharak; Akbarzadeh, Farzaneh

    2017-01-01

    Background The New compositions of white mineral trioxide aggregate (WMTA) or use of various additives like nanoparticles might affect MTA’s ideal characteristics This study was performed to evaluate the cytotoxicity of WMTA and WMTA with Titanium dioxide (TiO2) nanoparticles (1% weight ratio) at different storage times after mixing on human gingival fibroblasts (HGFs). Material and Methods HGFs were obtained from the attached gingiva of human premolars. HGFs were cultured in Dulbecco’s Modified Eagle medium, supplemented with 10% fetal calf serum, penicillin and streptomycin. The cells were exposed to WMTA (groups 1 and 2) and WMTA+TiO2 (groups 3 and 4). The fifth and sixth groups served as controls. Each group contained 15 wells. After 24h (groups 1, 3 and 5) and 48 h (groups 2, 4 and 6) of exposure, HGF viability was determined by Mosmann’s tetrazolium toxicity (MTT) assay. Statistical analysis of the data was performed by using one-way analysis of variance and Tukey post hoc test, with significance of p < 0.05. Results With both materials, the viability of HGFs significantly decrased with increasing the incubation time from 24h to 48 h (P<0.05). There was no significant difference between the materials regarding HGF viability (P>0.05). Conclusions Under the limitations of the present study, incorporation of TiO2 nanoparticles into MTA at 1 wt% had no negative effect on its biocompatibility. Key words:Cytotoxicity, fibroblast, MTA, MTT assay, nanoparticle, TiO2. PMID:28210432

  17. Biocompatibility of Accelerated Mineral Trioxide Aggregate on Stem Cells Derived from Human Dental Pulp.

    PubMed

    Kulan, Pinar; Karabiyik, Ozge; Kose, Gamze T; Kargul, Betul

    2016-02-01

    The aim of this study was to evaluate the effects of several additives on the setting time and cytotoxicity of accelerated-set mineral trioxide aggregate (MTA) on stem cells of human dental pulp. ProRoot white MTA (WMTA) (Dentsply Tulsa Dental, Johnson City, TN) was mixed with various additives including distilled water, 2.5% disodium hydrogen phosphate (Na2HPO4) (Merck, Darmstadt, Germany), K-Y Jelly (Johnson & Johnson, Markham, ON, Canada), and 5% and 10% calcium chloride (CaCl2) (Merck). The setting times were evaluated using a Vicat apparatus (Alsa Lab, Istanbul, Turkey). Human dental pulp stem cells were isolated and seeded into 48-well plates at 2 × 10(3) cells per well and incubated with MTA samples for 24 hours, 3 days, and 7 days. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. MTA mixed with 10% CaCl2 showed the lowest setting time (P < .05). According to the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium results on the 1st, 3rd, and 7th days, a statistically significant difference was found (P < .05) between MTA groups and the control group. MTA mixed with K-Y Jelly in all groups showed the lowest cell viability at all time points (P < .05). The cell viability of MTA mixed with distilled water, 5% CaCl2, 10% CaCl2, and Na2HPO4 increased significantly through time (P < .05). This in vitro study found MTA mixed with 5% and 10% CaCl2 and Na2HPO4 is biocompatible with dental pulp stem cells in terms of cell viability. Further in vitro and in vivo investigations are required to prove the clinical applications of MTA mixed with various additives.

  18. Rabbit IgG directed to a synthetic C-terminal peptide of the major grass pollen allergen Lol p I inhibits human basophil histamine release induced by natural Lol p I.

    PubMed

    van Ree, R; Aalberse, R C

    1995-03-01

    The potential role of allergen-specific IgG antibodies as 'blocking' antibodies in allergen-induced human basophil histamine release was investigated. This was studied in a model with the major grass pollen allergen Lol p I and polyclonal rabbit antisera directed against this allergen and against a synthetic peptide of its C terminus. When allergen and antibodies were allowed to preincubate, Lol p I induced histamine release was inhibited up to 85% by the antiserum against Lol p I. By omitting preincubation, and thereby more closely mimicking an in vivo situation, up to 55% inhibition was realized. This indicates that allergen-specific IgG can act as 'blocking' antibody without preincubation. Immunization of rabbits with a synthetic C-terminal peptide of Lol p I resulted in antibodies reactive with natural Lol p I. Despite their 100-fold lower avidity for Lol p I (as compared with antinatural Lol p I), these antibodies had the capacity to inhibit Lol p I induced histamine release for > 90% (up to 50% without preincubation). This indicates that it is possible to block histamine release induced by a major allergen with low-avidity IgG antibodies directed against a minor proportion of the allergen (25 amino acids). IgE antibodies from the donors studied were unreactive with this synthetic peptide, indicating that for blocking activity identical epitope specificity of IgE and IgG is not essential. This opens interesting perspectives for application of synthetic peptides in immunotherapy, distinct from their effects on T cell reactivity.

  19. Natural infection of baboons by Entamoeba histolytica elicits anti- gal-lectin heavy subunit IgA and IgG antibodies with shared epitope specificity to that of humans.

    PubMed

    Abd-Alla, Mohamed D; Wolf, Roman F; White, Gary L; Kosanke, Stanley D; Carey, David W; Verweij, Jaco J; El-Dessouky, Yasser M M; Zhang, Mie-Jie; Ravdin, Jonathan I

    2013-12-01

    Non-human primates, such as baboons (Papio hamadryas anubis), are natural hosts for Entamoeba species; infections can be asymptomatic or result in invasive lethal disease. It was sought to determine whether following natural infection by Entamoeba. histolytica, baboon anti-amebic antibodies recognized native Gallectin, a recombinant portion of the lectin heavy subunit (designated LC3) and specific heavy subunit epitopes; we compared the specificity of anti-amebic antibodies from baboons to that of humans following asymptomatic E. histolytica infection or cure of amebic liver abscess (ALA). Female baboons (n=54), aged one to three years of age and living in captivity were screened for infection by real time PCR. E. histolytica infection was found in 37 baboons and was associated with serum anti-LC3 IgG (73%) and anti-LC3 IgA (46%) or intestinal anti-Gal-Lectin IgA antibody responses (49%), p<0.021 for each compared to that observed with baboons having an E. dispar infection (n=10) or uninfected baboons (n=7). The ELISA OD reading for anti-LC3 or anti-lectin antibodies correlated strongly with the presence of a PCR CT value indicative of E. histolytica infection. In humans with asymptomatic E. histolytica infection or those recently cured of ALA, 63% and 57% had serum anti- LC3 IgA and 65% and 57% had serum anti-LC3 IgG antibodies respectively. Epitope- specific synthetic peptides were used as capture antigens in ELISA; for baboons that possessed anti-LC3 and anti-lectin antibodies, 74% had anti-peptide IgG or IgA antibodies, compared to 86% of asymptomatic humans and 92% of ALA subjects(P>0.05).

  20. Receptor-mediated toxicity of human amylin fragment aggregated by short- and long-term incubations with copper ions.

    PubMed

    Caruso, Giuseppe; Distefano, Donatella A; Parlascino, Paolo; Fresta, Claudia G; Lazzarino, Giuseppe; Lunte, Susan M; Nicoletti, Vincenzo G

    2017-01-01

    Human amylin (hA1-37) is a polypeptide hormone secreted in conjunction with insulin from the pancreatic β-cells involved in the pathogenesis of type 2 diabetes mellitus (T2DM). The shorter fragment hA17-29 than full-length peptide is capable to form amyloids "in vitro". Here, we monitored the time course of hA17-29 β-amyloid fibril and oligomer formation [without and with copper(II)], cellular toxicity of different amyloid aggregates, and involvement of specific receptors (receptor for advanced glycation end-products, RAGE; low-affinity nerve growth factor receptor, p75-NGFR) in aggregate toxicity. Fibril and oligomer formation of hA17-29 incubated at 37 °C for 0, 48, and 120 h, without or with copper(II), were measured by the thioflavin T fluorescence assay and ELISA, respectively. Toxicity of hA17-29 aggregates and effects of anti-RAGE and anti-p75-NGFR antibodies were evaluated on neuroblastoma SH-SY5Y viability. Fluorescence assay of hA17-29 indicates an initial slow rate of soluble fibril formation (48 h), followed by a slower rate of insoluble aggregate formation (120 h). The highest quantity of oligomers was recorded when hA17-29 was pre-aggregated for 48 h in the presence of copper(II) showing also the maximal cell toxicity (-44% of cell viability, p < 0.01 compared to controls). Anti-RAGE or anti-p75-NGFR antibodies almost abolished cell toxicity of hA17-29 aggregates. These results indicate that copper(II) influences the aggregation process and hA17-29 toxicities are especially attributable to oligomeric aggregates. hA17-29 aggregate toxicity seems to be mediated by RAGE and p75-NGFR receptors which might be potential targets for new drugs in T2DM treatment.

  1. Selection of single chain variable fragments (scFv) against the glycoprotein antigen of the rabies virus from a human synthetic scFv phage display library and their fusion with the Fc region of human IgG1

    PubMed Central

    Ray, K; Embleton, M J; Jailkhani, B L; Bhan, M K; Kumar, R

    2001-01-01

    We have prepared human recombinant antibody molecules against the glycoprotein antigen of the rabies virus (GPRV) based on the single chain variable fragment (scFv) format. Anti-GPRV scFvs were selected from a human synthetic scFv phage display library with a repertoire of approximately 109 specificities. After three rounds of selection against the PV11 strain of the virus, 40% of the clones tested recognized the rabies antigen. Of the 20 positive clones that were sequenced, five distinct sequences were identified. These distinct scFvs were cloned into a mammalian expression vector carrying the human IgG1 Fc region. The specificity of the resulting scFv-Fc molecules for GPRV was established by ELISA, dot blot and western blot analyses and membrane immunofluorescence. Two of the scFv-Fc fusion proteins neutralized the PV11 strain in a standard in vivo neutralization assay where the virus was incubated with the scFv-Fc molecules before intracranial inoculation in mice. These anti-GPRV scFv-Fc molecules have the potential to be used as an alternative to the presently available HRIG, for use in post-exposure preventive treatment. PMID:11472431

  2. Effects of ethanol on aggregation, serotonin release, and amyloid precursor protein processing in rat and human platelets.

    PubMed

    Ehrlich, Daniela; Humpel, Christian

    2014-01-01

    It is known that oxidative stress leads to amyloid precursor protein (APP) dysregulation in platelets. Ethanol (EtOH) is a vascular risk factor and induces oxidative stress. The aim of the present study was thus to investigate whether EtOH affects APP processing in rat and human platelets. Platelets were exposed to 50 mM EtOH with and without 2 mM calcium-chloride (CaCl₂) for 20 or 180 minutes at 37°C. Platelet aggregation, serotonin release and APP isoforms 130 and 106/110 kDa were analyzed. As a control, 100 mM H₂O₂ was tested in rat platelets. Our data show that EtOH alone did not affect any of the analyzed parameters, whereas CaCl₂ significantly increased aggregation of rat and human platelets. In addition, CaCl₂ alone enhanced serotonin release in rat platelets. EtOH counteracted CaCl₂-induced aggregation and serotonin release. In the presence of CaCl₂, EtOH reduced the 130 kDa APP isoform in rat and human platelets. In conclusion, this study shows that in the presence of CaCl₂, EtOH affects the platelet function and APP processing in rat and human platelets.

  3. High-affinity IgG antibodies develop naturally in Ig-knockout rats carrying germline human IgH/Igκ/Igλ loci bearing the rat CH region.

    PubMed

    Osborn, Michael J; Ma, Biao; Avis, Suzanne; Binnie, Ashleigh; Dilley, Jeanette; Yang, Xi; Lindquist, Kevin; Ménoret, Séverine; Iscache, Anne-Laure; Ouisse, Laure-Hélène; Rajpal, Arvind; Anegon, Ignacio; Neuberger, Michael S; Buelow, Roland; Brüggemann, Marianne

    2013-02-15

    Mice transgenic for human Ig loci are an invaluable resource for the production of human Abs. However, such mice often do not yield human mAbs as effectively as conventional mice yield mouse mAbs. Suboptimal efficacy in delivery of human Abs might reflect imperfect interaction between the human membrane IgH chains and the mouse cellular signaling machinery. To obviate this problem, in this study we generated a humanized rat strain (OmniRat) carrying a chimeric human/rat IgH locus (comprising 22 human V(H)s, all human D and J(H) segments in natural configuration linked to the rat C(H) locus) together with fully human IgL loci (12 Vκs linked to Jκ-Cκ and 16 Vλs linked to Jλ-Cλ). The endogenous Ig loci were silenced using designer zinc finger nucleases. Breeding to homozygosity resulted in a novel transgenic rat line exclusively producing chimeric Abs with human idiotypes. B cell recovery was indistinguishable from wild-type animals, and human V(D)J transcripts were highly diverse. Following immunization, the OmniRat strain performed as efficiently as did normal rats in yielding high-affinity serum IgG. mAbs, comprising fully human variable regions with subnanomolar Ag affinity and carrying extensive somatic mutations, are readily obtainable, similarly to conventional mAbs from normal rats.

  4. Biophysical and Functional Characterization of Rhesus Macaque IgG Subclasses

    PubMed Central

    Boesch, Austin W.; Osei-Owusu, Nana Yaw; Crowley, Andrew R.; Chu, Thach H.; Chan, Ying N.; Weiner, Joshua A.; Bharadwaj, Pranay; Hards, Rufus; Adamo, Mark E.; Gerber, Scott A.; Cocklin, Sarah L.; Schmitz, Joern E.; Miles, Adam R.; Eckman, Joshua W.; Belli, Aaron J.; Reimann, Keith A.; Ackerman, Margaret E.

    2016-01-01

    Antibodies raised in Indian rhesus macaques [Macaca mulatta (MM)] in many preclinical vaccine studies are often evaluated in vitro for titer, antigen-recognition breadth, neutralization potency, and/or effector function, and in vivo for potential associations with protection. However, despite reliance on this key animal model in translation of promising candidate vaccines for evaluation in first in man studies, little is known about the properties of MM immunoglobulin G (IgG) subclasses and how they may compare to human IgG subclasses. Here, we evaluate the binding of MM IgG1, IgG2, IgG3, and IgG4 to human Fc gamma receptors (FcγR) and their ability to elicit the effector functions of human FcγR-bearing cells, and unlike in humans, find a notable absence of subclasses with dramatically silent Fc regions. Biophysical, in vitro, and in vivo characterization revealed MM IgG1 exhibited the greatest effector function activity followed by IgG2 and then IgG3/4. These findings in rhesus are in contrast with the canonical understanding that IgG1 and IgG3 dominate effector function in humans, indicating that subclass-switching profiles observed in rhesus studies may not strictly recapitulate those observed in human vaccine studies. PMID:28018355

  5. 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.

  6. A Glycoform of Immunoglobulin G (IgG) as an Early Biomarker of Exposure to Nonhuman Substances

    DTIC Science & Technology

    2012-12-01

    were greatly elevated in the serum of human patients with liver diseases such as cirrhosis. This led to speculation that the IgGs were produced in...IgG subclasses (IgG1, IgG2, IgG3, and IgG4 ) or to the helper T cell response. Figure 8. Hypothetical models of (left) non-AAL-reactive...primebody contains a subset of fucosylated G0F or G1F IgGs. The AAL binding of primebody appears to be related to the conformation of the IgG CH2 domain

  7. 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.

  8. Comparison of the Effects of British Anti-Lewisite (BAL) and Beta Mercapto Ethanol on the Reduction and Cleavage of Disulfide Bonds in IgG and Human Keratinocyte Proteins

    DTIC Science & Technology

    1988-02-01

    34. Din1a.Z’atic representation of anticipated :-su ts I<•:;di.,’¢,i two-dimensionai e cto h rsswhen- sŕ,’ý" l s are trertet with a disulfide redu cer...Development Laboraliory A TTN: SGRD-UBG;-M l ;ort Detrick, Bldg 568 F~rederick, MD 21701-5010 2/88 S~WkWWW ~1W,$~/ b)AlEmm ...Cleavage of Disulfide Bonds in IgG and Human Keratinocyte Proteins Michael A . Deaton, PhD, CPT, MS Charles A . Barba, BS, SP4 Carlos R. Flores, BS, SP4

  9. Comparative Analysis of Human γD-Crystallin Aggregation under Physiological and Low pH Conditions

    PubMed Central

    Wu, Josephine W.; Chen, Mei-Er; Wen, Wen-Sing; Chen, Wei-An; Li, Chien-Ting; Chang, Chih-Kai; Lo, Chun-Hsien; Liu, Hwai-Shen; Wang, Steven S.-S.

    2014-01-01

    Cataract, a major cause of visual impairment worldwide, is the opacification of the eye’s crystalline lens due to aggregation of the crystallin proteins. The research reported here is aimed at investigating the aggregating behavior of γ-crystallin proteins in various incubation conditions. Thioflavin T binding assay, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, intrinsic (tryptophan) fluorescence spectroscopy, light scattering, and electron microscopy were used for structural characterization. Molecular dynamics simulations and bioinformatics prediction were performed to gain insights into the γD-crystallin mechanisms of fibrillogenesis. We first demonstrated that, except at pH 7.0 and 37°C, the aggregation of γD-crystallin was observed to be augmented upon incubation, as revealed by turbidity measurements. Next, the types of aggregates (fibrillar or non-fibrillar aggregates) formed under different incubation conditions were identified. We found that, while a variety of non-fibrillar, granular species were detected in the sample incubated under pH 7.0, the fibrillogenesis of human γD-crystallin could be induced by acidic pH (pH 2.0). In addition, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and intrinsic fluorescence spectroscopy were used to characterize the structural and conformational features in different incubation conditions. Our results suggested that incubation under acidic condition led to a considerable change in the secondary structure and an enhancement in solvent-exposure of the hydrophobic regions of human γD-crystallin. Finally, molecular dynamics simulations and bioinformatics prediction were performed to better explain the differences between the structures and/or conformations of the human γD-crystallin samples and to reveal potential key protein region involved in the varied aggregation behavior. Bioinformatics analyses revealed that

  10. Russell body inducing threshold depends on the variable domain sequences of individual human IgG clones and the cellular protein homeostasis.

    PubMed

    Stoops, Janelle; Byrd, Samantha; Hasegawa, Haruki

    2012-10-01

    Russell bodies are intracellular aggregates of immunoglobulins. Although the mechanism of Russell body biogenesis has been extensively studied by using truncated mutant heavy chains, the importance of the variable domain sequences in this process and in immunoglobulin biosynthesis remains largely unknown. Using a panel of structurally and functionally normal human immunoglobulin Gs, we show that individual immunoglobulin G clones possess distinctive Russell body inducing propensities that can surface differently under normal and abnormal cellular conditions. Russell body inducing predisposition unique to each immunoglobulin G clone was corroborated by the intrinsic physicochemical properties encoded in the heavy chain variable domain/light chain variable domain sequence combinations that define each immunoglobulin G clone. While the sequence based intrinsic factors predispose certain immunoglobulin G clones to be more prone to induce Russell bodies, extrinsic factors such as stressful cell culture conditions also play roles in unmasking Russell body propensity from immunoglobulin G clones that are normally refractory to developing Russell bodies. By taking advantage of heterologous expression systems, we dissected the roles of individual subunit chains in Russell body formation and examined the effect of non-cognate subunit chain pair co-expression on Russell body forming propensity. The results suggest that the properties embedded in the variable domain of individual light chain clones and their compatibility with the partnering heavy chain variable domain sequences underscore the efficiency of immunoglobulin G biosynthesis, the threshold for Russell body induction, and the level of immunoglobulin G secretion. We propose that an interplay between the unique properties encoded in variable domain sequences and the state of protein homeostasis determines whether an immunoglobulin G expressing cell will develop the Russell body phenotype in a dynamic cellular setting.

  11. Scaling up a chemically-defined aggregate-based suspension culture system for neural commitment of human pluripotent stem cells.

    PubMed

    Miranda, Cláudia C; Fernandes, Tiago G; Diogo, M Margarida; Cabral, Joaquim M S

    2016-12-01

    The demand of high cell numbers for applications in cellular therapies and drug screening requires the development of scalable platforms capable to generating highly pure populations of tissue-specific cells from human pluripotent stem cells. In this work, we describe the scaling-up of an aggregate-based culture system for neural induction of human induced pluripotent stem cells (hiPSCs) under chemically-defined conditions. A combination of non-enzymatic dissociation and rotary agitation was successfully used to produce homogeneous populations of hiPSC aggregates with an optimal (140 μm) and narrow distribution of diameters (coefficient of variation of 21.6%). Scalable neural commitment of hiPSCs as 3D aggregates was performed in 50 mL spinner flasks, and the process was optimized using a factorial design approach, involving parameters such as agitation rate and seeding density. We were able to produce neural progenitor cell cultures, that at the end of a 6-day neural induction process contained less than 3% of Oct4-positive cells and that, after replating, retained more than 60% of Pax6-positive neural cells. The results here presented should set the stage for the future generation of a clinically relevant number of human neural progenitors for transplantation and other biomedical applications using controlled, automated and reproducible large-scale bioreactor culture systems.

  12. The development of a modified human IFN-alpha2b linked to the Fc portion of human IgG1 as a novel potential therapeutic for the treatment of hepatitis C virus infection.

    PubMed

    Jones, Tim D; Hanlon, Marian; Smith, Beverley J; Heise, Charles T; Nayee, Prakash D; Sanders, Deborah A; Hamilton, Anita; Sweet, Clive; Unitt, Esther; Alexander, Graeme; Lo, Kin-Ming; Gillies, Stephen D; Carr, Frank J; Baker, Matthew P

    2004-09-01

    Interferon-alpha (IFN-alpha), in conjunction with ribavirin, is the current standard for the treatment of chronic hepatitis C virus (HCV) infection. This treatment requires frequent dosing, with a significant risk of the development of anti-IFN-alpha neutralizing antibodies that correlates with lack of efficacy or relapse. We have developed an IFN-alpha linked to the Fc region of human IgG1 for improved half-life and less frequent dosing. We have also identified, using a human T cell proliferation assay, three regions of IFN-alpha2b that are potentially immunogenic, and a variant containing a total of six mutations within these regions was made. This variant was made as a fusion to Fc either with or without a flexible linker between the fusion partners. Both configurations of the variant were less active than native IFN-alpha alone, although the variant containing the flexible linker had in vitro antiviral activity within the range of other modified IFN-alphas currently in clinical use. Peptides spanning the modified regions were tested in T cell proliferation assays and found to be less immunogenic than native controls when using peripheral blood mononuclear cells (PBMCs) from both healthy individuals and HCV-infected patients who had been treated previously with IFN-alpha2b.

  13. Non-specific pinocytosis by human endothelial cells cultured as multicellular aggregates: uptake of lucifer yellow and horse radish peroxidase.

    PubMed

    Catizone, A; Chiantore, M V; Andreola, F; Coletti, D; Medolago Albani, L; Alescio, T

    1996-12-01

    We have analyzed the pattern of time-dependent and concentration-dependent incorporation of Lucifer Yellow CH (LY) and Horseradish Peroxidase (HRP) by human umbilical vein endothelial cells cultured on a non-adhesive substratum, where they they become organized into stable, multicellular aggregates. The data were compared with those previously obtained from low-density cultures of non-growing endothelial cells adherent to plastic. While the linear trend of the incorporation kinetics is preserved, the rate of uptake with both time and concentrations is highly dependent on the culture conditions, namely typology of cell-cell and cell-substrate interactions. An at least two-fold increase of the rate of uptake was observed with both markers in the aggregated cells. The extracellular concentration of LY required to saturate the binding capacity of the cell surface shifts from approximately 0.25 mg/ml, with the adherent cells, to approximately 0.5 mg/ml in the aggregated cells; the rate of uptake of three different forms of HRP shows, besides a sharp quantitative increase, also qualitative variations, testified by differential changes of their incorporation rates. These results are entirely consistent with the assumption that the association of the endothelial cells into multicellular aggregates increases the rate of pinocytic uptake by modifying the physicochemical properties of the cell surface, thereby increasing its differential affinity for the extracellular markers.

  14. 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.

  15. Effect of the crude extract of Cestrum parqui on carrageenin-induced rat paw oedema and aggregation of human blood platelets.

    PubMed

    Shehnaz, D; Hamid, F; Baqai, F T; Uddin Ahmad, V

    1999-08-01

    An extract of Cestrum parqui aerial parts in methanol:water (1:1) showed inhibition of carrageenin-induced oedema. The aggregation of human blood platelets induced by adenosine diphosphate and platelet activating factor was also inhibited (IC(50)s were 3 and 2 mg/mL, respectively). On the contrary, the extract did not inhibit arachidonic acid-mediated platelet aggregation.

  16. 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

  17. [Effect of acetylsalicylic acid in complex with lipid nanostructures of various compositions on human platelet aggregation].

    PubMed

    Suslina, Z A; Prokhorov, D I; Shilova, A G; Kaplun, A P; Ionova, V G; Seĭfulla, R D

    2011-01-01

    The effect of lipid nanocomplexes loaded with acetylsalicylic acid (aspirin) on platelet aggregation in vitro was investigated. The antithrombotic effect of aspirin in complex with liposomes prepared from pig brain glycosphingolipids is not only significantly higher compared to control, but also accompanied by leveling of the development of proaggregant effects. It was shown that ADP-induced platelet aggregation is reduced by the introduction of electrostatic charge in the structure of lipid bilayer of liposomes. The effect achieved for the liposomes possessing a negative charge was more pronounced in comparison to the effect of positively charged liposomes.

  18. CD8+ T cells in human uterine endometrial lymphoid aggregates: evidence for accumulation of cells by trafficking

    PubMed Central

    Yeaman, G R; Collins, J E; Fanger, M W; Wira, C R

    2001-01-01

    Lymphoid aggregates (LA) develop during the proliferative phase of the menstrual cycle in the human uterine endometrium (EM). They contain mostly CD8+ T cells and B cells. As these LA are absent immediately following menses, they may arise by division of cells resident in the EM, or by division of a limited number of precursor cells that traffic into the EM during the early proliferative phase of the menstrual cycle. Alternatively, they may arise by the continuous trafficking of cells into the EM throughout the proliferative phase of the menstrual cycle. In this study we investigated the distribution and frequency of CD8+ T cells in the aggregates using expression of Vβ2 or Vβ8 as markers of clonality and Ki-67 as a marker of dividing cells. Confocal microscopic analysis of endometrial tissues showed the random distribution of CD8+ T cells within aggregates within the same sample and in aggregates from different samples. Furthermore, comparisons of the distribution of Vβ2 and Vb8 with expected values predicted from Poisson distribution values were not significantly different, suggesting that CD8+ T cells do not arise by division from single precursors. A low level of T-cell division within LAs was confirmed by positive staining for Ki-67. Dividing T cells were randomly dispersed throughout the LA and the frequency of dividing cells did not vary greatly between aggregates within the same tissue. Nearest-neighbour analysis of dividing cells showed no statistically significant deviations from a random distribution. Taken together, these results suggest that LA develop during the menstrual cycle largely by the trafficking of cells to nucleation sites within the EM, rather than by division of a limited number of precursor cells. PMID:11328377

  19. 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.

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

    PubMed Central

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

    2016-01-01

    ABSTRACT 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

  1. Triethylenetetramine prevents insulin aggregation and fragmentation during copper catalyzed oxidation.

    PubMed

    Torosantucci, Riccardo; Weinbuch, Daniel; Klem, Robin; Jiskoot, Wim

    2013-08-01

    Metal catalyzed oxidation via the oxidative system Cu(2+)/ascorbate is known to induce aggregation of therapeutic proteins, resulting in enhanced immunogenicity. Hence, inclusion of antioxidants in protein formulations is of great interest. In this study, using recombinant human insulin (insulin) as a model, we investigated the ability of several excipients, in particular triethylenetetramine (TETA), reduced glutathione(GSH) and ethylenediamine tetraacetic acid (EDTA), for their ability to prevent protein oxidation, aggregation, and fragmentation. Insulin (1mg/ml) was oxidized with 40 μM Cu(2+) and 4mM ascorbic acid in absence or presence of excipients. Among the excipients studied, 1mM of TETA, EDTA, or GSH prevented insulin aggregation upon metal catalyzed oxidation (MCO) for 3h at room temperature, based on size exclusion chromatography (SEC). At lower concentration (100 μM), for 72 h at +4 °C, TETA was the only one to inhibit almost completely oxidation-induced insulin aggregation, fragmentation, and structural changes, as indicated by SEC, nanoparticle tracking analysis, light obscuration particle counting, intrinsic/extrinsic fluorescence, circular dichroism, and chemical derivatization. In contrast, GSH had a slight pro-oxidant effect, as demonstrated by the higher percentage of aggregates and a more severe structural damage, whereas EDTA offered substantially less protection. TETA also protected a monoclonal IgG1 against MCO-induced aggregation, suggesting its general applicability. In conclusion, TETA is a potential candidate excipient for inclusion in formulations of oxidation-sensitive proteins.

  2. Maternofetal transplacental transport of recombinant IgG antibodies lacking effector functions.

    PubMed

    Mathiesen, Line; Nielsen, Leif K; Andersen, Jan Terje; Grevys, Algirdas; Sandlie, Inger; Michaelsen, Terje E; Hedegaard, Morten; Knudsen, Lisbeth E; Dziegiel, Morten Hanefeld

    2013-08-15

    The neonatal Fc receptor (FcRn) directs the transfer of maternal immunoglobulin G (IgG) antibodies across the placenta and thus provides the fetus and newborn with passive protective humoral immunity. Pathogenic maternal IgG antibodies will also be delivered via the placenta and can cause alloimmunity, which may be lethal. A novel strategy to control pathogenic antibodies would be administration of a nondestructive IgG antibody blocking antigen binding while retaining binding to FcRn. We report on 2 human IgG3 antibodies with a hinge deletion and a C131S point mutation (IgG3ΔHinge) that eliminate complement activation and binding to all classical Fcγ receptors (FcγRs) and to C1q while binding to FcRn is retained. Additionally, 1 of the antibodies has a single point mutation in the Fc (R435H) at the binding site for FcRn (IgG3ΔHinge:R435H). We compared transplacental transport with wild-type IgG1 and IgG3, and found transport across trophoblast-derived BeWo cells and ex vivo placenta perfusions with hierarchies as follows: IgG3ΔHinge:R435H>wild-type IgG1≥IgG3ΔHinge and IgG3ΔHinge:R435H=wild-type IgG1=wild-type IgG3>IgG3ΔHinge, respectively. Collectively, IgG3ΔHinge:R435H was transported efficiently from the maternal to the fetal placental compartment. Thus, IgG3ΔHinge:R435H may be a good candidate for transplacental delivery of a nondestructive antibody to the fetus to combat pathogenic antibodies.

  3. Fusion of the Fc part of human IgG1 to CD14 enhances its binding to gram-negative bacteria and mediates phagocytosis by Fc receptors of neutrophils.

    PubMed

    Vida, András; Bardoel, Bart; Milder, Fin; Majoros, László; Sümegi, Andrea; Bácsi, Attila; Vereb, György; van Kessel, Kok P M; van Strijp, Jos A G; Antal-Szalmás, Péter

    2012-08-30

    Microbial resistance to antimicrobial drugs is promoting a search for new antimicrobial agents that target highly conservative structures of pathogens. Human CD14 - a known pattern recognition receptor (PRR) which recognizes multiple ligands from different microbes might be a worthy candidate. The aim of our work was to create a CD14/Fc dimer protein and evaluate its whole bacteria binding and opsonizing capabilities. Fusion of CD14 with the fragment crystallisable (Fc) part of human IgG1 could not only lead to an artificial opsonin but the dimerization through the Fc part might also increase its affinity to different ligands. Human CD14 and the Fc part of human IgG1 was fused and expressed in HEK293 cells. A histidine tagged CD14 (CD14/His) was also expressed as control. Using flow cytometry we could prove that CD14/Fc bound to whole Gram-negative bacteria, especially to short lipopolysaccharide (Ra and Re) mutants, and weak interaction was observed between the fusion protein and Listeria monocytogenes. Other Gram-positive bacteria and fungi did not show any association with CD14/Fc. CD14/His showed about 50-times less potent binding to Gram-negative bacteria. CD14/Fc acted as an opsonin and enhanced phagocytosis of these bacteria by neutrophil granulocytes, monocyte-derived macrophages and dendritic cells. Internalization of bacteria was confirmed by trypan blue quenching and confocal microscopy. On neutrophils the Fc part of the fusion protein was recognized by Fc receptors (CD16, CD32), as determined by blocking experiments. CD14/Fc enhanced the killing of bacteria in an ex vivo whole blood assay. Our experiments confirm that PRR/Fc fusion proteins can give a boost to FcR dependent phagocytosis and killing provided the antimicrobial part binds efficiently to microbes.

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

    PubMed

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

    2015-08-01

    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 under 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.

  5. Migration-driven aggregate behaviors of human mesenchymal stem cells on a dendrimer-immobilized surface direct differentiation toward a cardiomyogenic fate commitment.

    PubMed

    Ogawa, Yuuki; Kim, Mee-Hae; Kino-Oka, Masahiro

    2016-11-01

    Dynamic behaviors of cell aggregates on a dendrimer surface were investigated to drive the directed differentiation of human mesenchymal stem cells (hMSCs) toward a cardiomyogenic lineage. Cell aggregates on the polyamidoamine dendrimer surface with fifth-generation (G5) of dendron structure showed dynamic changes in morphology associated with repetitive stretching and contracting during migration. Spatial-temporal observations revealed cellular movement in single aggregates by their morphological change through stretching and contracting on the G5 surface, suggesting that the dynamic behavior of aggregate causes mixing of cells. However, aggregates without cell-substrate adhesions on the low-binding culture surface sustained their spherical morphology without cellular movement within a single aggregate. Furthermore, β-catenin was observed at nuclei in aggregates on the G5 surface, and expression of the cardiomyocyte marker cardiac Troponin T (cTnT) was detected. However, β-catenin localized to the nuclei only in the outer region of the aggregate on the low-binding culture surface, and cTnT expression was restricted at the exterior surface of the aggregates. These observations indicate that cell mixing within aggregates on the G5 surface induced the directed differentiation of hMSCs toward a cardiomyogenic lineage by nuclear translocation of β-catenin through dissociation of cell-cell adhesions. These results suggest that migration-driven aggregate behaviors on the dendrimer surface caused repeated morphological changes of aggregate through stretching and contracting, leading to the directed differentiation of hMSCs toward a cardiomyogenic fate commitment.

  6. Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches.

    PubMed

    Abecasis, Bernardo; Aguiar, Tiago; Arnault, Émilie; Costa, Rita; Gomes-Alves, Patricia; Aspegren, Anders; Serra, Margarida; Alves, Paula M

    2017-03-20

    Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. We demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4%) and perfusion at 1.3day(-1) dilution rate to improve hiPSC growth as aggregates in a xeno-free medium. This strategy allowed for increased cell specific growth rate, maximum volumetric concentrations (4.7×10(6)cell/mL) and expansion factors (approximately 19 in total cells), resulting in a 2.6-fold overall improvement in cell yields. Extensive cell characterization, including whole proteomic analysis, was performed to confirm that cells' pluripotent phenotype was maintained during culture. A scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using mechanical dissociation for aggregate disruption and cell passaging. A total expansion factor of 1100 in viable cells was obtained in 11days of culture, while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability after 3 sequential passages in bioreactors.

  7. Continuity and discontinuity in the anti-V3 IgG response of human immunodeficiency virus type 1-infected persons in a cross-sectional and longitudinal study using synthetic peptides.

    PubMed

    Lawoko, A; Johansson, B; Dash, R; Falck, L; Dietrich, U; Pipkorn, R; Nilehn, B; Blomberg, J

    1995-09-01

    The principal neutralization domain (PND) of the V3 region of human immunodeficiency virus type 1 (HIV-1) gp120 is central to HIV pathogenesis. The IgG antibody response to PND was followed in 15 HIV-1-infected persons from southern Sweden over 2-5 years using 32 synthetic V3 peptides. Five peptides had amino acid sequences derived from isolates from each of 5 patients. Sera obtained simultaneously with isolate almost always reacted strongly with these cognate peptides; however, reactivity was undetectable in 1 patient's serum and short lived in the sera of another, indicating inducible holes in the antibody repertoire, which would facilitate dissemination of the corresponding virus strains. Reactivity to other V3 peptides correlated with sequence similarity to the cognate peptide. Strong, stable reactivity to peptides with sequences similar to a south Swedish V3-consensus was accompanied by transient activity to less similar ones. The latter may reflect viral variation, B lymphocyte clonal depletion, or both. Certain IgG responses appeared to preclude others, suggesting clonal dominance.

  8. Proteins that accumulate with age in human skeletal-muscle aggregates contribute to declines in muscle mass and function in Caenorhabditis elegans

    PubMed Central

    Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Suri, Pooja; Mackintosh, Samuel G.; Tackett, Alan J.; Sullivan, Dennis H.; Shmookler Reis, Robert J.; Dennis, Richard A.

    2016-01-01

    Protein aggregation increases with age in normal tissues, and with pathology and age in Alzheimer's hippocampus and mouse cardiac muscle. We now ask whether human skeletal muscle accumulates aggregates with age. Detergent-insoluble protein aggregates were isolated from vastus lateralis biopsies from 5 young (23–27 years of age) and 5 older (64–80 years) adults. Aggregates, quantified after gel electrophoresis, contain 2.1-fold more protein (P<0.0001) when isolated from older subjects relative to young. Of 515 proteins identified by liquid chromatography coupled to tandem mass spectrometry, 56 (11%) were significantly more abundant in older muscle, while 21 (4%) were depleted with age (each P<0.05). Orthologs to seven of these proteins were then targeted in C. elegans by RNA interference. Six of the seven knockdown treatments decreased protein aggregation (range 6–45%, P<0.01 to <0.0001) and increased muscle mass (range 1.5- to 1.85-fold, P<0.01 to <0.0001) in aged nematodes, and rescued mobility (range 1.4 to 1.65-fold, P≤0.0005 each) in a nematode amyloidopathy model. We conclude that specific aggregate proteins, discovered as differentially abundant in aging human muscle, have orthologs that contribute functionally to aggregation and age-associated muscle loss in nematodes, and thus can be considered potential drug targets for sarcopenia in humans. PMID:27992858

  9. 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 ...

  10. Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12.

    PubMed

    Nie, Jianhui; Zhao, Juan; Chen, Qingqing; Huang, Weijin; Wang, Youchun

    2014-10-01

    The CD4 binding site (CD4bs) of envelope glycoprotein (Env) is an important conserved target for anti-human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12 (b12) could recognize conformational epitopes that overlap the CD4bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4bs epitopes.

  11. In vitro functional characterization of feline IgGs.

    PubMed

    Strietzel, Catherine J; Bergeron, Lisa M; Oliphant, Theodore; Mutchler, Veronica T; Choromanski, Leszek J; Bainbridge, Graeme

    2014-04-15

    Very little is known about the functional properties of feline IgGs. Here we report the in vitro characterization of cloned feline IgGs. Rapid amplification of cDNA ends (RACE) and full-length PCR of cat splenic cDNA were used to identify feline sequences encoding IgG heavy chain constant regions (IGHC). Two of the sequences are possibly allelic and have been previously reported in the literature as the only feline IgG, IgG1. Although we confirmed these alleles to be highly abundant (∼98%), analysis of numerous amplification products revealed an additional sequence (∼2%). We cloned and characterized chimeric monoclonal antibodies with each of these heavy chains. Using RACE we revealed the sequences for feline Fc gamma receptor I (FcγRI) and feline Fc neonatal receptor (FcRn). We constructed these recombinant receptors as well as fFcγRIII and determined their binding affinities to the chimeras. All of the chimeras bound to Protein A but not to Protein G, and bound tightly to fFcRn (KD=2-5 nM). Both IgG1 alleles have a high affinity for fFcγRI (KD=10-20 nM), they bind to the low-affinity fFcγRIII receptor (2-4 μM), and also bind to human complement C1q. Thus, feline IgG1a and 1b are expected to induce strong effector function in vivo. The additional IgG detected does not bind to recombinant fFcγRI or fFcγRIII and has negligible binding to hC1q. Consequently, although this putative subclass is projected to have a similar serum half-life as the IgG1 alleles based on comparable in vitro affinity to FcRn, it may not elicit the effector responses mediated by fFcγRI or fFcγRIII. Further testing with native receptors and functional cell-based assays would confirm effector function capabilities of feline IgG subclasses; however this is the first report characterizing affinities of feline IgGs to their Fc receptors and helps pave the way for construction of feline-specific IgGs for therapeutic use.

  12. 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

  13. IgG4 subclass antibodies impair antitumor immunity in melanoma.

    PubMed

    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

    2013-04-01

    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.

  14. IgG4-Related Tubulointerstitial Nephritis.

    PubMed

    Zhang, Pingchuan; Cornell, Lynn D

    2017-03-01

    Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) is a fibroinflammatory disorder that can involve nearly any organ. The disorder has increasingly become known as a distinct clinical entity during the last decade. IgG4-related tubulointerstitial nephritis (IgG4-TIN) is the most common manifestation of IgG4-RD in the kidney. Many patients with IgG4-TIN are diagnosed after IgG4-RD has been recognized in other organ systems, but the kidney may also be the first or only site involved. The presenting clinical features of IgG4-TIN are most commonly kidney insufficiency, kidney mass lesion(s), or both. On biopsy, IgG4-TIN shows a dense lymphoplasmacytic infiltrate, increased IgG4+ plasma cells, storiform fibrosis, and often tubular basement membrane immune complex deposits. Elevation of serum IgG4 often accompanies IgG4-RD; however, it is not specific in reaching the diagnosis. Like IgG4-RD in other organs, IgG4-TIN characteristically responds promptly to steroids, although there is a high relapse rate on discontinuation of immunosuppression. The pathogenesis of IgG4-RD is not understood.

  15. 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.

  16. Tomographic reconstruction reveals the morphology of a unique cellular organelle, the aggregated macrotubules (Macrotubuli aggregati) of human retinal horizontal cells.

    PubMed

    Jastrow, Holger; Yarwood, Andrew; Majorovits, Endre; Harris, J Robin

    2015-04-01

    Horizontal cells of the human retina contain unique tubular organelles that have a diameter which is about 10 times larger than that of microtubules (~230 nm). These macrotubuli in most cases form regular aggregates. Therefore we propose to introduce them as Macrotubuli aggregati in the Terminologia histologica. Tomographic investigation of the structures revealed that the walls of the tubules most probably consist of intermediate filaments running nearly parallel to each other and show somewhat regularly attached ribosomes on their inner and also outer surface. About 2% of the organelles exhibit double- to multiple layered walls and less than 1% resemble large scrolls. The tubules may extend 10 to over 20 μm in the cytoplasm and are also encountered in soma-near processes extending into the outer plexiform layer. It remains unclear why these structures are only present in humans and few other species and why almost only in horizontal cells. Speculations on possible functions are discussed.

  17. Neuron-derived IgG protects neurons from complement-dependent cytotoxicity.

    PubMed

    Zhang, Jie; Niu, Na; Li, Bingjie; McNutt, Michael A

    2013-12-01

    Passive immunity of the nervous system has traditionally been thought to be predominantly due to the blood-brain barrier. This concept must now be revisited based on the existence of neuron-derived IgG. The conventional concept is that IgG is produced solely by mature B lymphocytes, but it has now been found to be synthesized by murine and human neurons. However, the function of this endogenous IgG is poorly understood. In this study, we confirm IgG production by rat cortical neurons at the protein and mRNA levels, with 69.0 ± 5.8% of cortical neurons IgG-positive. Injury to primary-culture neurons was induced by complement leading to increases in IgG production. Blockage of neuron-derived IgG resulted in more neuronal death and early apoptosis in the presence of complement. In addition, FcγRI was found in microglia and astrocytes. Expression of FcγR I in microglia was increased by exposure to neuron-derived IgG. Release of NO from microglia triggered by complement was attenuated by neuron-derived IgG, and this attenuation could be reversed by IgG neutralization. These data demonstrate that neuron-derived IgG is protective of neurons against injury induced by complement and microglial activation. IgG appears to play an important role in maintaining the stability of the nervous system.

  18. Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR.

    PubMed

    Suzuki, Takuo; Ishii-Watabe, Akiko; Tada, Minoru; Kobayashi, Tetsu; Kanayasu-Toyoda, Toshie; Kawanishi, Toru; Yamaguchi, Teruhide

    2010-02-15

    The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcgammaRI binding region of the Fc domain.

  19. [Aggregate shocks and investment in human capital: higher educational achievement during the lost decade in Mexico.

    PubMed

    Peña, Pablo A

    2013-01-01

    This article documents a negative aggregate response in the attainment of postsecondary education (more than 12 years of schooling) in Mexico to the recession of 1982-83 and the stagnation that followed. The response was not homogeneous across genders, regions or family backgrounds. Males experienced a drop in attainment and females experienced a slowdown in attainment growth. On average, states with greater pre-shock educational attainment experienced larger drops. There was no clear trend for the response by family background. However, a negative effect is found even between siblings. The evidence suggests a demand side story: the drop in household income seems to be the main determinant of the fall/slowing down in attainment. The conclusion is that the recession and the lack of growth that ensued had a sizeable and lasting negative impact on skill formation in Mexico.

  20. IgG Placental Transfer in Healthy and Pathological Pregnancies

    PubMed Central

    Palmeira, Patricia; Quinello, Camila; Silveira-Lessa, Ana Lúcia; Zago, Cláudia Augusta; Carneiro-Sampaio, Magda

    2012-01-01

    Placental transfer of maternal IgG antibodies to the fetus is an important mechanism that provides protection to the infant while his/her humoral response is inefficient. IgG is the only antibody class that significantly crosses the human placenta. This crossing is mediated by FcRn expressed on syncytiotrophoblast cells. There is evidence that IgG transfer depends on the following: (i) maternal levels of total IgG and specific antibodies, (ii) gestational age, (iii) placental integrity, (iv) IgG subclass, and (v) nature of antigen, being more intense for thymus-dependent ones. These features represent the basis for maternal immunization strategies aimed at protecting newborns against neonatal and infantile infectious diseases. In some situations, such as mothers with primary immunodeficiencies, exogenous IgG acquired by intravenous immunoglobulin therapy crosses the placenta in similar patterns to endogenous immunoglobulins and may also protect the offspring from infections in early life. Inversely, harmful autoantibodies may cross the placenta and cause transitory autoimmune disease in the neonate. PMID:22235228

  1. Trickle or clumped infection process? An analysis of aggregation in the weights of the parasitic roundworm of humans, Ascaris lumbricoides.

    PubMed

    Walker, Martin; Hall, Andrew; Basáñez, María-Gloria

    2010-10-01

    Studying the distribution of parasitic helminth body size across a population of definitive hosts can advance our understanding of parasite population biology. Body size is typically correlated with egg production. Consequently, inequalities in body size have been frequently measured to infer variation in reproductive success (VRS). Body size is also related to parasite age (time since entering the definitive host) and potentially provides valuable information on the mode of acquisition and establishment of immature (larval) parasites within the host: whether parasites tend to establish singly or in aggregates. The mode of acquisition of soil-transmitted helminths has been a theoretical consideration in the parasitological literature but has eluded data-driven investigation. In this paper, we analyse individual Ascaris lumbricoides weight data collected from a cohort of human hosts before and after re-infection following curative treatment, and explore its distribution within and among individuals in the population. Lorenz curves and Gini coefficients indicate that levels of weight inequality (a proxy for VRS) in A.lumbricoides are lower than other published estimates from animal-helminth systems. We explore levels of intra-host weight aggregation using statistical models to estimate the intraclass correlation coefficient (ICC) while adjusting for covariates using a flexible fractional polynomial transformation approach capable of handling non-linear functional relationships. The estimated ICCs indicate that weights are aggregated within hosts both at equilibrium and after re-infection, suggesting that parasites may establish within the host in clumps. The implications of a clumped infection process are discussed in terms of ascariasis transmission dynamics, control and anthelmintic resistance.

  2. 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

  3. 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

  4. Aggregated Alpha-Synuclein Transfer Efficiently between Cultured Human Neuron-Like Cells and Localize to Lysosomes

    PubMed Central

    Severinsson, Emelie; Agholme, Lotta; Bergström, Joakim; Ingelsson, Martin

    2016-01-01

    Parkinson’s disease and other alpha-synucleinopathies are progressive neurodegenerative diseases characterized by aggregates of misfolded alpha-synuclein spreading throughout the brain. Recent evidence suggests that the pathological progression is likely due to neuron-to-neuron transfer of these aggregates between neuroanatomically connected areas of the brain. As the impact of this pathological spreading mechanism is currently debated, we aimed to investigate the transfer and subcellular location of alpha-synuclein species in a novel 3D co-culture human cell model based on highly differentiated SH-SY5Y cells. Fluorescently-labeled monomeric, oligomeric and fibrillar species of alpha-synuclein were introduced into a donor cell population and co-cultured with an EGFP-expressing acceptor-cell population of differentiated neuron-like cells. Subsequent transfer and colocalization of the different species were determined with confocal microscopy. We could confirm cell-to-cell transfer of all three alpha-synuclein species investigated. Interestingly the level of transferred oligomers and fibrils and oligomers were significantly higher than monomers, which could affect the probability of seeding and pathology in the recipient cells. Most alpha-synuclein colocalized with the lysosomal/endosomal system, both pre- and postsynaptically, suggesting its importance in the processing and spreading of alpha-synuclein. PMID:28030591

  5. Hypothermia-induced platelet aggregation in human blood in an in vitro model: the dominant role of blood-material interactions.

    PubMed

    Hall, Matthew W; Solen, Kenneth A

    2002-03-05

    Hypothermia-induced platelet aggregation (HIPA) with or without neutrophil involvement may cause neurologic dysfunction during hypothermic surgery. We report the use of a previously developed model to study the contributions of several surfaces, surface area, shear rate, and blood-material exposure time to HIPA. Heparinized (1.5 u/mL) human blood was quenched to 24 degrees C and passed (0.5 mL/min) through a 75-cm long 1/32" ID tubing of polyvinylchloride (PVC), polyethylene (PE), polyurethane (PU), Teflon-FEP, or heparin (Duraflo)-coated PVC. The number of aggregates was measured by a light-scattering method, and the concentration of occlusive aggregates was assessed using constant-pressure filtration (50 mmHg). No differences were seen among PVC, PE, PU, or Teflon-FEP. The heparin-coated PVC tubing produced fewer occlusive aggregates, and heparin leaching from the coating was not the cause of the decrease in occlusive aggregates. Increasing surface area increased the number of aggregates, and increasing shear rates decreased the occlusiveness of those aggregates.

  6. Wild-type Human γD-crystallin Promotes Aggregation of Its Oxidation-mimicking, Misfolding-prone W42Q Mutant*

    PubMed Central

    Serebryany, Eugene; King, Jonathan A.

    2015-01-01

    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

  7. 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.

  8. Purification and characterization of L-amino acid oxidase from king cobra (Ophiophagus hannah) venom and its effects on human platelet aggregation.

    PubMed

    Li, Z Y; Yu, T F; Lian, E C

    1994-11-01

    Venoms of several snake species contain large amounts of L-amino acid oxidase but its effects on human plasma coagulation and platelet aggregation have not been explored. We have purified L-amino acid oxidase from king cobra venom through CM-Sephadex C-25, Sephadex G-100 and DEAE Sephadex A-50 chromatographies. The purified enzyme has a mol. wt of 135,000 as determined by gel filtration and 65,000 by SDS-PAGE under non-reducing and reducing conditions. Incubation of plasma with L-amino acid oxidase at 200 micrograms/ml did not affect prothrombin time, activated partial thromboplastin time, or thrombin time. Upon addition of L-amino acid oxidase, platelets in platelet-rich plasma were aggregated. The enzyme-induced aggregation was abolished by catalase. The aggregation was also inhibited by indomethacin, aspirin, ethylenediaminetetraacetate, sodium nitroprusside, prostaglandin E1, mepacrine and verapamil, but not by heparin, hirudin, creatine phosphate/creatine phosphokinase or antimycin/2-deoxy-D-glucose. These results suggest that L-amino acid oxidase induces human platelet aggregation through the formation of H2O2, and subsequent thromboxane A2 synthesis requiring Ca2+ but independent of ADP release. The platelet aggregation caused by L-amino acid oxidase is likely to contribute to toxicity inflicted by cobra venom.

  9. Human stefin B normal and patho-physiological role: molecular and cellular aspects of amyloid-type aggregation of certain EPM1 mutants

    PubMed Central

    Polajnar, Mira; Čeru, Slavko; Kopitar-Jerala, Nataša; Žerovnik, Eva

    2012-01-01

    Epilepsies are characterized by abnormal electrophysiological activity of the brain. Among various types of inherited epilepsies different epilepsy syndromes, among them progressive myoclonus epilepsies with features of ataxia and neurodegeneration, are counted. The progressive myoclonus epilepsy of type 1 (EPM1), also known as Unverricht-Lundborg disease presents with features of cerebellar atrophy and increased oxidative stress. It has been found that EPM1 is caused by mutations in human cystatin B gene (human stefin B). We first describe the role of protein aggregation in other neurodegenerative conditions. Protein aggregates appear intraneurally but are also excreted, such as is the case with senile plaques of amyloid-β (Aβ) that accumulate in the brain parenchyma and vessel walls. A common characteristic of such diseases is the change of the protein conformation toward β secondary structure that accounts for the strong tendency of such proteins to aggregate and form amyloid fibrils. Second, we describe the patho-physiology of EPM1 and the normal and aberrant roles of stefin B in a mouse model of the disease. Furthermore, we discuss how the increased protein aggregation observed with some of the mutants of human stefin B may relate to the neurodegeneration that occurs in rare EPM1 patients. Our hypothesis (Ceru et al., 2005) states that some of the EPM1 mutants of human stefin B may undergo aggregation in neural cells, thus gaining additional toxic function (apart from loss of normal function). Our in vitro experiments thus far have confirmed that four mutants undergo increased aggregation relative to the wild-type protein. It has been shown that the R68X mutant forms amyloid-fibrils very rapidly, even at neutral pH and forms perinuclear inclusions, whereas the G4R mutant exhibits a prolonged lag phase, during which the toxic prefibrillar aggregates accumulate and are scattered more diffusely over the cytoplasm. Initial experiments on the G50E and Q71P

  10. 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

  11. Relationship between native-state solubility and non-native aggregation of recombinant human granulocyte colony stimulating factor: practical implications for protein therapeutic development.

    PubMed

    Banks, Douglas D; Zhang, Jun; Siska, Christine C

    2014-10-06

    Prescreening methods are needed in the biotechnology industry for rapid selection of protein therapeutic candidates and formulations of low aggregation propensity. In recent reports solubility measurements have shown promise as one such method, although the connection between protein solubility and non-native aggregation is not well understood. In the present investigation, recombinant human granulocyte colony stimulating factor (rhGCSF) was used to explore this relationship since it was previously shown to rapidly undergo non-native aggregation/precipitation under physiological conditions in a reaction attenuated by the addition of sucrose [Krishnan, S.; et al. Biochemistry 2002, 41, 6422-6431]. Strong correlations were found between rhGCSF non-native aggregation and both solubility and thermal stability as a function of sucrose concentration. We believe these results make sense in the context of an rhGCSF aggregation mechanism where loss of monomer to insoluble aggregate is limited by association to an observable dimer from a less soluble (and aggregation competent) intermediate species that exists in a temperature sensitive pre-equilibrium with the native monomer. Both solubility and measures of conformational stability report on the position of this equilibrium and therefore the concentration of reactive intermediate. Interestingly, aggregation also correlated with rhGCSF solubility as a function of salting-in concentrations of phosphate since both are dependent on the colloidal stability of the reactive intermediate but not with conformational stability. In lieu of a complete understanding of the aggregation processes that limit protein therapeutic shelf life, these results highlight the potential of using simple solubility measurements as an additional tool in the biotechnology prescreening repertoire.

  12. 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

  13. Bioprocess development for mass production of size-controlled human pluripotent stem cell aggregates in stirred suspension bioreactor.

    PubMed

    Abbasalizadeh, Saeed; Larijani, Mehran Rezaei; Samadian, Azam; Baharvand, Hossein

    2012-11-01

    Current protocols for the scalable suspension culture of human pluripotent stem cells (hPSCs) are limited by multiple biological and technical challenges that need to be addressed before their use in clinical trials. To overcome these challenges, we have developed a novel bioprocess platform for large-scale expansion of human embryonic and induced pluripotent stem cell lines as three-dimensional size-controlled aggregates. This novel bioprocess utilizes the stepwise optimization of both static and dynamic suspension culture conditions. After screening eight xeno-free media in static suspension culture and optimizing single-cell passaging in dynamic conditions, the scale-up from a static to a dynamic suspension culture in the stirred bioreactor resulted in a two- to threefold improvement in expansion rates, as measured by cell counts and metabolic activity. We successfully produced size-specific aggregates through optimization of bioreactor hydrodynamic conditions by using combinations of different agitation rates and shear protectant concentrations. The expansion rates were further improved by controlling oxygen concentration at normoxic conditions, and reached a maximum eightfold increase for both types of hPSCs. Subsequently, we demonstrated a simple and rapid scale-up strategy that produced clinically relevant numbers of hPSCs (∼2×10(9) cells) over a 1-month period by the direct transfer of "suspension-adapted frozen cells" to a stirred suspension bioreactor. We omitted the required preadaptation passages in the static suspension culture. The cells underwent proliferation over multiple passages in the demonstrated xeno-free dynamic suspension culture while maintaining their self-renewal capabilities, as determined by marker expressions and in vitro spontaneous differentiation. In conclusion, suspension culture protocols of hPSCs could be used to mass produce homogenous and pluripotent undifferentiated cells by identification and optimization of key bioprocess

  14. IgG subclasses to food antigens.

    PubMed

    Quinti, I; Papetti, C; D'Offizi, G; Cavagni, G; Panchor, M L; Lunardi, C; Paganelli, R

    1988-02-01

    Involvement of sub-classes of IgG that are specific for food allergens in anaphylactoid reactions and some manifestations of atopy no longer needs to be shown. Accordingly, sub-classes of IgG specific for ovalbumin (OVA) and beta-lactoglobulin (BLG) were compared in healthy subjects and those who presented with an intolerance or food allergy to OVA and BLG to decide whether a restrictive diet was necessary. The four sub-classes of IgG1, IgG2, IgG3 and IgG4 were isolated in all the groups. IgG4 was highest in the allergic subjects and the IgG sub-class values were modified by the diet differently in each group. Unfortunately, the small number of subjects does not allow the formation of a definite conclusion to this study.

  15. Development of IgG responses to mycobacterial antigens.

    PubMed Central

    Pilkington, C; Costello, A M; Rook, G A; Stanford, J L

    1993-01-01

    Recent studies link mycobacterial and human heat shock protein antigens with autoimmune diseases. Little is known about the development of antibody responses to these antigens in children. IgG responses to mycobacterial antigens were studied in children living in the UK (an environment low in mycobacteria) who had not received BCG vaccination. Age curves of IgG response to sonicates from different species of mycobacteria were similar suggesting that the greater part of the developing IgG response is to the common antigens shared by all mycobacteria. The major part of the IgG response was to carbohydrate antigens: lipoarabinomannan is a mycobacterial cell wall carbohydrate and was confirmed as a major immunodominant antigen. Infants showed a marked early response to the mycobacterial 65 kilodalton (kDa) and 70 kDa heat shock proteins, but not to the human 65 kDa heat shock protein. The early IgG response to heat shock proteins may reflect cross reactivity to proteins released by a wide variety of bacteria (possibly from breakdown in the gut) or recognition of other immunodominant antigens with high levels of cross reactivity to self. PMID:8285775

  16. Lysine methylation is an endogenous post-translational modification of tau protein in human brain and a modulator of aggregation propensity

    PubMed Central

    Funk, Kristen E.; Thomas, Stefani N.; Schafer, Kelsey N.; Cooper, Grace L.; Liao, Zhongping; Clark, David J.; Yang, Austin J.; Kuret, Jeff

    2015-01-01

    In Alzheimer disease, the microtubule-associated protein tau dissociates from the neuronal cytoskeleton and aggregates to form cytoplasmic inclusions. Although hyper-phosphorylation of tau Ser and Thr residues is an established trigger of tau misfunction and aggregation, tau modifications extend to Lys residues as well, raising the possibility that different modification signatures depress or promote aggregation propensity depending on site occupancy. To identify Lys-residue modifications associated with normal tau function, soluble tau proteins isolated from four cognitively normal human brains were characterized by mass spectrometry methods. The major detectable Lys modification was found to be methylation, which appeared in the form of mono- and di-methyl Lys residues distributed among at least eleven sites. Unlike tau phosphorylation sites, the frequency of Lys methylation was highest in the microtubule binding repeat region that mediates both microtubule binding and homotypic interactions. When purified recombinant human tau was modified in vitro through reductive methylation, its ability to promote tubulin polymerization was retained, whereas its aggregation propensity was greatly attenuated at both nucleation and extension steps. These data establish Lys methylation as part of the normal tau post-translational modification signature in human brain, and suggest that it can function in part to protect against pathological tau aggregation. PMID:24869773

  17. T15 group A streptococcal Fc receptor binds to the same location on IgG as staphylococcal protein A and IgG rheumatoid factors.

    PubMed

    Nardella, F A; Schröder, A K; Svensson, M L; Sjöquist, J; Barber, C; Christensen, P

    1987-02-01

    Previous work has shown that IgG rheumatoid factors (RF) bind to the C gamma 2-C gamma 3 interface region of human IgG in the same area that binds staphylococcal protein A (SPA). Group A, C, and G strains of Streptococci possess Fc receptors that bind to IgG but not to fragments containing only the C gamma 2 or C gamma 3 domains. This work describes the binding site location on human IgG for the binding of the isolated Fc receptor from the T15 strain of a Group A streptococcus and its relationship to the site that binds SPA and the IgG RF. The isolated T15 Fc receptor (T15) with a molecular mass of 29.5 kD inhibited the binding of IgG RF to IgG. The binding of T15 itself to IgG was strongly inhibited by SPA (42.0 kD) and its monovalent fragment D (7 kD). Human IgG fragments consisting of the C gamma 3 domains did not inhibit the binding of T15 to IgG, whereas those with both domains were effective inhibitors. T15 did not bind to rabbit IgG fragments consisting of either the C gamma 2 or C gamma 3 domains, but did bind to those with both domains. An IgG3 myeloma protein was a poor inhibitor and has been shown to bind poorly to the IgG RF. Most IgG3 myeloma proteins did not bind to SPA. The substitution of Arg and Phe for His 435 and Tyr 436 is responsible for the poor binding of IgG3 to SPA and to the IgG RF. Chemical modification of His or Tyr on IgG reduced its ability to inhibit the binding of T15 to IgG. Reversal of the chemical modifications with hydroxylamine resulted in near complete restoration of inhibitory capacity. This information, collectively, coupled with the known positions in space of the His and Tyr residues in the C gamma 2-C gamma 3 interface region, verified that both His 435 and Tyr 436, and possibly His 310 and 433, are involved. These residues are also involved in binding SPA and the IgG RF. These data therefore indicate that the T15 Group A Streptococcal Fc receptor binds to the same location on the Fc of IgG as SPA and the IgG RF. The

  18. Human ecology, crime, and crime control: linking individual behavior and aggregate crime.

    PubMed

    Savage, Joanne; Vila, Bryan

    2003-01-01

    The paper extends previous research published by Cohen, Machalek, Vila, and others on the evolutionary-ecological paradigm for understanding criminal behavior. After reviewing literature related to human ecology and crime, the paper focuses on elements relevant to human ecology-biology, development, and ecological factors--and their role in criminal behavior. Major emphasis is placed on the linkages between individual factors and macro-level crime using chronic offending as a case in point. The principles of evolutionary ecology then are used to discuss counterstrategies to crime, and the prospects for protection/avoidance, deterrent, and nurturant strategies in light of evidence on chronic offending.

  19. 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.

  20. 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

  1. 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

  2. Identification of the site on IgG Fc for interaction with streptococci of groups A, C and G.

    PubMed

    Schröder, A K; Nardella, F A; Mannik, M; Johansson, P J; Christensen, P

    1987-12-01

    The interaction between living groups A, C and G streptococci and IgG Fc was studied using human IgG, IgG Fc and IgG Fc-intermediate (Fci) fragments, chemically modified human IgG and fragment D of staphylococcal protein A (SPA). Diethylpyrocarbonate modification of His or N-acetylimidazole modification of Tyr of human IgG resulted in the loss of its capacity to inhibit the binding of radiolabelled human IgG Fc to the group A streptococci types M1 and M55, and to the group C strain SC-1, indicating that the amino acids His and Tyr are involved in the binding. Lys seems not to participate in the binding of IgG to these bacteria, however, since reductive methylation of Lys did not reduce its inhibitory capacity. Fragment D of SPA also inhibited the binding of radiolabelled human IgG Fc to strains M1, M55 and SC-1. We have previously shown that these bacteria do not bind to IgG fragments consisting of only the C gamma 2 or C gamma 3 domains. On the basis of these results, and the known relative positions in space of the His and Tyr residues on IgG Fc, it is speculated whether streptococci with IgG Fc receptors, like SPA and rheumatoid factors, interact with IgG in the interface between the C gamma 2 and C gamma 3 domains and involve His 435 and one or more of Tyr 436, His 433 and His 310. The similarities in binding sites on IgG for RFs and these bacterial Fc binding proteins suggest structural similarities between them that may be relevant to the production of rheumatoid factors in rheumatoid arthritis.

  3. 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.

  4. IgG rheumatoid factors and staphylococcal protein A bind to a common molecular site on IgG.

    PubMed

    Nardella, F A; Teller, D C; Barber, C V; Mannik, M

    1985-12-01

    The antigenic determinant on the Fc region of human IgG for two IgG rheumatoid factors (IgG-RF) from patients with rheumatoid arthritis were investigated in detail. The RF did not interact with IgG fragments that contained the C gamma 2 or C gamma 3 region alone, but required the presence of both regions for binding. The RF binding to solid-phase IgG were poorly inhibited by the IgG3 subclass and strongly inhibited by staphylococcal protein A (SPA) (42 kD), and fragment D of SPA (7 kD), indicating that the binding site is most likely the same as the Ga antigenic determinant described for IgM-RF, and is in the same location as the site on IgG that binds SPA. pH titration studies of the RF binding to IgG indicated the involvement of histidine and lysine or tyrosine side chains. Chemical modification studies showed the histidines were involved on the Fc side of the interactions, and tyrosines were involved on both the antigenic and antibody sides of the interactions. Lysines were not involved. The above information, and the knowledge of the number and position in space of the amino acid residues involved in the C gamma 2-C gamma 3 interface region of IgG, the binding site for SPA, and the amino acid substitutions in IgG3 that account for its inability to bind protein A, allowed the identification of the site on IgG that bind IgG-RF. This binding site involves some of the same amino acid side chains, His 435, Tyr 436, and one or both His 433 and 310, and is in the same location as the site that binds SPA. The same site is likely to be a common antigenic determinant for other RF. Furthermore, the described molecular mimicry suggests a biological relationship between bacterial Fc-binding proteins and the production of RF in rheumatoid arthritis.

  5. Inability of probiotic bacterial strains Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019 to induce human platelet aggregation in vitro.

    PubMed

    Zhou, J S; Rutherfurd, K J; Gill, H S

    2005-11-01

    Platelet aggregation contributes to the pathogenesis of infective endocarditis, and aggregation of platelets induced by lactobacilli is thought to be an important contributory factor in the development and progression of Lactobacillus endocarditis. The main purpose of this study was to examine the effect of immunity-enhancing probiotic strains Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019 on the activation and aggregation of human blood platelets. Whole blood samples from healthy individuals were incubated in vitro with HN001 or HN019 and subsequently labeled with platelet-specific monoclonal antibodies, fluorescein isothiocyanate-conjugated anti-CD41a (expressed on normal platelets), and phycoerythrin-streptavidin-conjugated anti-CD62p (expressed on activated platelets) before analysis by flow cytometry. Platelet-rich plasma was used to assist the gating of the platelet cluster. ADP and epinephrine were used as the physiological platelet activation agonists. Platelet aggregation-inducing strain Streptococcus sanguis 133-79 was used as a positive control strain. The mean fluorescence intensity of phycoerythrin and the percentage of platelets expressing the CD62p marker were used to assess the degree of platelet activation. The percentage of CD62p-positive platelets and the light scatter profiles of the agonist-activated platelets were used to identify the occurrence and degree of platelet aggregation. HN001 and HN019 had no effect on spontaneous platelet activation and aggregation; they also failed to exacerbate the platelet aggregation activity induced by ADP and epinephrine. Therefore, these test probiotic strains HN001 and HN019 are less likely to participate in the pathogenesis of infective endocarditis or other thrombotic disorders with regard to platelet aggregation factors.

  6. Role of cosolutes in the aggregation kinetics of monoclonal antibodies.

    PubMed

    Nicoud, Lucrèce; Sozo, Margaux; Arosio, Paolo; Yates, Andrew; Norrant, Edith; Morbidelli, Massimo

    2014-10-16

    We propose a general strategy based on kinetic analysis to investigate how cosolutes affect the aggregation behavior of therapeutic proteins. We apply this approach to study the impact of NaCl and sorbitol on the aggregation kinetics of two monoclonal antibodies, an IgG1 and an IgG2. By using a combination of size exclusion chromatography and light scattering techniques, we study the impact of the cosolutes on the monomer depletion, as well as on the formation of dimers, trimers, and larger aggregates. We analyze these macroscopic effects in the frame of a kinetic model based on Smoluchowski's population balance equations modified to account for nucleation events. By comparing experimental data with model simulations, we discriminate the effect of cosolutes on the elementary steps which contribute to the global aggregation process. In the case of the IgG1, it is found that NaCl accelerates the kinetics of aggregation by promoting specifically aggregation events, while sorbitol delays the kinetics of aggregation by specifically inhibiting protein unfolding. In the case of the IgG2, whose monomer depletion kinetics is limited by dimer formation, NaCl and sorbitol are found respectively to accelerate and inhibit conformational changes and aggregation events to the same extent.

  7. Overexpression of human alpha-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion

    PubMed Central

    1992-01-01

    Human lysosomal alpha-galactosidase A (alpha-Gal A) was stably overexpressed in CHO cells and its biosynthesis and targeting were investigated. Clone AGA5.3-1000Mx, which was the highest enzyme overexpressor, produced intracellular alpha-Gal A levels of 20,900 U/mg (approximately 100 micrograms of enzyme/10(7) cells) and secreted approximately 13,000 U (or 75 micrograms/10(7) cells) per day. Ultrastructural examination of these cells revealed numerous 0.25-1.5 microns crystalline structures in dilated trans-Golgi network (TGN) and in lysosomes which stained with immunogold particles using affinity- purified anti-human alpha-Gal A antibodies. Pulse-chase studies revealed that approximately 65% of the total enzyme synthesized was secreted, while endogenous CHO lysosomal enzymes were not, indicating that the alpha-Gal A secretion was specific. The recombinant intracellular and secreted enzyme forms were normally processed and phosphorylated; the secreted enzyme had mannose-6-phosphate moieties and bound the immobilized 215-kD mannose-6-phosphate receptor (M6PR). Thus, the overexpressed enzyme's selective secretion did not result from oversaturation of the M6PR-mediated pathway or abnormal binding to the M6PR. Of note, the secreted alpha-Gal A was sulfated and the percent of enzyme sulfation decreased with increasing amplification, presumably due to the inaccessibility of the enzyme's tyrosine residues for the sulfotransferase in the TGN. Overexpression of human lysosomal alpha-N-acetylgalactosaminidase and acid sphingomyelinase in CHO cell lines also resulted in their respective selective secretion. In vitro studies revealed that purified secreted alpha-Gal A was precipitated as a function of enzyme concentration and pH, with 30% of the soluble enzyme being precipitated when 10 mg/ml of enzyme was incubated at pH 5.0. Thus, it is hypothesized that these overexpressed lysosomal enzymes are normally modified until they reach the TGN where the more acidic environment of

  8. Movement responses of caribou to human-induced habitat edges lead to their aggregation near anthropogenic features.

    PubMed

    Fortin, Daniel; Buono, Pietro-Luciano; Fortin, André; Courbin, Nicolas; Tye Gingras, Christian; Moorcroft, Paul R; Courtois, Réhaume; Dussault, Claude

    2013-06-01

    The assessment of disturbance effects on wildlife and resulting mitigation efforts are founded on edge-effect theory. According to the classical view, the abundance of animals affected by human disturbance should increase monotonically with distance from disturbed areas to reach a maximum at remote locations. Here we show that distance-dependent movement taxis can skew abundance distributions toward disturbed areas. We develop an advection-diffusion model based on basic movement behavior commonly observed in animal populations and parameterize the model from observations on radio-collared caribou in a boreal ecosystem. The model predicts maximum abundance at 3.7 km from cutovers and roads. Consistently, aerial surveys conducted over 161,920 km(2) showed that the relative probability of caribou occurrence displays nonmonotonic changes with the distance to anthropogenic features, with a peak occurring at 4.5 km away from these features. This aggregation near disturbed areas thus provides the predators of this top-down-controlled, threatened herbivore species with specific locations to concentrate their search. The edge-effect theory developed here thus predicts that human activities should alter animal distribution and food web properties differently than anticipated from the current paradigm. Consideration of such nonmonotonic response to habitat edges may become essential to successful wildlife conservation.

  9. von Willebrand factor fibers promote cancer-associated platelet aggregation in malignant melanoma of mice and humans

    PubMed Central

    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

    2015-01-01

    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

  10. 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.

  11. Selective IgG subclass deficiency: quantification and clinical relevance.

    PubMed Central

    Jefferis, R; Kumararatne, D S

    1990-01-01

    Each of the four human IgG subclasses exhibits a unique profile of effector functions relevant to the clearance and elimination of infecting microorganisms. The quantitative response within each IgG subclass varies with the nature of the antigen, its route of entry and, presumably, the form in which it is presented to the immune system. This results in antibody responses to certain antigens being predominantly or exclusively of a single IgG subclass. An inability to produce antibody of the optimally protective isotype can result in a selective immunodeficiency state. This is particularly apparent for responses to certain bacterial carbohydrate antigens that are normally of IgG2 isotype. A failure to produce the appropriate specific antibody response may result in recurrent upper and/or lower respiratory tract infection. Careful patient investigation can identify such deficiencies and suggest appropriate clinical management. In this review we outline the biology and clinical relevance of the IgG subclasses and summarize current rational treatment approaches. PMID:2204502

  12. IgG4-related kidney disease.

    PubMed

    Cornell, Lynn D

    2012-11-01

    IgG4-related kidney disease is a term that refers to any form of renal involvement by IgG4-related disease (IgG4-RD), a recently recognized systemic immune-mediated disease. The most common renal manifestation is IgG4-related tubulointerstitial nephritis (IgG4-TIN), which presents as acute or chronic renal insufficiency, renal mass lesions, or both. On biopsy, IgG4-TIN shows a plasma cell-rich interstitial inflammatory infiltrate with increased IgG4+ plasma cells, along with expansile interstitial fibrosis; tubular basement membrane immune complex deposits are common. IgG4-TIN usually shows a brisk response to immunosuppressive therapy. Glomeruli may be affected by IgG4-RD, usually in the form of membranous glomerulonephritis. Other patterns of glomerular disease include IgA nephropathy, membranoproliferative glomerulonephritis, and endocapillary or mesangioproliferative immune complex glomerulonephritis. IgG4-related plasma cell arteritis has also been observed in the kidney. This review describes the histopathologic and immunophenotypic patterns of renal involvement by IgG4-RD, with associated clinical, radiographic, and serologic features.

  13. Quantification of IgG on erythrocytes of patients and normals by a radio-ligand-binding assay.

    PubMed

    Giles, C M; Davies, K A; Loizou, S; Moulds, J J; Walport, M J

    1991-12-01

    A monoclonal IgG anti-human IgG, 1B12, was used in a radio-ligand-binding assay to quantify IgG on erythrocytes of patients and normals. The assay detected a range of 10-700 IgG molecules. Good correlation was achieved between the number of molecules and the strength of agglutination in antiglobulin tests performed in capillary tubes. The assay was capable of detecting subagglutinating immune bound IgG on erythrocytes from patients with systemic lupus erythematosus (SLE).

  14. 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.

  15. 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

  16. MRP4 knockdown enhances migration, suppresses apoptosis, and produces aggregated morphology in human retinal vascular endothelial cells.

    PubMed

    Tagami, Mizuki; Kusuhara, Sentaro; Imai, Hisanori; Uemura, Akiyoshi; Honda, Shigeru; Tsukahara, Yasutomo; Negi, Akira

    2010-10-01

    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.

  17. Enterococcus faecalis Bearing Aggregation Substance Is Resistant to Killing by Human Neutrophils despite Phagocytosis and Neutrophil Activation

    PubMed Central

    Rakita, Robert M.; Vanek, Natalie N.; Jacques-Palaz, Karen; Mee, Mee; Mariscalco, M. Michele; Dunny, Gary M.; Snuggs, Mark; Van Winkle, W. Barry; Simon, Scott I.

    1999-01-01

    Enterococcus faecalis aggregation substance (AS) mediates efficient bacterium-bacterium contact to facilitate plasmid exchange as part of a bacterial sex pheromone system. We have previously determined that AS promotes direct, opsonin-independent binding of E. faecalis to human neutrophils (PMNs) via complement receptor type 3 and other receptors on the PMN surface. We have now examined the functional consequences of this bacterium-host cell interaction. AS-bearing E. faecalis was phagocytosed and internalized by PMNs, as determined by deconvolution fluorescence microscopy. However, these bacteria were not killed by PMNs, and internalized bacteria excluded propidium iodide, indicating intact bacterial membranes. Resistance to killing occurred despite activation of PMNs, as indicated by an increase in both functional and total surface Mac-1 expression, shedding of l-selectin, and an increase in PMN extracellular superoxide and phagosomal oxidant production. Deconvolution fluorescence microscopy also revealed that phagosomes containing AS-bearing bacteria were markedly larger than phagosomes containing opsonized E. faecalis, suggesting that some modification of phagosomal maturation may be involved in AS-induced resistance to killing. PMN phagosomal pH was significantly higher after ingestion of nonopsonized AS-bearing E. faecalis than after that of opsonized bacteria. The novel ability of AS to promote intracellular survival of E. faecalis inside PMNs suggests that AS may be a virulence factor used by strains of E. faecalis. PMID:10531268

  18. BabA dependent binding of Helicobacter pylori to human gastric mucins cause aggregation that inhibits proliferation and is regulated via ArsS

    PubMed Central

    Skoog, Emma C.; Padra, Médea; Åberg, Anna; Gideonsson, Pär; Obi, Ikenna; Quintana-Hayashi, Macarena P.; Arnqvist, Anna; Lindén, Sara K.

    2017-01-01

    Mucins in the gastric mucus layer carry a range of glycan structures, which vary between individuals, can have antimicrobial effect or act as ligands for Helicobacter pylori. Mucins from various individuals and disease states modulate H. pylori proliferation and adhesin gene expression differently. Here we investigate the relationship between adhesin mediated binding, aggregation, proliferation and adhesin gene expression using human gastric mucins and synthetic adhesin ligand conjugates. By combining measurements of optical density, bacterial metabolic activity and live/dead stains, we could distinguish bacterial aggregation from viability changes, enabling elucidation of mechanisms behind the anti-prolific effects that mucins can have. Binding of H. pylori to Leb-glycoconjugates inhibited the proliferation of the bacteria in a BabA dependent manner, similarly to the effect of mucins carrying Leb. Furthermore, deletion of arsS lead to a decrease in binding to Leb-glycoconjugates and Leb-decorated mucins, accompanied by decreased aggregation and absence of anti-prolific effect of mucins and Leb-glycoconjugates. Inhibition of proliferation caused by adhesin dependent binding to mucins, and the subsequent aggregation suggests a new role of mucins in the host defense against H. pylori. This aggregating trait of mucins may be useful to incorporate into the design of adhesin inhibitors and other disease intervention molecules. PMID:28106125

  19. CD40 Ligand and Appropriate Cytokines Induce Switching to IgG, IgA, and IgE and Coordinated Germinal Center and Plasmacytoid Phenotypic Differentiation in a Human Monoclonal IgM+IgD+ B Cell Line1

    PubMed Central

    Cerutti, Andrea; Zan, Hong; Schaffer, Andras; Bergsagel, Leif; Harindranath, Nagaradona; Max, Edward E.; Casali, Paolo

    2015-01-01

    B lymphocytes are induced to undergo Ig class switching and a complex phenotypic differentiation by the milieu of the germinal center. Partly as a result of the lack of a suitable in vitro B cell model, the relationship between these processes in the humans has never been formally established in vitro. We have identified a human monoclonal B cell line, CL-01, that expresses surface IgM and IgD and, upon induction with CD40 ligand, IL-4, and IL-10, switches to all seven downstream isotypes, showing typical DNA switch recombination preceded by germline transcription of targeted CH regions. In CL-01 cells, switch-inducing stimuli trigger concomitant changes in expression of surface IgD, CD23, CD38, and CD77 that parallel those reported in ex vivo isolated tonsillar centroblasts, centrocytes, and memory B cells. Eventually, in the presence of IL-6, CL-01 cells express CD56 and accumulate cytoplasmic IgG and IgA, both traits of plasmacytoid differentiation. Analysis of transcription and recombination of the Ig H locus in sorted CL-01 cells suggest that Ig class switching begins in centroblasts, it extends to all isotypes in centrocytes, and it is extinct in memory B cells. Thus, we have induced coordinated Ig class switching, progression through germinal center phenotypic stages, and differentiation to memory B cells and plasma cells at the level of a single B clonotype. Our data suggest that these processes are likely regulated by a common maturation program, the activation of which may require CD40 ligand, IL-4, IL-10, and IL-6 only. PMID:9498752

  20. The immunoglobulin-binding Eib proteins from Escherichia coli are receptors for IgG Fc.

    PubMed

    Leo, Jack C; Goldman, Adrian

    2009-05-01

    The immunoglobulin-binding proteins from Escherichia coli (Eibs) comprise a family of six proteins homologous to the Yersinia adhesin YadA. These proteins are postulated to bind to the Fc portion of immunoglobulin G (IgG) in a non-immune manner. However, a recent study [Ghumra, A., Pleass, R.J., 2007. Escherichia coli do not express Fc-receptors for human immunoglobulin G (IgG). Mol. Immunol. 44, 2144-2146] appeared to show that these proteins do not bind Fc and suggested that the binding seen in earlier studies is due to the polyclonal preparations used in the assays containing antibodies specific to epitopes in the Eib proteins. To resolve this matter, we produced purified, recombinant Eibs for the first time and investigated their binding to intact antibodies and Fc fragments by immunoblot and ELISA techniques. We were able to purify four members of the family, EibA, -C, -D and -F, and show conclusively that these bind IgG Fc. We were also able to block the binding of full-length antibody with IgG Fc, but not with IgG Fab. Binding to IgG Fab was not detectable by surface plasmon resonance, whereas the affinities of Eibs to IgG and IgG Fc were in the range of 50-200 nM. We further demonstrate that deglycosylating IgG Fc does not affect Eib binding. Our results show that the Eib proteins do indeed bind human IgG Fc and that IgG Fc receptors are present in E. coli.

  1. 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

  2. Engraftment of human induced pluripotent stem cell-derived hepatocytes in immunocompetent mice via 3D co-aggregation and encapsulation.

    PubMed

    Song, Wei; Lu, Yen-Chun; Frankel, Angela S; An, Duo; Schwartz, Robert E; Ma, Minglin

    2015-11-23

    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.

  3. 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.

  4. Advanced analyses of kinetic stabilities of iggs modified by mutations and glycosylation

    PubMed Central

    Sedlák, Erik; Schaefer, Jonas V; Marek, Jozef; Gimeson, Peter; Plückthun, Andreas

    2015-01-01

    The stability of Immunoglobulin G (IgG) affects production, storage and usability, especially in the clinic. The complex thermal and isothermal transitions of IgGs, especially their irreversibilities, pose a challenge to the proper determination of parameters describing their thermodynamic and kinetic stability. Here, we present a reliable mathematical model to study the irreversible thermal denaturations of antibody variants. The model was applied to two unrelated IgGs and their variants with stabilizing mutations as well as corresponding non-glycosylated forms of IgGs and Fab fragments. Thermal denaturations of IgGs were analyzed with three transitions, one reversible transition corresponding to CH2 domain unfolding followed by two consecutive irreversible transitions corresponding to Fab and CH3 domains, respectively. The parameters obtained allowed us to examine the effects of these mutations on the stabilities of individual domains within the full-length IgG. We found that the kinetic stability of the individual Fab fragment is significantly lowered within the IgG context, possibly because of intramolecular aggregation upon heating, while the stabilizing mutations have an especially beneficial effect. Thermal denaturations of non-glycosylated variants of IgG consist of more than three transitions and could not be analyzed by our model. However, isothermal denaturations demonstrated that the lack of glycosylation affects the stability of all and not just of the CH2 domain, suggesting that the partially unfolded domains may interact with each other during unfolding. Investigating thermal denaturation of IgGs according to our model provides a valuable tool for detecting subtle changes in thermodynamic and/or kinetic stabilities of individual domains. PMID:25966898

  5. Equilibrium studies of protein aggregates and homogeneous nucleation in protein formulation.

    PubMed

    Kiese, Sylvia; Pappenberger, Astrid; Friess, Wolfgang; Mahler, Hanns-Christian

    2010-02-01

    Shaking or heat stress may induce protein aggregates. Aggregation behavior of an IgG1 stressed by shaking or heat following static storage at 5 and 25 degrees C was investigated to determine whether protein aggregates exist in equilibrium. Aggregates were detected using different analytical methods including visual inspection, turbidity, light obscuration, size exclusion chromatography, and dynamic light scattering. Significant differences were evident between shaken and heated samples upon storage. Visible and subvisible particles (insoluble aggregates), turbidity and z-average diameter decreased whilst soluble aggregate content increased in shaken samples over time. Insoluble aggregates were considered to be reversible and dissociate into soluble aggregates and both aggregate types existed in equilibrium. Heat-induced aggregates had a denatured protein structure and upon static storage, no significant change in insoluble aggregates content was shown, whilst changes in soluble aggregates content occurred. This suggested that heat-induced insoluble aggregates were irreversible and not in equilibrium with soluble aggregates. Additionally, the aggregation behavior of unstressed IgG1 after spiking with heavily aggregated material (shaken or heat stressed) was studied. The aggregation behavior was not significantly altered, independent of the spiking concentration over time. Thus, neither mechanically stressed native nor temperature-induced denatured aggregates were involved in nucleating or propagating aggregation.

  6. 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.

  7. 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.

  8. 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

  9. 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).

  10. Efficient production of bispecific IgG of different isotypes and species of origin in single mammalian cells

    PubMed Central

    Dillon, Michael; Zhou, Jianhui; McCarty, Luke; Ellerman, Diego; Slaga, Dionysos; Junttila, Teemu T.; Sandoval, Wendy; Ovacik, Meric A.; Lin, Kedan; Hu, Zhilan; Spiess, Christoph

    2017-01-01

    ABSTRACT Bispecific IgG production in single host cells has been a much sought-after goal to support the clinical development of these complex molecules. Current routes to single cell production of bispecific IgG include engineering heavy chains for heterodimerization and redesign of Fab arms for selective pairing of cognate heavy and light chains. Here, we describe novel designs to facilitate selective Fab arm assembly in conjunction with previously described knobs-into-holes mutations for preferential heavy chain heterodimerization. The top Fab designs for selective pairing, namely variants v10 and v11, support near quantitative assembly of bispecific IgG in single cells for multiple different antibody pairs as judged by high-resolution mass spectrometry. Single-cell and in vitro-assembled bispecific IgG have comparable physical, in vitro biological and in vivo pharmacokinetics properties. Efficient single-cell production of bispecific IgG was demonstrated for human IgG1, IgG2 and IgG4 thereby allowing the heavy chain isotype to be tailored for specific therapeutic applications. Additionally, a reverse chimeric bispecific IgG2a with humanized variable domains and mouse constant domains was generated for preclinical proof-of-concept studies in mice. Efficient production of a bispecific IgG in stably transfected mammalian (CHO) cells was shown. Individual clones with stable titer and bispecific IgG composition for >120 days were readily identified. Such long-term cell line stability is needed for commercial manufacture of bispecific IgG. The single-cell bispecific IgG designs developed here may be broadly applicable to biotechnology research, including screening bispecific IgG panels, and to support clinical development. PMID:27929752

  11. IgG4-related nephropathy.

    PubMed

    Quattrocchio, Giacomo; Roccatello, Dario

    2016-08-01

    IgG4-related disease (IgG4-RD) is a recently recognized disorder, often with multiple organ involvement, characterized by dense tissue infiltration of IgG4-positive plasma cells, storiform fibrosis, obliterative phlebitis and frequently elevated serum IgG4 concentration. The kidney can be involved either directly or indirectly. The most frequent direct renal manifestations of IgG4-RD are IgG4-related tubulointerstitial nephritis (TIN) and membranous glomerulonephropathy. Retroperitoneal fibrosis (RPF) is another condition that is frequently IgG4-related and that can indirectly affect the kidney causing ureteral obstruction and hydronephrosis. Contrast-enhanced computerized tomography, magnetic resonance imaging and (18)F-fluorodeoxyglucose positron emission tomography/computed tomography show different imaging findings and are useful tools for monitoring therapeutic response. Steroid treatment is the first line of therapy, but relapsing or refractory forms of the disease are frequently observed and require more aggressive therapeutic approaches. At our centre, we treated three cases of aggressive IgG4-related TIN and two cases of IgG4-related RPF with an intensified, immune suppressive protocol, obtaining good results without severe adverse effects.

  12. Charge variants in IgG1

    PubMed Central

    Goswami, Sirj; Hutchinson, Ryan; Kwong, Zephania W; Yang, Jihong; Wang, Xiangdan; Yao, Zhenling; Sreedhara, Alavattam; Cano, Tony; Tesar, Devin; Nijem, Ihsan; Allison, David E; Wong, Pin Yee; Kao, Yung-Hsiang; Quan, Cynthia; Joshi, Amita; Harris, Reed J; Motchnik, Paul

    2010-01-01

    Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7–9.1 and was composed of about 20% acidic variants, 12% basic variants and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity or the PK properties in rats. PMID:20818176

  13. Hsp72 (HSPA1A) Prevents Human Islet Amyloid Polypeptide Aggregation and Toxicity: A New Approach for Type 2 Diabetes Treatment.

    PubMed

    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.

  14. 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

  15. 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.

  16. [IgG4-related disease].

    PubMed

    Sato, Yasuharu; Yoshino, Tadashi

    2012-02-01

    IgG4-related disease is a recently recognized systemic syndrome characterized by mass-forming lesions, mainly in exocrine tissue, that consist of lymphoplasmacytic infiltrates and sclerosis. There are numerous IgG4+ plasma cells in the affected tissues, and the serum IgG4 level is elevated in these patients. Ocular adnexal IgG4-related disease frequently involves bilateral lacrimal glands, and obliterative phlebitis is rare. Moreover, some malignant lymphomas, especially mucosa-associated lymphoid tissue lymphoma, arise from ocular adnexal IgG4-related disease. It is known that hyper IL-6 syndromes, such as multicentric Castleman's disease, rheumatoid arthritis, and other autoimmune diseases, fulfill the histological diagnostic criteria for IgG4-related disease; therefore, hyper IL-6 syndromes and IgG4-related disease cannot be differentially diagnosed by immunohistochemical staining alone. However, upon laboratory examination, hype IL-6 syndromes show elevation of the CRP level, polyclonal hyper gamma-globulinemia, anemia, and hypoalbuminemia. These findings are quite different from IgG4-related disease, which is not characterized by elevated serum IgA, IgM, and CRP levels. Therefore, laboratory findings are crucial for the differential diagnosis.

  17. Elevated IgG4 in patient circulation is associated with the risk of disease progression in melanoma

    PubMed Central

    Karagiannis, Panagiotis; Villanova, Federica; Josephs, Debra H; Correa, Isabel; Van Hemelrijck, Mieke; Hobbs, Carl; Saul, Louise; Egbuniwe, Isioma U; Tosi, Isabella; Ilieva, Kristina M; Kent, Emma; Calonje, Eduardo; Harries, Mark; Fentiman, Ian; Taylor-Papadimitriou, Joyce; Burchell, Joy; Spicer, James F; Lacy, Katie E; Nestle, Frank O; Karagiannis, Sophia N

    2015-01-01

    Emerging evidence suggests pathological and immunoregulatory functions for IgG4 antibodies and IgG4+ B cells in inflammatory diseases and malignancies. We previously reported that IgG4 antibodies restrict activation of immune effector cell functions and impair humoral responses in melanoma. Here, we investigate IgG4 as a predictor of risk for disease progression in a study of human sera (n = 271: 167 melanoma patients; 104 healthy volunteers) and peripheral blood B cells (n = 71: 47 melanoma patients; 24 healthy volunteers). IgG4 (IgG4/IgGtotal) serum levels were elevated in melanoma. High relative IgG4 levels negatively correlated with progression-free survival (PFS) and overall survival. In early stage (I–II) disease, serum IgG4 was independently negatively prognostic for progression-free survival, as was elevation of IgG4+ circulating B cells (CD45+CD22+CD19+CD3−CD14−). In human tissues (n = 256; 108 cutaneous melanomas; 56 involved lymph nodes; 60 distant metastases; 32 normal skin samples) IgG4+ cell infiltrates were found in 42.6% of melanomas, 21.4% of involved lymph nodes and 30% of metastases, suggesting inflammatory conditions that favor IgG4 at the peripheral and local levels. Consistent with emerging evidence for an immunosuppressive role for IgG4, these findings indicate association of elevated IgG4 with disease progression and less favorable clinical outcomes. Characterizing immunoglobulin and other humoral immune profiles in melanoma might identify valuable prognostic tools for patient stratification and in the future lead to more effective treatments less prone to tumor-induced blockade mechanisms. PMID:26451312

  18. Extensive Chemical Modifications in the Primary Protein Structure of IgG1 Subvisible Particles Are Necessary for Breaking Immune Tolerance.

    PubMed

    Boll, Björn; Bessa, Juliana; Folzer, Emilien; Ríos Quiroz, Anacelia; Schmidt, Roland; Bulau, Patrick; Finkler, Christof; Mahler, Hanns-Christian; Huwyler, Jörg; Iglesias, Antonio; Koulov, Atanas V

    2017-03-07

    A current concern with the use of therapeutic proteins is the likely presence of aggregates and submicrometer, subvisible, and visible particles. It has been proposed that aggregates and particles may lead to unwanted increases in the immune response with a possible impact on safety or efficacy. The aim of this study was thus to evaluate the ability of subvisible particles of a therapeutic antibody to break immune tolerance in an IgG1 transgenic mouse model and to understand the particle attributes that might play a role in this process. We investigated the immunogenic properties of subvisible particles (unfractionated, mixed populations, and well-defined particle size fractions) using a transgenic mouse model expressing a mini-repertoire of human IgG1 (hIgG1 tg). Immunization with proteinaceous subvisible particles generated by artificial stress conditions demonstrated that only subvisible particles bearing very extensive chemical modifications within the primary amino acid structure could break immune tolerance in the hIgG1 transgenic mouse model. Protein particles exhibiting low levels of chemical modification were not immunogenic in this model.

  19. 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.

  20. 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

  1. 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

  2. Development of a sensitive size exclusion HPLC method with fluorescence detection for the quantitation of recombinant human erythropoietin (r-HuEPO) aggregates.

    PubMed

    Gunturi, Srinivas R; Ghobrial, Ibrahim; Sharma, Basant

    2007-01-04

    Human erythropoietin produced by recombinant DNA technology, is now marketed worldwide for the treatment of anemias associated with chronic renal failure and chemotherapy. No sensitive methods, which can determine r-HuEPO dimer or oligomer aggregate content in formulated products, have been published to date. This report describes the development and validation of a sensitive size exclusion high performance liquid chromatography (HPLC) method for the quantitation of r-HuEPO aggregates in formulations containing 0.03% polysorbate 80. A Waters Alliance 2690 HPLC system connected to a TosoHaas TSKgel G3000 SWxl (7.8 mm x 30 cm, 250 A pore size, 5 microm particle size) column and a Waters 474 fluorescence detector was used. The mobile phase for the SEC-HPLC method consists of isopropyl alcohol-potassium phosphate (0.1 M)/potassium chloride buffer (pH 6.8+/-0.1, 0.2 M) (25:75, v/v). The flow rate was 0.3 mL/min and the method run time was 60 min. The SEC-HPLC method presented here was shown to be specific for r-HuEPO total aggregates (dimer and oligomers) and allows for their quantitation at 80 ng/mL or 4 ngs/injection, in the presence of r-HuEPO monomer and the pharmaceutical excipients, glycine (5 mg/mL), sodium chloride (4.3 mg/mL), and 0.03% polysorbate 80. The finalized method is stability-indicating and is suitable for determining r-HuEPO aggregates between 0.2 and 0.5% levels in the formulated product of r-HuEPO. This method offers a robust way to measure total aggregates on a routine basis with a high sensitivity for use in product quality control.

  3. Shaken, not stirred: mechanical stress testing of an IgG1 antibody.

    PubMed

    Kiese, Sylvia; Papppenberger, Astrid; Friess, Wolfgang; Mahler, Hanns-Christian

    2008-10-01

    Protein aggregation is known to occur under different stress conditions and displays a wide variety of morphologies. In this work, the aggregation behavior of a monoclonal antibody (IgG1) was investigated using two different mechanical stress methods namely stirring and shaking at two temperatures, various fill volumes and headspaces and different amounts of polysorbate present in the formulation. The detection of aggregates in terms of size and number was carried out using various analytical techniques including visible particle inspection, turbidity, sub-visible particle analysis, size exclusion chromatography and dynamic light scattering. The data showed that shaking and stirring resulted in different species of aggregates both qualitatively and quantitatively, where stirring was found more stressful than shaking on the IgG1 formulation. Mechanical stress testing performed at 5 and 25 degrees C only showed a difference on samples stressed by shaking and not by stirring. The headspace in the vials had great influence on the stability of the protein formulation when stressed by shaking. The presence of polysorbate had a protective effect on the antibody, however certain polysorbate concentrations even resulted in increased protein aggregation. An array of analytical methods was essential in order to cover the vast aggregate morphologies, which occurred during agitation.

  4. Staphylococcus aureus protein A activates TNFR1 signaling through conserved IgG binding domains.

    PubMed

    Gómez, Marisa I; O'Seaghdha, Maghnus; Magargee, Mariah; Foster, Timothy J; Prince, Alice S

    2006-07-21

    Staphylococcus aureus continues to be a major cause of infection in normal as well as immunocompromised hosts, and the increasing prevalence of highly virulent community-acquired methicillin-resistant strains is a public health concern. A highly expressed surface component of S. aureus, protein A (SpA), contributes to its success as a pathogen by both activating inflammation and by interfering with immune clearance. SpA is known to bind to IgG Fc, which impedes phagocytosis. SpA is also a potent activator of tumor necrosis factor alpha (TNF-alpha) receptor 1 (TNFR1) signaling, inducing both chemokine expression and TNF-converting enzyme-dependent soluble TNFR1 (sTNFR1) shedding, which has anti-inflammatory consequences, particularly in the lung. Using a collection of glutathione S-transferase fusions to the intact IgG binding region of SpA and to each of the individual binding domains, we found that the SpA IgG binding domains also mediate binding to human airway cells. TNFR1-dependent CXCL8 production could be elicited by any one of the individual SpA IgG binding domains as efficiently as by either the entire SpA or the intact IgG binding region. SpA induction of sTNFR1 shedding required the entire IgG binding region and tolerated fewer substitutions in residues known to interact with IgG. Each of the repeated domains of the IgG binding domain can affect multiple immune responses independently, activating inflammation through TNFR1 and thwarting opsonization by trapping IgG Fc domains, while the intact IgG binding region can limit further signaling through sTNFR1 shedding.

  5. 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

  6. 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

  7. Kinetic analysis of the multistep aggregation mechanism of monoclonal antibodies.

    PubMed

    Nicoud, Lucrèce; Arosio, Paolo; Sozo, Margaux; Yates, Andrew; Norrant, Edith; Morbidelli, Massimo

    2014-09-11

    We investigate by kinetic analysis the aggregation mechanism of two monoclonal antibodies belonging to the IgG1 and IgG2 subclass under thermal stress. For each IgG, we apply a combination of size exclusion chromatography and light scattering techniques to resolve the time evolution of the monomer, dimer, and trimer concentrations, as well as the average molecular weight and the average hydrodynamic radius of the aggregate distribution. By combining the detailed experimental characterization with a theoretical kinetic model based on population balance equations, we extract relevant information on the contribution of the individual elementary steps on the global aggregation process. The analysis shows that the two molecules follow different aggregation pathways under the same operating conditions. In particular, while the monomer depletion of the IgG1 is found to be rate-limited by monomeric conformational changes, bimolecular collision is identified as the rate-limiting step in the IgG2 aggregation process. The measurement of the microscopic rate constants by kinetic analysis allows the quantification of the protein-protein interaction potentials expressed in terms of the Fuchs stability ratio (W). It is found that the antibody solutions exhibit large W values, which are several orders of magnitude larger than the values computed in the frame of the DLVO theory. This indicates that, besides net electrostatic repulsion, additional effects delay the aggregation kinetics of the antibody solutions with respect to diffusion-limited conditions. These effects likely include the limited efficiency of the collision events due to the presence of a limited number of specific aggregation-prone patches on the heterogeneous protein surface, and the contribution of additional repulsive non-DLVO forces to the protein-protein interaction potential, such as hydration forces.

  8. Ectoine and hydroxyectoine inhibit thermal-induced aggregation and increase thermostability of recombinant human interferon Alfa2b.

    PubMed

    Salmannejad, Faranak; Nafissi-Varcheh, Nastaran

    2017-01-15

    This study is to investigate whether ectoines (ectoine and hydroxyectoine) can reduce aggregation of rhIFNα2b in aqueous solutions on thermal stress. The effect of thermal stress condition on the stability was therefore investigated using size exclusion-high performance liquid chromatography (SE-HPLC), different spectroscopic measurements, dynamic light scattering (DLS), electrophoresis, and differential scanning calorimetry (DSC). All experiments were performed in a sodium phosphate buffer system (100mM, pH7). The protein samples (100μg/ml) were incubated at 50°C for 14days in the absence or presence (1, 10, 20, and 100mM) of ectoines. In summary, thermal-induced aggregation was reduced in the presence of ectoines, regardless of the ectoines concentration in different periods of incubation time by analyzing with SE-HPLC and turbidity measurement. The inhibitory effect of ectoines on the aggregation was shown by other techniques used. The optimal ectoines concentration was 10mM for aggregation reduction, so samples containing of 10mM of ectoines were selected for further evaluation. Secondary structural and conformational stability increased in presence of ectoines as measured by far-UV circular dichroism and fluorescence spectroscopy, respectively. DSC showed slight increase in Tm of interferon in the presence of ectoines. Hydroxyectoine had superior protein-stabilizing properties than ectoine. In conclusion, this study demonstrates that ectoine and hydroxyectoine are highly effective excipients which can significantly reduce the thermal-induced aggregation of rhIFNα2b at low concentration.

  9. Aggregation and disaggregation kinetics of human blood platelets: Part I. Development and validation of a population balance method.

    PubMed Central

    Huang, P Y; Hellums, J D

    1993-01-01

    Hydrodynamic shear stress of sufficient intensity is known to cause platelet activation and aggregation and to alter the effects of biochemical platelet agonists and antagonists. In this work, a population balance equation (PBE) model is developed for analysis of platelet aggregation and disaggregation kinetics under the influence of a shear field. The model incorporates both aggregation and disaggregation by splitting and/or erosion mechanisms. This paper, the first of a series of three, deals with the formulation, simplification, and validation of the PBE and with the estimation of parameters involved in the PBE. These population parameters include collision efficiency, void fraction (related to the particle collision diameter), and the breakage rate coefficient. The platelet particle size distribution is determined experimentally, both initially and at some later times. The PBE can then be used to match satisfactorily the observed particle histograms, by appropriate choice of parameters of the model as functions of time, platelet size, and magnitude of physical or chemical stimuli. Besides providing information on adhesive forces and on the rates of aggregation and disaggregation, these parameters infer the physical properties of platelets and platelet aggregates. These properties are of potential value in increasing our understanding of the processes involved in thrombotic disease and/or therapy. A numerical procedure for solving the PBE is validated by application to simple cases for which analytical solutions are available. The model is applied to analysis of experiments, and parameter sensitivity studies are used to order the importance of the parameters and to reduce the complexity of the model. The simplified model is shown to give good agreement with experimental observations. PMID:8369441

  10. IgG4-related prostatitis progressed from localized IgG4-related lymphadenopathy

    PubMed Central

    Li, Dujuan; Kan, Yunzhen; Fu, Fangfang; Wang, Shuhuan; Shi, Ligang; Liu, Jie; Kong, Lingfei

    2015-01-01

    Immunoglobulin G4-related disease (IgG4-RD) is a recently described inflammatory disease involving multiple organs. Prostate involvement with IgG4-RD is very rare. In this report, we describe a case of IgG4-related prostatitis progressed from localized IgG4-related lymphadenopathy. This patient was present with urine retention symptoms. MRI and CT examination revealed the prostatic enlargement and the multiple lymphadenopathy. Serum IgG4 levels were elevated. Prostatic tissue samples resected both this time and less than 1 year earlier showed the same histological type of prostatitis with histopathologic and immunohistochemical findings characteristic of IgG4-RD. The right submandibular lymph nodes excised 2 years earlier were eventually proven to be follicular hyperplasia-type IgG4-related lymphadenopathy. This is the first case of IgG4-RD that began as localized IgG4-related lymphadenopathy and progressed into a systemic disease involving prostate and multiple lymph nodes. This patient showed a good response to steroid therapy. This leads us to advocate a novel pathogenesis of prostatitis, and a novel therapeutic approach against prostatitis. Pathologists and urologists should consider this disease entity in the patients with elevated serum IgG4 levels and the symptoms of prostatic hyperplasia to avoid ineffective medical or unnecessary surgical treatment. PMID:26617921

  11. IgG4-related prostatitis progressed from localized IgG4-related lymphadenopathy.

    PubMed

    Li, Dujuan; Kan, Yunzhen; Fu, Fangfang; Wang, Shuhuan; Shi, Ligang; Liu, Jie; Kong, Lingfei

    2015-01-01

    Immunoglobulin G4-related disease (IgG4-RD) is a recently described inflammatory disease involving multiple organs. Prostate involvement with IgG4-RD is very rare. In this report, we describe a case of IgG4-related prostatitis progressed from localized IgG4-related lymphadenopathy. This patient was present with urine retention symptoms. MRI and CT examination revealed the prostatic enlargement and the multiple lymphadenopathy. Serum IgG4 levels were elevated. Prostatic tissue samples resected both this time and less than 1 year earlier showed the same histological type of prostatitis with histopathologic and immunohistochemical findings characteristic of IgG4-RD. The right submandibular lymph nodes excised 2 years earlier were eventually proven to be follicular hyperplasia-type IgG4-related lymphadenopathy. This is the first case of IgG4-RD that began as localized IgG4-related lymphadenopathy and progressed into a systemic disease involving prostate and multiple lymph nodes. This patient showed a good response to steroid therapy. This leads us to advocate a novel pathogenesis of prostatitis, and a novel therapeutic approach against prostatitis. Pathologists and urologists should consider this disease entity in the patients with elevated serum IgG4 levels and the symptoms of prostatic hyperplasia to avoid ineffective medical or unnecessary surgical treatment.

  12. IgG4-related skin manifestations in patients with IgG4-related disease.

    PubMed

    Ikeda, Tetsuya; Oka, Masahiro; Shimizu, Hideki; Hatakeyama, Mayumi; Kanki, Haruhisa; Kunisada, Makoto; Tsuji, Goh; Morinobu, Akio; Kumagai, Shunichi; Azumi, Atsushi; Negi, Akira; Nishigori, Chikako

    2013-04-01

    We describe two cases of IgG4-related disease associated with skin manifestations with IgG4-positive plasma cells. The first patient was a 52-year-old woman with a 3-year history of IgG4-related sialadenitis who presented with pruritic, indurated erythematous lesions on the auricle, postauricular and submandibular regions and neck. A skin biopsy showed infiltration of IgG4-positive plasma cells in the subcutaneous tissue. The second patient was a 53-year-old woman with IgG4-related lesions in the ocular adnexal tissues and nasal cavity who presented with pruritic, indurated erythema on the cheek and submandibular region. Histopathological examination of a skin biopsy revealed a dense, patchy infiltrate comprised of lymphocytes, IgG4-positive plasma cells and eosinophils around blood vessels and sweat glands in the entire dermis and subcutis. The skin lesions in these cases were considered to be skin manifestations of IgG4-related disease. The findings of these two cases together with the three reported cases of IgG4-related disease with skin manifestations in the literature suggest that IgG4-related skin lesions may appear on the scalp, face, neck, auricle and postauricular regions during the course of IgG4-related disease.

  13. Reverse enzyme-linked immunosorbent assay using monoclonal antibodies against SAG1-related sequence, SAG2A, and p97 antigens from Toxoplasma gondii to detect specific immunoglobulin G (IgG), IgM, and IgA antibodies in human sera.

    PubMed

    Carvalho, Fernando R; Silva, Deise A O; Cunha-Júnior, Jair P; Souza, Maria A; Oliveira, Taísa C; Béla, Samantha R; Faria, Gabriele G; Lopes, Carolina S; Mineo, José R

    2008-08-01

    The present study aimed to evaluate the performance of three monoclonal antibodies (MAbs) in reverse enzyme-linked immunosorbent assays (ELISAs) for detecting immunoglobulin G (IgG), IgM, and IgA antibodies against Toxoplasma gondii in 175 serum samples from patients at different stages of T. gondii infection, as defined by both serological and clinical criteria, as follows: recent (n = 45), transient (n = 40), and chronic (n = 55) infection as well as seronegative subjects (n = 35). The results were compared with those obtained by indirect ELISA using soluble Toxoplasma total antigen (STAg). Our data demonstrated that MAb A3A4 recognizes a conformational epitope in SAG1-related-sequence (SRS) antigens, while A4D12 and 1B8 recognize linear epitopes defined as SAG2A surface antigen and p97 cytoplasmatic antigen, respectively. Reverse ELISA for IgG with A3A4 or A4D12 MAbs was highly correlated with indirect ELISA for anti-STAg IgG, whereas only A4D12 reverse ELISA showed high correlation with indirect ELISA for IgM and IgA isotypes. To our knowledge, this is the first report analyzing the performance of a reverse ELISA for simultaneous detection of IgG, IgM, and IgA isotypes active toward native SAG2A, SRS, and p97 molecules from STAg, using a panel of human sera from patients with recent and chronic toxoplasmosis. Thus, reverse ELISA based on the capture of native SAG2A and SRS antigens of STAg by MAbs could be an additional approach for strengthening the helpfulness of serological tests assessing the stage of infection, particularly in combination with highly sensitive and specific assays that are frequently used nowadays for diagnosis of toxoplasmosis during pregnancy or congenital infection in newborns.

  14. 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.

  15. IgG purification by bentonite-acrylamide-histidine microcomposite.

    PubMed

    Akkaya, Birnur

    2012-04-01

    In this work, a new microcomposite composed of bentonite, acrylamide and histidine, as a pseudospecific ligand, was synthesized by bulk polymerization. The aim of this study was to improve IgG adsorption capacity of bentonite by incorporating histidine. The surface areas of the bentonite and bentonite-acrylamide-histidine microcomposites were 33.4 and 1.42 m(2)/g, respectively. The amount of histidine was found to be 50 μmol/g bentonite via elemental analysis. Adsorption capacity was at the value of 100mg/g from aqueous solution while adsorption capacity was 108 mg/g from human plasma with a purity of 90%. IgG biomolecules were able to be adsorbed and desorbed five times by using the same microcomposites without significant loss in their adsorption capacity.

  16. Removal of sialic acid from the surface of human MCF-7 mammary cancer cells abolishes E-cadherin-dependent cell-cell adhesion in an aggregation assay.

    PubMed

    Deman, J J; Van Larebeke, N A; Bruyneel, E A; Bracke, M E; Vermeulen, S J; Vennekens, K M; Mareel, M M

    1995-09-01

    MCF-7 human breast cancer cells express E-cadherin and show, at least in some circumstances, E-cadherin-dependent cell-cell adhesion (Bracke et al., 1993). The MCF-7/AZ variant spontaneously displays E-cadherin-dependent fast aggregation; in the MCF-7/6 variant, E-cadherin appeared not to be spontaneously functional in the conditions of the fast aggregation assay, but function could be induced by incubation of the suspended cells in the presence of insulinlike growth factor I (IGF-I) (Bracke et al., 1993). E-cadherin from MCF-7 cells was shown to contain sialic acid. Treatment with neuraminidase was shown to remove this sialic acid, as well as most of the sialic acid present at the cell surface. Applied to MCF-7/AZ, and MCF-7/6 cells, pretreatment with neuraminidase abolished spontaneous as well as IGF-I induced, E-cadherin-dependent fast cell-cell adhesion of cells in suspension, as measured in the fast aggregation assay. Treatment with neuraminidase did not, however, inhibit the possibly different, but equally E-cadherin-mediated, process of cell-cell adhesion of MCF-7 cells on a flat plastic substrate as assessed by determining the percentage of cells remaining isolated (without contact with other cells) 24 h after plating.

  17. Igg Subclasses Targeting the Flagella of Salmonella enterica Serovar Typhimurium Can Mediate Phagocytosis and Bacterial Killing.

    PubMed

    Goh, Yun Shan; Armour, Kathryn L; Clark, Michael R; Grant, Andrew J; Mastroeni, Pietro

    2016-05-30

    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.

  18. 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

  19. 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.

  20. 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

  1. Effects of polyethylene glycol and hydroxyethyl starch in University of Wisconsin preservation solution on human red blood cell aggregation and viscosity.

    PubMed

    Mosbah, I B; Franco-Gou, R; Abdennebi, H B; Hernandez, R; Escolar, G; Saidane, D; Rosello-Catafau, J; Peralta, C

    2006-06-01

    University of Wisconsin (UW) preservation solution is considered an effective flush and cold storage liquid. However, recent studies have provided evidence of the hyperaggregating effect on human red blood cells (RBC) of hydroxyethyl starch (HES), one of the components of the UW solution. In contrast, preservation solutions containing polyethylene glycol (PEG) have been found to be effective for organ preservation. The aim of this study was to compare the effects of HES (50 g/L); PEG 20 kDa (50 and 30 g/L), and PEG35 kDa (1.05 g/L) added to UW on the rheologic parameters of human RBC at 4 degrees C. Sedimentation rate was measured by the Westergren procedure and blood viscosity evaluated at high shear rates using a cone/plate viscometer. Alterations in RBC morphology and aggregation were evaluated by light microscopy. RBC sedimentation and viscosity were not affected by the inversion of Na+ and K+ concentrations in UW, but were increased by HES. PEGs appeared to reduce RBC deformability with concomitant inhibition of RBC aggregation. These results were consistent with reduced viscosity for PEG-containing solutions. In conclusion, the use of PEG did not change the physiologic function of human RBCs and thus may be an alternative to HES in UW liquids.

  2. Inferring Aggregated Functional Traits from Metagenomic Data Using Constrained Non-negative Matrix Factorization: Application to Fiber Degradation in the Human Gut Microbiota

    PubMed Central

    Raguideau, Sébastien; Plancade, Sandra; Pons, Nicolas; Leclerc, Marion

    2016-01-01

    Whole Genome Shotgun (WGS) metagenomics is increasingly used to study the structure and functions of complex microbial ecosystems, both from the taxonomic and functional point of view. Gene inventories of otherwise uncultured microbial communities make the direct functional profiling of microbial communities possible. The concept of community aggregated trait has been adapted from environmental and plant functional ecology to the framework of microbial ecology. Community aggregated traits are quantified from WGS data by computing the abundance of relevant marker genes. They can be used to study key processes at the ecosystem level and correlate environmental factors and ecosystem functions. In this paper we propose a novel model based approach to infer combinations of aggregated traits characterizing specific ecosystemic metabolic processes. We formulate a model of these Combined Aggregated Functional Traits (CAFTs) accounting for a hierarchical structure of genes, which are associated on microbial genomes, further linked at the ecosystem level by complex co-occurrences or interactions. The model is completed with constraints specifically designed to exploit available genomic information, in order to favor biologically relevant CAFTs. The CAFTs structure, as well as their intensity in the ecosystem, is obtained by solving a constrained Non-negative Matrix Factorization (NMF) problem. We developed a multicriteria selection procedure for the number of CAFTs. We illustrated our method on the modelling of ecosystemic functional traits of fiber degradation by the human gut microbiota. We used 1408 samples of gene abundances from several high-throughput sequencing projects and found that four CAFTs only were needed to represent the fiber degradation potential. This data reduction highlighted biologically consistent functional patterns while providing a high quality preservation of the original data. Our method is generic and can be applied to other metabolic processes in

  3. Proteomic Investigation on Grp94-IgG Complexes Circulating in Plasma of Type 1 Diabetic Subjects

    PubMed Central

    Roveri, Antonella; Zaccarin, Mattia; Pagetta, Andrea; Tramentozzi, Elisa; Finotti, Paola

    2015-01-01

    The glucose-regulated protein94 (Grp94) has been found in complexes with IgG in plasma of Type 1 (T1) diabetic subjects; however, the pathogenetic meaning of Grp94-IgG complexes has not yet been elucidated. To shed light on the nature and structure of these complexes in vivo, we conducted a proteomic analysis on plasma of both T1 diabetic subjects and healthy control subjects. IgG purified from plasma was submitted to 2D PAGE followed by Western blotting and mass analysis. Grp94 was detected in plasma of all diabetic but not control subjects and found linked with its N-terminus to the IgG heavy chain. Mass analysis of heavy chain of IgG that binds Grp94 also in vitro, forming stable complexes with characteristics similar to those of native ones, permitted identifying CH2 and CH3 regions as those involved in binding Grp94. At the electron microscopy, IgG from diabetic plasma appeared as fibrils of various lengthes and dimensions, suggestive of elevated aggregating tendency conferred to IgG by Grp94. The nonimmune nature of complexes turned out to be responsible for the particular stability and structure adopted by complexes in plasma of diabetic subjects. Results are of relevance to understanding the pathogenetic mechanisms underlying diabetes and its complications. PMID:26167512

  4. Subclass distribution of IgG antibodies to the rat oesophagus stratum corneum (so-called anti-keratin antibodies) in rheumatoid arthritis.

    PubMed Central

    Vincent, C; Serre, G; Basile, J P; Lestra, H C; Girbal, E; Sebbag, M; Soleilhavoup, J P

    1990-01-01

    Serum IgG, labelling the stratum corneum of the rat oesophagus epithelium, so-called anti-keratin antibodies (AKA) constitute the most specific marker for the diagnosis of rheumatoid arthritis. In this study, we investigated 31 IgG AKA-positive rheumatoid sera and 21 control sera from patients with non-rheumatoid inflammatory rheumatic diseases. The serum level of IgG1,2,3 and 4 was determined by radial immunodiffusion and the subclass distribution of IgG AKA by a three-step semi-quantitative immunofluorescence assay using standard monoclonal antibodies specific for each of the four human IgG subclasses. In the rheumatoid sera, the serum level of IgG1 was found to be significantly increased and the level of IgG2 significantly decreased with regard to the control sera, while the levels of IgG3 and 4 as well as total IgG were in the normal range. IgG1,2,3, and 4 AKA were detected in 27 (87%), 6 (19%), 4 (13%) and 11 (35%) of the 31 rheumatoid sera, respectively, and were found to be independent of the clinical and biological indices of the disease. In spite of inter-individual heterogeneity, two predominant profiles were distinguished: IgG1 (alone) and IgG(1 + 4), which together represented 18 sera (58%). The large predominance of IgG1 AKA and the quasi-absence of IgG2 AKA suggest that the recognized antigen may be partly comprised of protein. Moreover, the high frequency of occurrence of IgG4 AKA might result from chronic exposure to the eliciting antigen, which could be a genuine autoantigen since we demonstrated that it is also present in the stratum corneum of human epidermis. Images Fig. 1 PMID:1696185

  5. Targeting human prostate cancer with 111In-labeled D2B IgG, F(ab')2 and Fab fragments in nude mice with PSMA-expressing xenografts.

    PubMed

    Lütje, Susanne; van Rij, Catharina M; Franssen, Gerben M; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J; Colombatti, Marco; Boerman, Otto C

    2015-01-01

    D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing subcutaneous prostate cancer xenografts. The optimal time point for imaging was determined in biodistribution and microSPECT imaging studies with (111)In-D2B IgG, (111)In-capromab pendetide, (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments in mice with PSMA-expressing LNCaP and PSMA-negative PC3 tumors at several time points after injection. All (111)In-labeled antibody formats specifically accumulated in the LNCaP tumors, with highest uptake of (111)In-D2B IgG and (111)In-capromab pendetide at 168 h p.i. (94.8 ± 19.2% injected dose per gram (ID/g) and 16.7 ± 2.2% ID/g, respectively), whereas uptake of (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments peaked at 24 h p.i. (12.1 ± 3.0% ID/g and 15.1 ± 2.9% ID/g, respectively). Maximum LNCaP tumor-to-blood ratios were 13.0 ± 2.3 (168 h p.i.), 6.2 ± 0.7 (24 h p.i.), 23.0 ± 4.0 (24 h p.i.) and 4.5 ± 0.6 (168 h p.i.) for (111)In-D2B IgG, (111)In-F(ab')2, (111)In-Fab and (111)In-capromab pendetide, respectively. LNCaP tumors were clearly visualized with microSPECT with all antibody formats. This study demonstrates the feasibility of D2B IgG, F(ab')2 and Fab fragments for targeting PSMA-expressing prostate cancer xenografts.

  6. IgG subclasses and DNA detection of HHV-6 and HHV-7 in healthy individuals.

    PubMed

    Biganzoli, Patricia; Ferreyra, Leonardo; Sicilia, Paola; Carabajal, Claudia; Frattari, Susana; Littvik, Ana; Nates, Silvia; Pavan, Jorge

    2010-10-01

    Human herpesvirus 6 (HHV-6) and 7 (HHV-7) are common opportunistic agents in immunocompromised hosts, although infection with HHV-6 and HHV-7 can also be observed in immunocompetent hosts. Despite similar biology and epidemiology, this study evaluated differences in the IgG subclass distribution associated with HHV-6 and HHV-7 in seropositive, healthy persons. The identified subclasses were also compared with the detection of HHV-6 and HHV-7 DNA. For these assays, sera, plasma, and saliva samples were obtained from 40 healthy blood donors in Argentina who were seropositive for both HHV-6 and HHV-7. HHV-6 and HHV-7 DNA were detected in saliva and plasma samples using nested PCR, and specific IgG subclasses were determined using immunofluorescent assays of sera samples. HHV-7 DNA was detected in 90% of all plasma samples and in 100% of saliva samples. In contrast, HHV-6 DNA was not detected in any of the plasma samples, and it was detected in only 6 of 40 saliva samples. Determination of IgG subclass distributions showed that HHV-6 was restricted to IgG1, whereas HHV-7 IgG subclasses included two groups, one restricted only to IgG1 and the other to IgG1 and IgG3. These results demonstrate the differences between HHV-6 and HHV-7 DNA range detection in saliva and plasma samples, as well as the IgG subclass patterns for each virus type, in healthy persons in Argentina.

  7. Naturally Acquired Human Immunity to Pneumococcus Is Dependent on Antibody to Protein Antigens

    PubMed Central

    Reglinski, Mark; Jose, Ricardo J.; Marshall, Helina; de Vogel, Corné; Gordon, Stephen; Petersen, Fernanda C.; Baxendale, Helen

    2017-01-01

    Naturally acquired immunity against invasive pneumococcal disease (IPD) is thought to be dependent on anti-capsular antibody. However nasopharyngeal colonisation by Streptococcus pneumoniae also induces antibody to protein antigens that could be protective. We have used human intravenous immunoglobulin preparation (IVIG), representing natural IgG responses to S. pneumoniae, to identify the classes of antigens that are functionally relevant for immunity to IPD. IgG in IVIG recognised capsular antigen and multiple S. pneumoniae protein antigens, with highly conserved patterns between different geographical sources of pooled human IgG. Incubation of S. pneumoniae in IVIG resulted in IgG binding to the bacteria, formation of bacterial aggregates, and enhanced phagocytosis even for unencapsulated S. pneumoniae strains, demonstrating the capsule was unlikely to be the dominant protective antigen. IgG binding to S. pneumoniae incubated in IVIG was reduced after partial chemical or genetic removal of bacterial surface proteins, and increased against a Streptococcus mitis strain expressing the S. pneumoniae protein PspC. In contrast, depletion of type-specific capsular antibody from IVIG did not affect IgG binding, opsonophagocytosis, or protection by passive vaccination against IPD in murine models. These results demonstrate that naturally acquired protection against IPD largely depends on antibody to protein antigens rather than the capsule. PMID:28135322

  8. 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).

  9. The Emerging Importance of IgG Fab Glycosylation in Immunity.

    PubMed

    van de Bovenkamp, Fleur S; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

    2016-02-15

    Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity.

  10. IgG4 related sclerosing mastitis: expanding the morphological spectrum of IgG4 related diseases.

    PubMed

    Chougule, Abhijit; Bal, Amanjit; Das, Ashim; Singh, Gurpreet

    2015-01-01

    IgG4 related disease (IgG4RD) is a recently recognised condition characterised by mass forming lesions associated with storiform fibrosis, obliterative phlebitis, lymphoplasmacytic infiltrate rich in IgG4 positive plasma cells and elevated serum IgG4 levels. Although rare, mammary involvement has been reported as IgG4 related sclerosing mastitis, the morphological counterpart of a growing family of IgG4 related diseases. A total of 17 cases belonging to mass forming benign inflammatory breast lesions such as plasma cell mastitis, granulomatous lobular mastitis, non-specific mastitis and inflammatory pseudotumour were investigated as a possible member of IgG4 related sclerosing mastitis. Clinical, radiological, histopathological and immunohistochemistry findings were noted in all cases. Cases diagnosed as inflammatory pseudotumour showed all the histopathological features of IgG4RD along with increased number of IgG4 positive plasma cells and IgG4/IgG ratio >40%. However, only a few IgG4 positive cells were seen in plasma cell mastitis, granulomatous lobular mastitis and non-specific mastitis cases. These cases also did not fulfill the morphological criteria for the diagnosis of IgG4 related diseases. IgG4RD should be excluded in plasma cell rich lesions diagnosed on core biopsies by IgG4 immunostaining. This can avoid unnecessary surgery as IgG4 related diseases respond to simple and effective steroid treatment.

  11. IgG Subclasses and Isotypes of VH4-34 Encoded Antibodies.

    PubMed

    Bhat, Neelima M; Kshirsagar, Mihir A; Bieber, Marcia M; Teng, Nelson N H

    2015-01-01

    VH4-34 gene encoded autoantibodies are elevated in systemic lupus erythematosus (SLE) and in other diseases associated with B-cell hyperproliferation/dysfunction. One of the autoantigens recognized by VH4-34-encoded antibodies are branched/linear poly N-acetyl lactosamine chains. Since the anti-carbohydrate response in humans is dominated by the IgG2 subclass, here we tested whether VH4-34 encoded IgG showed similar subclass segregation. Serum samples from SLE, infectious mononucleosis, nasopharyngeal carcinoma and hepatitis-C were analyzed. Levels of VH4-34-encoded IgM and IgA isotypes were also tested. VH4-34-IgM and IgA were elevated in all four clinical conditions. VH4-34-IgG was detected in the IgG1 and IgG3 subclass but not in the IgG2 and IgG4 subclass. Interestingly, VH4-34-IgG3 was also detected in serum samples of normal healthy adults. These observations are discussed in context of the VH4-34 gene regulation. VH4-34 repertoire development is of interest since it is the only human VH gene profoundly overrepresented in the naïve repertoire but counter-selected for antibody secretion. VH4-34 B-cell could thus become a unique tool to inspect germinal center independent/dependent pathways of subclass and isotype-specific antibody secretion.

  12. Serum IgG subclasses in autoimmune diseases.

    PubMed

    Zhang, Haoze; Li, Ping; Wu, Di; Xu, Dong; Hou, Yong; Wang, Qian; Li, Mengtao; Li, Yongzhe; Zeng, Xiaofeng; Zhang, Fengchun; Shi, Qun

    2015-01-01

    To characterize serum IgG subclass levels in several autoimmune diseases, including primary Sjogren syndrome (pSS), systemic sclerosis (SSc), systemic lupus erythematosus (SLE), and primary biliary cirrhosis (PBC). We aimed to analyze serum IgG subclass distribution and to test whether serum IgG4 levels are elevated in these diseases. Serum IgG subclass levels from 102 pSS, 102 SSc, 100 SLE, and 59 PBC patients, as well as 40 healthy controls (HCs), were measured using the immunonephelometric assay. The distribution of IgG subclasses among these autoimmune diseases was analyzed. In this cross-sectional study, serum IgG1 (IgG1/IgG) and/or IgG3 (IgG3/IgG) were significantly increased, compared with those in HCs. Only 6.34% of patients had levels of serum IgG4 >135 mg/dL. There were no significant differences in the frequency of elevated serum IgG4 levels between patients and HC. In pSS, serum IgG1 levels were much higher than those in other disease groups, whereas serum IgG2 and IgG3 levels were most prominently increased in PBC. A strikingly different serum IgG subclass distribution was detected in patients with autoimmune diseases compared with HCs. Serum IgG subclass levels also showed distinct characteristics among different autoimmune diseases. Serum IgG4 levels in these patients were lower or not much higher than those in HCs, which differed from IgG4-related diseases.

  13. IgG4-related kidney disease--A review.

    PubMed

    Pradhan, Dinesh; Pattnaik, Niharika; Silowash, Russell; Mohanty, Sambit Kumar

    2015-10-01

    IgG4-related disease (IgG4-RD) is a recently recognized systemic autoimmune disorder characterized by high levels of serum IgG4 and dense infiltration of IgG4-positive plasma cells in multiple organs. The condition was first described as a disease of the pancreas, and has since been recognized in various organ systems including the kidneys. IgG4 related kidney disease (IgG4-RKD) signifies any form of renal involvement by IgG4-RD. The most common renal involvement by IgG4-RD is tubulointerstitial nephritis. Glomerular disease, in particular membranous glomerulonephritis, may also be seen. Other co-existent glomerular diseases such as IgA nephropathy, membranoproliferative glomerulonephritis, and mesangioproliferative immune complex glomerulonephritis may be identified. IgG4-related plasma cell arteritis has also been noted in the kidney. As with IgG4-RD in general, IgG4 related kidney disease (IgG4-RKD) usually occurs in middle-aged to elderly men. Common findings in IgG4-RKD are plasma cell-rich interstitial inflammatory infiltrate either in a focal or diffuse pattern with increased IgG4+ plasma cells, expansile swirling interstitial fibrosis, high levels of serum IgG and IgG4, hypocomplementemia, high serum IgE levels and/or peripheral blood eosinophilia. By immunofluorescence, most of the cases show IgG4 dominant tubular basement membrane immune complex deposits. Similar to IgG4-RD, IgG4-RKD often shows a rapid response to steroid therapy. In this review, we discuss the current knowledge on IgG4-RKD and its clinical relevance.

  14. ELISA detection of specific functional antibodies in human serum to Escherichia coli, tetanus toxoid, and diphtheria-tetanus toxoids: normal values for IgG, IgA, and IgM.

    PubMed

    Moen, R C; Oemichen, S L; Kiggens, A J; Hong, R

    1986-01-01

    An inexpensive, easily performed enzyme-linked immunosorbent assay (ELISA) was developed to measure specific IgG, IgA, and IgM antibodies to the common antigens Escherichia coli, diphtheria-tetanus toxoid, and tetanus toxoid. Normal values were established. Classical antibody deficiency disease states were confirmed and delineated by these assays. Additionally, several instances were discovered when functional antibody levels were abnormal when the serum immunoglobulin levels were normal. The use of ELISA assays for antibodies to common antigens provides a useful technique to measure and monitor isotype responses of the humoral immune system.

  15. Engineered antibody CH2 domains binding to nucleolin: Isolation, characterization and improvement of aggregation.

    PubMed

    Li, De-Zhi; Gong, Rui; Zheng, Jun; Chen, Xi-Hai; Dimitrov, Dimiter S; Zhao, Qi

    2017-02-12

    Smaller recombinant antibody fragments are now emerging as alternatives of conventional antibodies. Especially, immunoglobulin (Ig) constant CH2 domain and engineered CH2 with improved stability are promising as scaffolds for selection of specific binders to various antigens. We constructed a yeast display library based on an engineered human IgG1 CH2 scaffold with diversified loop regions. A group of CH2 binders were isolated from this yeast display library by panning against nucleolin, which is a tumor-associated antigen involved in cell proliferation, tumor cell growth and angiogenesis. Out of 20 mutants, we selected 3 clones exhibiting relatively high affinities to nucleolin on yeasts. However, recombinant CH2 mutants aggregated when they were expressed. To find the mechanism of the aggregation, we employed computational prediction approaches through structural homology models of CH2 binders. The analysis of potential aggregation prone regions (APRs) and solvent accessible surface areas (ASAs) indicated two hydrophobic residues, Val264 and Leu309, in the β-sheet, in which replacement of both charged residues led to significantly decrease of the protein aggregation. The newly identified CH2 binders could be improved to use as candidate therapeutics or research reagents in the future.

  16. IgG4 autoantibodies against muscle-specific kinase undergo Fab-arm exchange in myasthenia gravis patients.

    PubMed

    Koneczny, Inga; Stevens, Jo A A; De Rosa, Anna; Huda, Saif; Huijbers, Maartje G; Saxena, Abhishek; Maestri, Michelangelo; Lazaridis, Konstantinos; Zisimopoulou, Paraskevi; Tzartos, Socrates; Verschuuren, Jan; van der Maarel, Silvère M; van Damme, Philip; De Baets, Marc H; Molenaar, Peter C; Vincent, Angela; Ricciardi, Roberta; Martinez-Martinez, Pilar; Losen, Mario

    2017-02-01

    Autoimmunity mediated by IgG4 subclass autoantibodies is an expanding field of research. Due to their structural characteristics a key feature of IgG4 antibodies is the ability to exchange Fab-arms with other, unrelated, IgG4 molecules, making the IgG4 molecule potentially monovalent for the specific antigen. However, whether those disease-associated antigen-specific IgG4 are mono- or divalent for their antigens is unknown. Myasthenia gravis (MG) with antibodies to muscle specific kinase (MuSK-MG) is a well-recognized disease in which the predominant pathogenic IgG4 antibody binds to extracellular epitopes on MuSK at the neuromuscular junction; this inhibits a pathway that clusters the acetylcholine (neurotransmitter) receptors and leads to failure of neuromuscular transmission. In vitro Fab-arm exchange-inducing conditions were applied to MuSK antibodies in sera, purified IgG4 and IgG1-3 sub-fractions. Solid-phase cross-linking assays were established to determine the extent of pre-existing and inducible Fab-arm exchange. Functional effects of the resulting populations of IgG4 antibodies were determined by measuring inhibition of agrin-induced AChR clustering in C2C12 cells. To confirm the results, κ/κ, λ/λ and hybrid κ/λ IgG4s were isolated and tested for MuSK antibodies. At least fifty percent of patients had IgG4, but not IgG1-3, MuSK antibodies that could undergo Fab-arm exchange in vitro under reducing conditions. Also MuSK antibodies were found in vivo that were divalent (monospecific for MuSK). Fab-arm exchange with normal human IgG4 did not prevent the inhibitory effect of serum derived MuSK antibodies on AChR clustering in C2C12 mouse myotubes. The results suggest that a considerable proportion of MuSK IgG4 could already be Fab-arm exchanged in vivo. This was confirmed by isolating endogenous IgG4 MuSK antibodies containing both κ and λ light chains, i.e. hybrid IgG4 molecules. These new findings demonstrate that Fab-arm exchanged antibodies

  17. Prevalence of IgG Antibodies against Human Papillomavirus (HPV) type 6, 11, 16, and 18 Virus-Like Particles in Women of Childbearing Age in Port Harcourt, Nigeria.

    PubMed

    Okonko, I O; Ofoedu, V

    2015-01-01

    Most HPV prevalence studies have been carried out in high-resource countries with few studies focused on low-resource regions where highest HPV prevalence in the world occurs. This study reports on prevalence of IgG antibodies against HPVs among women of childbearing age in Port Harcourt, Nigeria. One hundred and eighty-two consented women (age-range 19-45 years) were consecutively recruited. Demographic/behavioral data and 5 mL blood samples were collected from each woman. Plasma of each sample was assayed for HPV-6/11/16/18 virus-like particles using a HPV IgG ELISA kit. The overall anti-HPV prevalence was 4.9% while 7.7% with itching/wound in the private part tested positive. Most (88.9%) of the seropositive women were sexually active. Group-specific seropositivity was low (0.0-10.0%). It also showed that all the 9(100.0%) who tested positive to the HPV responded "yes" to no information on the source of HPV information. Being younger, married, high educational level, religion, and lack of information on HPV were the main correlates of HPV positivity among these women. None was vaccinated and would have been naturally exposed to at least one of HPV-6/11/16/18. With 4.9% seropositivity and lack of information regarding HPV among these women, this study recommends a statewide enlightenment campaign and vaccination.

  18. IgG1 and IgG4 Antibody Responses to the Anopheles gambiae Salivary Protein gSG6 in the Sympatric Ethnic Groups Mossi and Fulani in a Malaria Hyperhendemic Area of Burkina Faso

    PubMed Central

    Lombardo, Fabrizio; Mangano, Valentina; Sirima, Sodiomon Bienvenu; Nèbiè, Issa; Fiorentino, Gabriella; Troye-Blomberg, Marita; Modiano, David; Arcà, Bruno

    2014-01-01

    Human antibody response to the Anopheles gambiae salivary protein gSG6 has recently emerged as a potentially useful tool for malaria epidemiological studies and for the evaluation of vector control interventions. However, the current understanding of the host immune response to mosquito salivary proteins and of the possible crosstalk with early response to Plasmodium parasites is still very limited. We report here the analysis of IgG1 and IgG4 subclasses among anti-gSG6 IgG responders belonging to Mossi and Fulani from Burkina Faso, two ethnic groups which are known for their differential humoral response to parasite antigens and for their different susceptibility to malaria. The IgG1 antibody response against the gSG6 protein was comparable in the two groups. On the contrary, IgG4 titers were significantly higher in the Fulani where, in addition, anti-gSG6 IgG4 antibodies appeared in younger children and the ratio IgG4/IgG1 stayed relatively stable throughout adulthood. Both gSG6-specific IgG1 and IgG4 antibodies showed a tendency to decrease with age whereas, as expected, the IgG response to the Plasmodium circumsporozoite protein (CSP) exhibited an opposite trend in the same individuals. These observations are in line with the idea that the An. gambiae gSG6 salivary protein induces immune tolerance, especially after intense and prolonged exposure as is the case for the area under study, suggesting that gSG6 may trigger in exposed individuals a Th2-oriented immune response. PMID:24760038

  19. 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.

  20. 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.

  1. Oligomeric TTR V30M aggregates compromise cell viability, erythropoietin gene expression and promoter activity in the human hepatoma cell line Hep3B.

    PubMed

    Moreira, Luciana; Beirão, João Melo; Beirão, Idalina; Pinho e Costa, Paulo

    2015-01-01

    Familial amyloidotic polyneuropathy, ATTRV30M (p. TTRV50M) amyloidosis, is a neurodegenerative disease characterized by systemic extracellular amyloid deposition of a mutant transthyretin, TTR V30M. Anemia, with low erythropoietin (EPO) levels and spared kidney function, affects about 25% of symptomatic patients, suggesting a blockage of EPO-producing cells. Early non-fibrillar TTR aggregates are highly cytotoxic, inducing oxidative stress, the expression of apoptosis-related molecules and secretion of pro-inflammatory cytokines, factors capable of inhibiting EPO production. Low EPO levels in these patients are not related to renal amyloid deposition or the presence of circulating TTR V30M. However, the role of early non-fibrillar TTR aggregates remains unexplored. We used the EPO producing Hep3B human hepatoma cell line to study the effect of TTR oligomeric aggregates on EPO expression. Hep3B cells were incubated with soluble and oligomeric TTR V30M, and cell proliferation as well as caspase 3/7 activation was evaluated. Relative quantification of EPO mRNA transcripts was performed by real-time PCR. Significant reductions in cell viability (13 ± 7.3%) and activation of caspases 3/7 were seen after 24 h in the presence of oligomeric TTR V30M. Also, EPO expression was significantly reduced (50 ± 2.8%), in normoxic conditions. A reporter assay was constructed with a PCR fragment of the EPO promoter linked to the luciferase gene to evaluate the role of transcription factors targeting the promoter. A significant reduction of EPO promoter activity (53 ± 6.5%) was observed in transfected cells exposed to TTR oligomers. Our results show that oligomeric TTR V30M reduces EPO expression, at least in part through inhibition of promoter activity.

  2. [IgG4-related disease].

    PubMed

    González-Moreno, Juan; Losada López, Inés; Ortego Centeno, Norberto

    2015-12-21

    IgG4-related disease is a recently described clinicopathological entity showing a wide spectrum of clinical manifestations that share a common pathology. Its most characteristic feature is the formation of inflammatory tumors in different organs, which makes differentiation mainly with neoplastic diseases fundamental. The inflammatory process is typically comprised of IgG4 lymphoplasmacytic cells. The pathophysiological role of the immunoglobulin is not clear. The treatment of choice is corticosteroids. This article aims to summarize the main features of the disease.

  3. Embryo-fetal distribution of a biopharmaceutical IgG2 during rat organogenesis.

    PubMed

    Bowman, C J; King, L E; Stedman, D B

    2012-08-01

    Embryo-fetal biodistribution of a maternally administered humanized IgG2 in rats was evaluated by enzyme-linked immunosorbent assay in dose response and time course studies. Fetal and maternal plasma IgG2 levels increased with dose from 10 to 300mg/kg but fetal:maternal ratio decreased with increasing dose. Plasma IgG2 levels decreased in fetal rat with increasing time post-dose but more slowly than maternal levels. This difference in post-dose kinetics resulted in an increased fetal:maternal ratio with increasing days since last dose. Lastly, IgG2 in embryo-fetal tissue was detected at very low levels on gestation day (GD) 10-12 and levels increased over 100-fold by GD 17. The profile of increasing IgG2 levels as gestation progressed continued in extra-embryonic fluid (GD 12-19) until the end of gestation in fetal plasma (GD 19-21). Based on the current study, there is a potential for direct effects on rat embryo-fetal development following maternal administration of a biopharmaceutical IgG2.

  4. Reverse single radial immunodiffusion for estimation of titre of anti IgG antibody.

    PubMed

    Thakar, Y S; Kulkarni, C; Pande, S; Dhanvijay, A G; Shrikhande, A V; Saoji, A M

    1993-05-01

    Reverse Single Radial Immunodiffusion (SRID) for estimating titre of anti IgG antisera is reported. Unlike the conventional radial immunodiffusion, the antigen (IgG) is held immobile in the gel while the antibody (Anti IgG) diffuses radially from the well (7 microliters) and the diameter of the resulting immuneprecipitates after immunodiffusion at 4 degrees C for 24 hr, represents a linear correlation with the antibody titre. The procedure was standardised by an extensive trial and error employing different concentrations of human IgG in the gel (60-240 micrograms) against varying dilutions of the standard antibody (titre: 3.8 mg/ml). The best results were obtained at 80 micrograms of IgG in the gel. The locally raised rabbit anti IgG antisera displayed a distinctive titre pattern under optimised conditions. Technical reproducibility, high-sensitivity threshold (0.25 mg/ml), simultaneous visual scrutiny of several antibody batches at a glance and ability to assess the shelf life of the stored antisera are its distinct assets.

  5. IgG dimers in multidonor-derived immunoglobulins: aspects of generation and function.

    PubMed

    Gronski, P

    2006-01-01

    Immunoglobulin G (IgG) concentrates for therapeutic purposes, like passive immunotherapy, supplementation in inherited or acquired deficiencies or immunomodulation, are prepared from multidonor-derived plasma pools. They usually contain varying amounts of dimeric IgG. The essential factor influencing dimer formation is the pool size; in addition, molecular properties of IgG and a variety of production process- and formulation-specific parameters are important. Numerous experimental findings suggest that dimers are predominantly generated by interactions of idiotypic and anti-idiotypic antibodies (Ids, anti-Ids). Ab-inherent crossreactivity, frequency distribution of both the affinities for particular Id-anti-Id interactions and the corresponding dimer concentrations still have to be elucidated. All these parameters influencing molecular features and functional activity of IgG dimers hamper the assay-dependent measurement of biological efficacy and correlation of total IgG content. A more detailed understanding may help to better control the dual nature of dimer-dependent biological activity comprising both undesirable (e.g., hypotension) and desirable effects of dimeric IgG (blockade of the reticuloendothelial system, RES, in immune thrombocytopenic purpura, ITP). These effects are detectable in in vitro and in vivo models and are thought to be of relevance for humans.

  6. IgG4-related kidney disease – an update

    PubMed Central

    Kawano, Mitsuhiro; Saeki, Takako

    2015-01-01

    Purpose of review IgG4-related disease (IgG4-RD) is a recently recognized systemic inflammatory disorder that can affect most organs/tissues such as sarcoidosis. The kidney is a frequently affected organ with tubulointerstitial nephritis (TIN), the representative lesion of IgG4-RD. This review focuses on the latest knowledge of IgG4-related kidney disease (IgG4-RKD). Recent findings A wide range of renal manifestations of IgG4-RD, that is TIN, membranous glomerulonephritis (MGN) and other glomerular lesions, and pyelitis, are collectively referred to as IgG4-RKD. Clinically, decreased renal function, or characteristic imaging findings such as multiple low-density lesions on contrast-enhanced computed tomography or diffuse thickening of the renal pelvic wall, are typical presenting features. Although a rapid response to corticosteroid therapy is a very important feature of IgG4-TIN, in cases in which renal function is moderately to severely decreased before therapy, only partial recovery of renal function is obtained. Summary TIN with characteristic imaging findings is a typical manifestation of IgG4-RKD in the interstitium, while MGN is a representative manifestation of the glomerular lesions. Although IgG4 is a central feature of IgG4-RD, the recent discovery of IgG4-negative IgG4-RD raises questions about the causative role of the IgG4 molecule in this context. PMID:25594543

  7. ITC-derived binding affinity may be biased due to titrant (nano)-aggregation. Binding of halogenated benzotriazoles to the catalytic domain of human protein kinase CK2.

    PubMed

    Winiewska, Maria; Bugajska, Ewa; Poznański, Jarosław

    2017-01-01

    The binding of four bromobenzotriazoles to the catalytic subunit of human protein kinase CK2 was assessed by two complementary methods: Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC). New algorithm proposed for the global analysis of MST pseudo-titration data enabled reliable determination of binding affinities for two distinct sites, a relatively strong one with the Kd of the order of 100 nM and a substantially weaker one (Kd > 1 μM). The affinities for the strong binding site determined for the same protein-ligand systems using ITC were in most cases approximately 10-fold underestimated. The discrepancy was assigned directly to the kinetics of ligand nano-aggregates decay occurring upon injection of the concentrated ligand solution to the protein sample. The binding affinities determined in the reverse ITC experiment, in which ligands were titrated with a concentrated protein solution, agreed with the MST-derived data. Our analysis suggests that some ITC-derived Kd values, routinely reported together with PDB structures of protein-ligand complexes, may be biased due to the uncontrolled ligand (nano)-aggregation, which may occur even substantially below the solubility limit.

  8. 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

  9. A Spectroscopic Study of the Aggregation State of the Human Antimicrobial Peptide LL-37 in Bacterial versus Host Cell Model Membranes.

    PubMed

    Bonucci, Alessio; Caldaroni, Elena; Balducci, Enrico; Pogni, Rebecca

    2015-11-17

    The LL-37 antimicrobial peptide is the only cathelicidin peptide found in humans that has antimicrobial and immunomodulatory properties. Because it exerts also chemotactic and angiogenetic activity, LL-37 is involved in promoting wound healing, reducing inflammation, and strengthening the host immune response. The key to the effectiveness of antimicrobial peptides (AMPs) lies in the different compositions of bacterial versus host cell membranes. In this context, antimicrobial peptide LL-37 and two variants were studied in the presence of model membranes with different lipid compositions and charges. The investigation was performed using an experimental strategy that combines the site-directed spin labeling-electron paramagnetic resonance technique with circular dichroism and fluorescence emission spectroscopies. LL-37 interacts with negatively charged membranes forming a stable aggregate, which can likely produce toroidal pores until the amount of bound peptide exceeds a critical concentration. At the same time, we have clearly detected an aggregate with a higher oligomeric degree for interaction of LL-37 with neutral membranes. These data confirm the absence of cell selectivity of the peptide and a more complex role in stimulating host cells.

  10. ITC-derived binding affinity may be biased due to titrant (nano)-aggregation. Binding of halogenated benzotriazoles to the catalytic domain of human protein kinase CK2

    PubMed Central

    Winiewska, Maria; Bugajska, Ewa

    2017-01-01

    The binding of four bromobenzotriazoles to the catalytic subunit of human protein kinase CK2 was assessed by two complementary methods: Microscale Thermophoresis (MST) and Isothermal Titration Calorimetry (ITC). New algorithm proposed for the global analysis of MST pseudo-titration data enabled reliable determination of binding affinities for two distinct sites, a relatively strong one with the Kd of the order of 100 nM and a substantially weaker one (Kd > 1 μM). The affinities for the strong binding site determined for the same protein-ligand systems using ITC were in most cases approximately 10-fold underestimated. The discrepancy was assigned directly to the kinetics of ligand nano-aggregates decay occurring upon injection of the concentrated ligand solution to the protein sample. The binding affinities determined in the reverse ITC experiment, in which ligands were titrated with a concentrated protein solution, agreed with the MST-derived data. Our analysis suggests that some ITC-derived Kd values, routinely reported together with PDB structures of protein-ligand complexes, may be biased due to the uncontrolled ligand (nano)-aggregation, which may occur even substantially below the solubility limit. PMID:28273138

  11. 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

  12. 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

  13. 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

  14. A hydrophobic patch surrounding Trp154 in human neuroserpin controls the helix F dynamics with implications in inhibition and aggregation

    PubMed Central

    Ali, Mohammad Farhan; Kaushik, Abhinav; Kapil, Charu; Gupta, Dinesh; Jairajpuri, Mohamad Aman

    2017-01-01

    Neuroserpin (NS) mediated inhibition of tissue-type plasminogen activator (tPA) is important for brain development, synapse formation and memory. Aberrations in helix F and β-sheet A movement during inhibition can directly lead to epilepsy or dementia. Conserved W154 residue in a hydrophobic patch between helix F and β-sheet A is ideally placed to control their movement during inhibition. Molecular Dynamics (MD) simulation on wild type (WT) NS and its two variants (W154A and W154P) demonstrated partial deformation in helix F and conformational differences in strands 1A and 2A only in W154P. A fluorescence and Circular Dichroism (CD) analysis with purified W154 variants revealed a significant red-shift and an increase in α-helical content in W154P as compared to W154A and WT NS. Kinetics of tPA inhibition showed a decline in association rates (ka) for W154A as compared to WT NS with indication of complex formation. Appearance of cleaved without complex formation in W154P indicates that the variant acts as substrate due to conformational misfolding around helix F. Both the variants however showed increased rate of aggregation as compared to WT NS. The hydrophobic patch identified in this study may have importance in helix F dynamics of NS. PMID:28230174

  15. A hydrophobic patch surrounding Trp154 in human neuroserpin controls the helix F dynamics with implications in inhibition and aggregation.

    PubMed

    Ali, Mohammad Farhan; Kaushik, Abhinav; Kapil, Charu; Gupta, Dinesh; Jairajpuri, Mohamad Aman

    2017-02-23

    Neuroserpin (NS) mediated inhibition of tissue-type plasminogen activator (tPA) is important for brain development, synapse formation and memory. Aberrations in helix F and β-sheet A movement during inhibition can directly lead to epilepsy or dementia. Conserved W154 residue in a hydrophobic patch between helix F and β-sheet A is ideally placed to control their movement during inhibition. Molecular Dynamics (MD) simulation on wild type (WT) NS and its two variants (W154A and W154P) demonstrated partial deformation in helix F and conformational differences in strands 1A and 2A only in W154P. A fluorescence and Circular Dichroism (CD) analysis with purified W154 variants revealed a significant red-shift and an increase in α-helical content in W154P as compared to W154A and WT NS. Kinetics of tPA inhibition showed a decline in association rates (ka) for W154A as compared to WT NS with indication of complex formation. Appearance of cleaved without complex formation in W154P indicates that the variant acts as substrate due to conformational misfolding around helix F. Both the variants however showed increased rate of aggregation as compared to WT NS. The hydrophobic patch identified in this study may have importance in helix F dynamics of NS.

  16. A hydrophobic patch surrounding Trp154 in human neuroserpin controls the helix F dynamics with implications in inhibition and aggregation

    NASA Astrophysics Data System (ADS)

    Ali, Mohammad Farhan; Kaushik, Abhinav; Kapil, Charu; Gupta, Dinesh; Jairajpuri, Mohamad Aman

    2017-02-01

    Neuroserpin (NS) mediated inhibition of tissue-type plasminogen activator (tPA) is important for brain development, synapse formation and memory. Aberrations in helix F and β-sheet A movement during inhibition can directly lead to epilepsy or dementia. Conserved W154 residue in a hydrophobic patch between helix F and β-sheet A is ideally placed to control their movement during inhibition. Molecular Dynamics (MD) simulation on wild type (WT) NS and its two variants (W154A and W154P) demonstrated partial deformation in helix F and conformational differences in strands 1A and 2A only in W154P. A fluorescence and Circular Dichroism (CD) analysis with purified W154 variants revealed a significant red-shift and an increase in α-helical content in W154P as compared to W154A and WT NS. Kinetics of tPA inhibition showed a decline in association rates (ka) for W154A as compared to WT NS with indication of complex formation. Appearance of cleaved without complex formation in W154P indicates that the variant acts as substrate due to conformational misfolding around helix F. Both the variants however showed increased rate of aggregation as compared to WT NS. The hydrophobic patch identified in this study may have importance in helix F dynamics of NS.

  17. 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.

  18. 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

  19. Clinical outcomes after IVF or ICSI using human blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum.

    PubMed

    Itoi, Fumiaki; Asano, Yukiko; Shimizu, Masashi; Nagai, Rika; Saitou, Kanako; Honnma, Hiroyuki; Murata, Yasutaka

    2017-01-25

    In this study the clinical and neo-natal outcomes after transfer of blastocysts derived from oocytes containing aggregates of smooth endoplasmic reticulum (SER) were compared between IVF and intracytoplasmic sperm injection (ICSI) cycles. Clinical and neo-natal outcomes of blastocysts in cycles with at least one SER metaphase II oocyte (SER + MII; SER + cycles) did not significantly differ between the two insemination methods. When SER + MII were cultured to day 5/6, fertilization, embryo cleavage and blastocyst rates were not significantly different between IVF and ICSI cycles. In vitrified-warmed blastocyst transfer cycles, the clinical pregnancy rates from SER + MII in IVF and ICSI did not significantly differ. In this study, 52 blastocysts (27 IVF and 25 ICSI) derived from SER + MII were transferred, yielding 15 newborns (5 IVF and 10 ICSI) and no malformations. Moreover, 300 blastocysts (175 IVF and 125 ICSI) derived from SER-MII were transferred, yielding 55 newborns (24 IVF and 31 ICSI cycles). Thus, blastocysts derived from SER + cycles exhibited an acceptable ongoing pregnancy rate after IVF (n = 125) or ICSI (n = 117) cycles. In conclusion, blastocysts from SER + MII in both IVF and ICSI cycles yield adequate ongoing pregnancy rates with neo-natal outcomes that do not differ from SER-MII.

  20. 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

  1. 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.

  2. Uric acid increases erythrocyte aggregation: Implications for cardiovascular disease.

    PubMed

    Sloop, Gregory D; Bialczak, Jessica K; Weidman, Joseph J; St Cyr, J A

    2016-10-05

    Uric acid may be a risk factor for atherosclerotic cardiovascular disease, although the data conflict and the mechanism by which it may cause cardiovascular disease is uncertain. This study was performed to test the hypothesis that uric acid, an anion at physiologic pH, can cause erythrocyte aggregation, which itself is associated with cardiovascular disease. Normal erythrocytes and erythrocytes with a positive direct antiglobulin test for surface IgG were incubated for 15 minutes in 14.8 mg/dL uric acid. Erythrocytes without added uric acid were used as controls. Erythrocytes were then examined microscopically for aggregation. Aggregates of up to 30 erythrocytes were noted when normal erythrocytes were incubated in uric acid. Larger aggregates were noted when erythrocytes with surface IgG were incubated in uric acid. Aggregation was negligible in controls. These data show that uric acid causes erythrocyte aggregation. The most likely mechanism is decreased erythrocyte zeta potential. Erythrocyte aggregates will increase blood viscosity at low shear rates and increase the risk of atherothrombosis. In this manner, hyperuricemia and decreased zeta potential may be risk factors for atherosclerotic cardiovascular disease.

  3. 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.

  4. 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.

  5. Study of natural IgG antibodies against vascular endothelial growth factor receptor 1 in hepatocellular carcinoma

    PubMed Central

    Wang, Yilai; Yan, Zhaoping; Huang, Yile; Qiu, Cailing; Chen, Xiangyun; Hu, Ying; Meng, Qingyong; Wei, Jun

    2017-01-01

    Natural antibodies have been found to have anti-tumorigenic function. This study was designed to investigate whether natural IgG antibodies against vascular endothelial growth factor receptor 1 (VEGFR1) could suppress the growth of hepatocellular carcinoma (HCC) cells. Three HCC cell lines and A549 lung cancer cells were used for this study. They were grown, respectively, with human plasma positive or negative for anti-VEGFR1 IgG. Cell viability, apoptosis and VEGFR1 gene expression were examined. Three patients with HCC were recruited for a case study. The results showed that plasma anti-VEGFR1 IgG significantly inhibited the proliferation of all three HCC cell lines but not A549 cell line; the proportions of apoptotic cells were significantly higher in HCC cells treated with anti-VEGFR1 IgG positive plasma than those treated with IgG negative plasma. The expression of the VEGFR1 gene was significantly higher in HCC cells than A549 cells. Of three HCC patients who received transfusion of anti-VEGFR1 IgG positive plasma, two cases with stage B showed a good response to the treatment but one with distant metastasis did not. Human plasma IgG against VEGFR1 may be a promising agent for anti-HCC therapy.

  6. 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

  7. IgG1 Is Pathogenic in Leishmania mexicana Infection

    PubMed Central

    Chu, Niansheng; Thomas, Bolaji N.; Patel, Supriya R.; Buxbaum, Laurence U.

    2010-01-01

    There are over 2 million new cases of leishmaniasis annually, and no effective vaccine has been developed to prevent infection. In murine infection, Leishmania mexicana, which lives intracellularly in host macrophages, has developed pathways to hijack host IgG to induce a suppressive IL-10 response through FcγRs, the cell-surface receptors for IgG. To guide vaccine development away from detrimental Ab responses, which can accompany attempts to induce cell-mediated immunity, it is crucial to know which isotypes of IgG are pathogenic in this infection. We have found that IgG1 and IgG2a/c induce IL-10 from macrophages in vitro equally well but through different FcγR subtypes: IgG1 through FcγRIII, and IgG2a/c through FcγRI primarily, but also through FcγRIII. In sharp contrast, mice lacking IgG1 develop earlier and stronger IgG2a/c, IgG3, and IgM responses to L. mexicana infection and yet are more resistant to the infection. Thus, IgG1, but not IgG2a/c or IgG3, is pathogenic in vivo, in agreement with prior studies indicating that FcγRIII is required for chronic disease. This calls into question the assumption that macrophages, which should secrete IL-10 in response to both IgG1 and IgG2a/c immune complexes, are the most important source of IL-10 generated by IgG-FcγR engagement in L. mexicana infection. Further investigations are required to better determine the cell type responsible for this immunosuppressive FcγRIII-induced IL-10 pathway and whether IgG2a/c is protective. PMID:21037092

  8. 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

  9. Plant-based production of two chimeric monoclonal IgG antibodies directed against immunodominant epitopes of Vibrio cholerae lipopolysaccharide.

    PubMed

    Levinson, Kara J; Giffen, Samantha R; Pauly, Michael H; Kim, Do H; Bohorov, Ognian; Bohorova, Natasha; Whaley, Kevin J; Zeitlin, Larry; Mantis, Nicholas J

    2015-07-01

    We have produced and characterized two chimeric human IgG1 monoclonal antibodies that bind different immunodominant epitopes on Vibrio cholerae lipopolysaccharide (LPS). MAb 2D6 IgG1 recognizes Ogawa O-polysaccharide antigen, while mAb ZAC-3 IgG1 recognizes core/lipid A moiety of Ogawa and Inaba LPS. Both antibodies were expressed using a Nicotiana benthamiana-based rapid antibody-manufacturing platform (RAMP) and evaluated in vitro for activities associated with immunity to V. cholerae, including vibriocidal activity, bacterial agglutination and motility arrest.

  10. Immunological properties of isolated IgG and IgM anti-gamma-globulins (rheumatoid factors)*

    PubMed Central

    Bianco, N. E.; Dobkin, Linda W.; Schur, P. H.

    1974-01-01

    Anti-gamma-globulins of the IgG and IgM classes have been isolated from sera of normal individuals and from patients with osteoarthritis, rheumatoid arthritis and juvenile rheumatoid arthritis. All of the isolated antibodies gave precipitin curves with heat-aggregated, reduced and alkylated gamma-globulin. IgM anti-gamma-globulins gave a positive latex fixation test at 4°C and 37°C while IgG anti-gamma-globulins generally gave a positive test only at 4°C. Anti-gamma-globulins from normals did not fix complement but IgG and IgM anti-gamma-globulins from rheumatoids fixed complement to a similar degree. This in vitro complement fixation could account at least in part for the diminished complement levels seen in many rheumatoid synovial effusions. PMID:4466600

  11. Rapid assessment of human amylin aggregation and its inhibition by copper(II) ions by laser ablation electrospray ionization mass spectrometry with ion mobility separation

    SciTech Connect

    Li, Hang; Ha, Emmeline; Donaldson, Robert P.; Jeremic, Aleksandar M.; Vertes, Akos

    2015-09-09

    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. In this paper, 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

  12. Rapid assessment of human amylin aggregation and its inhibition by copper(II) ions by laser ablation electrospray ionization mass spectrometry with ion mobility separation

    DOE PAGES

    Li, Hang; Ha, Emmeline; Donaldson, Robert P.; ...

    2015-09-09

    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 pancreasmore » 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. In this paper, 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

  13. Sialylation of E-cadherin does not change the spontaneous or ET-18-OMe-mediated aggregation of MCF-7 human breast cancer cells.

    PubMed

    Steelant, W F; Recchi, M A; Noë, V T; Boilly-Marer, Y; Bruyneel, E A; Verbert, A; Mareel, M M; Delannoy, P

    1999-05-01

    We have investigated the role of sialylation on cell-cell adhesion mediated by E-cadherin. Two MCF-7 human breast cancer cell variants were studied: MCF-7/AZ cells showed a spontaneous cell-cell adhesion in the fast and slow aggregation assay. whereas the adhesion deficient MCF-7/6 cell variant failed to form larger aggregates, suggesting that E-cadherin was not functional under the conditions of both assays. We measured the sialyltransferase activities using Galbeta1-3GalNAcalpha-O-benzyl and Galbeta1-4GlcNAcalpha-O-benzyl as acceptor substrates as well as mRNA levels of four sialyltransferases, ST3Gal I, ST3Gal III, ST3Gal IV, ST6Gal I, using multiplex RT-PCR in MCF-7 cell variants. The alpha2-6 and alpha2-3 sialylation of E-cadherin was investigated by immuno-blot using Sambucus nigra agglutinin and Maackia amurensis agglutinin. Compared to the adhesion-proficient MCF-7/AZ cells, the adhesion-deficient MCF-7/6 cell line apparently lacks ST6Gal I mRNA, has a lower ST3Gal I mRNA, a lower ST3Gal I sialyltransferase activity, and no alpha2-3 linked sialic acid moieties on E-cadherin. The potential anti-cancer drug 1-O-octadecyl-2-O-methylglycero-3-phosphocholine (ET-18-OMe, 48 h, 25 microg/ml) belonging to the class of alkyllysophospholipids restored the E-cadherin function in the adhesion-deficient MCF-7/6 cells as evidenced by an increased aggregation. ET-18-OMe caused loss of ST6Gal I mRNA in MCF-7/AZ cells but no changes of sialyltransferase activities or sialic acid moieties on E-cadherin could be observed. We conclude that Ca2+-dependent, E-cadherin-specific homotypic adhesion of MCF-7/AZ or MCF-7/6 cells treated with ET-18-OMe was not affected by sialylation of E-cadherin.

  14. 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

  15. Structural characteristics of articular cartilage proteoglycan in IgG induced experimental immune synovitis.

    PubMed Central

    Malemud, C J; Yoo, J U; Goldberg, V M; Kresina, T F

    1987-01-01

    The early changes (five weeks) in the structure of newly synthesised and endogenous articular cartilage sulphated proteoglycans were studied in lapine IgG induced experimental immune synovitis. Rabbits with immune synovitis (IS-IgG) were compared with animals with a developed hypersensitivity to IgG (I-IgG) and with non-treated normal weight matched controls. Medial and lateral femoral condyle and tibial plateau cartilage was pooled and radiolabelled for 24 h in vitro with 35SO4. The samples constituted tissue from regions underlying pannus and from pannus free sites. Cartilage from animals with IS-IgG showed a significantly diminished amount of newly synthesised and endogenous proteoglycan aggregate and an increased amount of hydrodynamically small proteoglycans. Newly synthesised (obtained by in vivo radiosulphate labelling) and endogenous proteoglycans showed a similar profile. The proteoglycan monomer fraction from animals with IS-IgG failed to form proteoglycan aggregates in the presence of excess hyaluronic acid. In the group with IS-IgG linear regression analysis showed a statistically significant relationship between the synovial pathology scores (but not cartilage pathology score) and diminished newly synthesised and endogenous proteoglycan aggregate. PMID:3662640

  16. 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.

  17. 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.

  18. 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

  19. Identification of Anti-Long Chain Saturated Fatty Acid IgG Antibodies in Serum of Patients with Type 2 Diabetes.

    PubMed

    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.

  20. 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

  1. Simultaneous Quantification of Anticardiolipin IgG and IgM by Time Resolved Fluoroimmunoassay

    PubMed Central

    Liu, Jie; Li, Mei; Ye, Yan; Chen, Yu

    2016-01-01

    The autoimmune disease antiphospholipid syndrome (APS) is characterized by the presence of anticardiolipin antibodies (aCL), along with anti-β2-glycoprotein I (β2GPI) antibodies and lupus anticoagulant (LA). In this study, we developed a time-resolved fluoroimmunoassay (TRFIA) system for simultaneous quantification of aCL IgG and IgM. A 96-well microtiter plate precoated with the complex of cardiolipin from bovine heart and bovine β2GPI was incubated with the anticardiolipin IgG and IgM standard substance or serum, and the conjugate of Eu3+-labeled anti-human IgG and Sm3+-labeled anti-human IgM was pipetted to the wells to form a tipical double-antibody-sandwich immunoreactions; finally the fluorescent intensity of Eu3+ and Sm3+ was detected to reflect the quantity of anticardiolipin IgG and IgM. This assay showed a good relationship between fluorescence intensities and the concentration of anticardiolipin antibody(aCL) IgG and IgM, with a low-end sensitivity of 0.1 U/ml for IgG and 0.1 U/ml for IgM, respectively. The intra- and inter-assay coefficients of variation (CV) of the calibrators was 3.0% and 4.51% for IgG, and 2.76% and 4.45% for IgM. The average recovery was 100.38% for aCL IgG and 100.45% for aCL IgM. For serum samples, the results of our method showed a good correlation with those obtained with ELISA kit. Simultaneous detection of aCL-IgG and aCL-IgM in the same reaction well can optimize assay performance by avoiding potential influence of different reaction conditions-timing, and well-to-well difference in concentration and characteristics of cardiolipin antigen. The results of a combo aCL-IgG and aCL-IgM assay for the same sample are more consistent and more reliable. This dual-label time-resolved fluoroimmunoassay is sensitive for detecting aCL IgG and IgM across a wide concentration range with stable reagents and may assist in the clinical diagnosis of antiphospholipid syndrome. PMID:27661084

  2. Group G streptococcal IgG binding molecules FOG and protein G have different impacts on opsonization by C1q.

    PubMed

    Nitsche-Schmitz, D Patric; Johansson, Helena M; Sastalla, Inka; Reissmann, Silvana; Frick, Inga-Maria; Chhatwal, Gursharan S

    2007-06-15

    Recent epidemiological data on diseases caused by beta-hemolytic streptococci belonging to Lancefield group C and G (GCS, GGS) underline that they are an emerging threat to human health. Among various virulence factors expressed by GCS and GGS isolates from human infections, M and M-like proteins are considered important because of their anti-phagocytic activity. In addition, protein G has been implicated in the accumulation of IgG on the bacterial surface through non-immune binding. The function of this interaction, however, is still unknown. Using isogenic mutants lacking protein G or the M-like protein FOG (group G streptococci), respectively, we could show that FOG contributes substantially to IgG binding. A detailed characterization of the interaction between IgG and FOG revealed its ability to bind the Fc region of human IgG and its binding to the subclasses IgG1, IgG2, and IgG4. FOG was also found to bind IgG of several animal species. Surface plasmon resonance measurements indicate a high affinity to human IgG with a dissociation constant of 2.4 pm. The binding site was localized in a central motif of FOG. It has long been speculated about anti-opsonic functions of streptococcal Fc-binding proteins. The presented data for the first time provide evidence and, furthermore, indicate functional differences between protein G and FOG. By obstructing the interaction between IgG and C1q, protein G prevented recognition by the classical pathway of the complement system. In contrast, IgG that was bound to FOG remained capable of binding C1q, an effect that may have important consequences in the pathogenesis of GGS infections.

  3. IgG4-related pleuritis with chylothorax.

    PubMed

    Kato, Eisuke; Takayanagi, Noboru; Ishiguro, Takashi; Kagiyama, Naho; Shimizu, Yoshihiko; Sugita, Yutaka

    2014-01-01

    Presently, 6 cases of IgG4-related pleuritis have been reported. We encountered a patient who developed chylothorax due to IgG4-related disease. To our knowledge, such patients have not been reported. This patient developed right-sided chylothorax and left-sided non-chylothorax lymphocyte-predominant pleuritis. Elevated serum and pleural IgG4 concentrations and histopathological analysis of pleural biopsy confirmed the diagnosis of IgG4-related pleuritis. Left-sided pleuritis improved with corticosteroid therapy, but right-sided chylothorax persists. IgG4-related disease can be one cause of chylothorax.

  4. Detection of Serum IgG4 Levels in Patients with IgG4-Related Disease and Other Disorders

    PubMed Central

    Wang, Chenqiong; Wu, Xuefen; Miao, Ye; Xiong, Hui; Bai, Lin; Dong, Lingli

    2015-01-01

    Objective Elevated serum IgG4 levels are an important hallmark for diagnosing IgG4-related disease (IgG4-RD), but can also be observed in other diseases. This study aimed to compare two different testing methods for IgG4: ELISA and nephelometric assay. Both assays were used to measure serum IgG4 concentrations, and to assess the prevalence of high serum IgG4 levels in both IgG4-RD and non-IgG4-RD diseases. Methods A total of 80 serum samples were tested using the nephelometric assay and ELISA method that we established. Serum IgG4 concentrations were determined by ELISA for 957 patients with distinct diseases, including 12 cases of IgG4-RD and 945 cases of non-IgG4-RD. Results IgG4 levels from 80 selected serum samples examined by ELISA were in agreement with those detected using the nephelometry assay. Meanwhile, the serum IgG4 concentrations measured by ELISA were also consistent with the clinical diagnoses of patients with IgG4-RD during the course of disease. The Elevated levels of serum IgG4 (>1.35 g/L) were detected in all IgG4-RD (12/12) patients, and the prevalence of high IgG4 serum levels was 3.39% in non-IgG4-RD cases. Among them, the positive rates of serum IgG4 were 2.06% in patients with carcinoma and 6.3% in patients with other non-IgG4 autoimmune diseases. Conclusion Our established ELISA method is a reliable and convenient technique, which could be extensively used in the clinic to measure serum IgG4 levels. High levels of IgG4 were observed in IgG4-RD. However, this phenomenon could also be observed in other diseases, such as carcinomas and other autoimmune diseases. Thus, a diagnosis of IgG4 disease cannot only be dependent on the detection of elevated serum IgG4 levels. PMID:25885536

  5. Processing of IgA aggregates in a rat model of chronic liver disease.

    PubMed

    Amore, A; Roccatello, D; Picciotto, G; Emancipator, S N; Ropolo, R; Cacace, G; Suriani, A; Gianoglio, B; Sena, L M; Cirina, P; Mazzucco, G; Alfieri, V; Piccoli, G; Coppo, R; De Filippi, P G

    1997-08-01

    Heavy alcohol intake and/or lipotrope-deficient diet induced hepatocellular injury and mesangial deposition of IgA and often IgG in Lewis rats. The experimental animals showing more severe urinary abnormalities and histologic damage in the glomeruli had increased levels of IgA antibodies to dietary antigens and altered intestinal permeability. Based on human studies, the prolonged circulation of IgA-containing complexes associated with the liver disease could be envisaged as important for the development of mesangial IgA deposits. In order to verify this hypothesis, four groups (G) of Lewis rats were studied: G1 received thrice a weak an intragastric infusion of 1.5 ml/100 g body wt of whiskey; G2 rats were nourished with lipotrope-deficient diet; G3 rats were given both whiskey and LD diet; G4 rats were nourished with regular chow. After 12 weeks, heat-aggregated rat monomeric IgA was labeled with 133I and intravenously injected. Three control subgroups of rats, one given whiskey, one nourished with LD diet, and one with regular chow, were injected with radiolabeled heat-aggregated rat IgG. A large field-of-view digital gamma camera, equipped with an ultra-high-resolution collimator and interfaced to a dedicated computer, was used to analyze tracer kinetics and fate. The liver was the main organ involved in clearance of both test probes. The hepatic mean transit (MTT) was 11.4 +/- 11 min in G1 (proteinuria of 6.9 +/- 1.41 mg/day and hematuria +/+2), 221 +/- 19 min in G2 (proteinuria 9.1 +/- 0.64 mg/day and hematuria +2/+3), and 230 +/- 15 min in G3 (proteinuria 9.5 +/- 0.58 mg/day and hematuria +2/+3). In each case MTT value was found to be significantly prolonged compared to G4 (85 +/- 4 min). The multiple regression analysis showed that MTT values, proteinuria, and hematuria were significantly correlated (P < 0.01). Controls had trace amount proteinuria (0.82 +/- 0.17 mg/day, significantly lower than for each study group, P < 0.08) and undetectable hematuria

  6. Proteolytic activity of IgGs from blood serum of Wistar rats at experimental rheumatoid arthritis.

    PubMed

    Kit, Yu Ya; Myronovsky, S L; Kril, I I; Havrylyuk, A M; Chop'yak, V V; Stoika, R S

    2014-01-01

    The aim of this work was to study the proteolytic activity of IgGs purified from blood serum of Wistar rats at experimental rheumatoid arthritis (ERA) induced by an injection of bovine collagen of type II. Twenty rats were immunized with a preparation of bovine collagen II (Sigma-Aldrich, USA) in the presence of complete Freund's adjuvant. ERA development was determined by inflammation in limbs of treated animals. IgG preparations were isolated from blood serum of immunized and non-immunized animals by precipitation of antibodies with 33% ammonium sulfate followed by chromatography on the Protein G-Sepharose column. Human histone H1, bovine collagen II, calf thymus histones, myelin basic protein (MBP), bovine serum albumin (BSA), and bovine casein were used as substrates of the proteolytic activity of IgGs. It was found that IgG preparations from blood serum of rats with ERA were capable of cleaving histone H1 and MBP, however, they were catalytically inactive towards collagen II, casein, BSA, and core histones. IgGs from blood serum of non-immunized rats were proteolytically inactive towards all used protein substrates. Thus, we demonstrated that immunization of rats with bovine collagen II induced IgG-antibodies possessing the proteolytic activity towards histone H1 and MBP. This activity might be associated with the development of inflammatory processes in the immunized rats.

  7. [Histopathology of IgG4-related disease].

    PubMed

    Detlefsen, S; Klöppel, G

    2016-09-01

    At an international consensus conference in 2011, multifocal chronic fibrosing inflammatory processes, which are associated with elevated IgG4 serum levels and/or tissue infiltration with IgG4 positive plasma cells, were recognized as a distinct disease entity called IgG4-related disease (IgG4-RD). As IgG4-RD responds well to steroid treatment but imitates a tumor in many organs, particularly in the pancreas, a biopsy for confirmation of the diagnosis is often warranted. The histological criteria for IgG4-RD as defined in 2011 are based on the following main features: 1) dense lymphoplasmacytic infiltrate, 2) storiform fibrosis and 3) obliterative phlebitis. The diagnosis is further supported by immunohistochemical demonstration of an increased infiltration of IgG4-positive plasma cells and an elevated IgG4/IgG ratio. The morphological criteria of IgG4-RD are in most cases detectable in biopsies and can significantly contribute to the diagnosis of this disease, in concert with clinical, serological (elevated serum IgG4 level) and radiological features.

  8. 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.

  9. 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.

  10. 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. )