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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. 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

  3. 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

  4. 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

  5. Interference of IgG, IgG aggregates and immune complexes in tests for platelet autoantibodies.

    PubMed

    Helmerhorst, F M; Smeenk, R J; Hack, C E; Engelfriet, C P; von dem Borne, A E

    1983-11-01

    Three techniques, based on the antiglobulin principle, used for the detection of autoantibodies against platelets, were compared; the antiglobulin consumption assay (QACA), the platelet radioactive antiglobulin test (PRAT) and the platelet suspension immunofluorescence test (PSIFT). Upon incubation of normal donor platelets with purified IgG, in concentrations higher than that in serum, an increased amount of platelet-associated IgG was demonstrated only in the QACA. Upon incubation with aggregated IgG, all three tests became positive, but the PSIFT only with high concentrations of aggregates. Binding of soluble C1q-binding immune complexes (IC), which consisted of tetanus toxoid and IgG antitetanus antibodies (TaT) to normal donor platelets, was only detectable in the QACA. However, a positive result was obtained in all three tests with platelets incubated with soluble DNA-IgG-antiDNA antibodies (DaD) IC. Fixation of the platelets with paraformaldehyde prevented the binding and the detection of the DaD-IC, but not of IgG, aggregated IgG or TaT-IC. Eluates from platelets incubated with aggregated IgG, TaT- or DaD-IC did not react with normal donor platelets in the three techniques, in contrast to eluates from platelets sensitized with platelet antibodies.

  6. Formation of C3-IgG complexes in serum by aggregated IgG and by non-immunoglobulin activators of complement.

    PubMed

    van Dam, A P; Hack, C E

    1987-06-01

    We studied the generation of C3-IgG complexes during the activation of C3 in serum by aggregated human IgG (AHG), zymosan or cobra venom factor (CVF). C3-IgG complexes were detected by specific radioimmunoassays: samples to be tested were incubated with anti-IgG Sepharose, and complexes that had bound to the Sepharose were detected by incubation with either 125I-anti-C3c or 125I-anti-C3d, g. Incubation of serum with as little as 6 micrograms AHG per ml, for 30 min at 37 degrees, resulted in the generation of C3-IgG complexes. When serum was incubated with zymosan or CVF, C3-IgG complexes were also generated. AHG appeared to be more effective in the generation of C3-IgG complexes than CVF. We calculated that AHG (2 mg/ml) caused about 36% of the C3 to be fixed to IgG, CVF (400 micrograms/ml) about 14%. Finally, the presence of C3 fixed to IgG in serum incubated with CVF was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting. This study indicates that the formation of C3-IgG complexes in serum is not only induced by immune complexes but also by non-immunoglobulin activators. Therefore, C3-IgG complexes might be considered as complement activation products, and their detection in patients' samples should not be considered as conclusive evidence for the presence of immune complexes.

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

    PubMed

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

    1988-07-01

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

  8. 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

  9. Human IgG2 can form covalent dimers.

    PubMed

    Yoo, Esther M; Wims, Letitia A; Chan, Lisa A; Morrison, Sherie L

    2003-03-15

    Unlike IgA and IgM, IgG has not yet been shown to form covalent polymers. However in the presence of specific Ag, murine IgG3 has been shown to polymerize through noncovalent interactions. In contrast to the noncovalent oligomers found with murine IgG3, we have detected covalent dimers in three different recombinant human IgG2 Abs produced in myeloma cells. Both IgG2,kappa and IgG2,lambda can form dimers. In addition, analysis of pooled human gamma globulin and several normal sera revealed the presence of IgG2 dimers. The IgG2 dimers are in contrast to the noncovalent IgG dimers found in pooled sera of multiple donors resulting from idiotype/anti-idiotype (Id/anti-Id) interactions. Cyanogen bromide cleavage analysis suggests that one or more Cys residues in the gamma 2 hinge are involved in dimer assembly. The potential role of IgG2 dimers in immunity against carbohydrate Ags is discussed.

  10. 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.

  11. 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.

  12. 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

  13. Estimation of polyclonal IgG4 hybrids in normal human serum

    PubMed Central

    Young, Elizabeth; Lock, Emma; Ward, Douglas G; Cook, Alexander; Harding, Stephen; Wallis, Gregg L F

    2014-01-01

    The in vivo or in vitro formation of IgG4 hybrid molecules, wherein the immunoglobulins have exchanged half molecules, has previously been reported under experimental conditions. Here we estimate the incidence of polyclonal IgG4 hybrids in normal human serum and comment on the existence of IgG4 molecules with different immunoglobulin light chains. Polyclonal IgG4 was purified from pooled or individual donor human sera and sequentially fractionated using light-chain affinity and size exclusion chromatography. Fractions were analysed by SDS–PAGE, immunoblotting, ELISA, immunodiffusion and matrix-assisted laser-desorption mass spectrometry. Polyclonal IgG4 purified from normal serum contained IgG4κ, IgG4λ and IgG4κ/λ molecules. Size exclusion chromatography showed that IgG4 was principally present in monomeric form (150 000 MW). SDS–PAGE, immunoblotting and ELISA showed the purity of the three IgG4 samples. Immunodiffusion, light-chain sandwich ELISA and mass spectrometry demonstrated that both κ and λ light chains were present on only the IgG4κ/λ molecules. The amounts of IgG4κ/λ hybrid molecules ranged from 21 to 33% from the five sera analysed. Based on the molecular weight these molecules were formed of two IgG4 heavy chains plus one κ and one λ light chain. Polyclonal IgG (IgG4-depleted) was similarly fractionated according to light-chain specificity. No evidence of hybrid IgG κ/λ antibodies was observed. These results indicate that hybrid IgG4κ/λ antibodies compose a substantial portion of IgG4 from normal human serum. PMID:24512211

  14. 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.

  15. Human platelet Fc (IgG) receptor and its modulation

    SciTech Connect

    King, M.; McDermott, P.; Schreiber, A.D.

    1986-03-01

    The authors demonstrated that IgG oligomers bind to washed human platelets (P) by an Fc dependent process optimally at low ionic strength (/sup +/0.07) in 3 hrs at 4/sup 0/, while IgG monomer binds immeasurably. The authors studied the modulation of this Fc (IgG) binding site (Rc) on P by measuring /sup 125/I-IgG trimer binding to P at equilibrium and assessing Rc number of affinity. At ..mu.. = 0.07, P expressed 2 fold more Rc than at ..mu.. = 0.15, without a change in affinity; this effect was reversed upon re-exposure of P to ionic strength ..mu.. = 0.15. Equal numbers and affinities of Rc were observed in the presence of either 2mM EDTA, 2 mM EGTA or 2 mM EGTA + 2 mM Mg/sup + +/. Cytochalasin B (10 ..mu..g/ml) did not alter Rc (4987 sites/P, Ka = 0.9 x 10/sup 7/M/sup -1/ vs 5098 sites/P, Ka = 1.1 x 10/sup 7/M/sup -1/). Incubation with P alloreactive plasma at a concentration which depleted 33% of plasma C3, decreased Rc by 50%. However, activation of P by 10..mu..M ADP with Ca/sup + +/, Mg/sup + +/ and 100 ..mu..g/ml fibrinogen did not affect Rc number of affinity (2825 sites/P, Ka = 1.1 x 10/sup 7/M/sup -1/ vs 2551 sites/P, Ka = 0.9 x 10/sup 7/M/sup -1/). Thrombin (0.01 - 10 U/ml) also did not alter the number or affinity of Rc. P from 2 patients with thrombastenia expressed normal Rc number and affinity. Binding of IgG trimer to P occurs independent of actin filament interaction, Mg/sup + +/, modulation of P by ADP or thrombin, and of GPIIb/IIIa orGPIIb/IIIa-fibrogen interaction.

  16. Probing Specific Interaction Forces Between Human IgG and Rat Anti-Human IgG by Self-Assembled Monolayer and Atomic Force Microscopy

    PubMed Central

    2010-01-01

    Interaction forces between biological molecules such as antigen and antibody play important roles in many biological processes, but probing these forces remains technically challenging. Here, we investigated the specific interaction and unbinding forces between human IgG and rat anti-human IgG using self assembled monolayer (SAM) method for sample preparation and atomic force microscopy (AFM) for interaction force measurement. The specific interaction force between human IgG and rat anti-human IgG was found to be 0.6–1.0 nN, and the force required for unbinding a single pair of human IgG and rat anti-human IgG was calculated to be 144 ± 11 pN. The results are consistent with those reported in the literatures. Therefore, SAM for sample preparation combined with AFM for interaction measurement is a relatively simple, sensitive and reliable technique to probe specific interactions between biological molecules such as antigen and antibody. PMID:20671785

  17. 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.

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

    PubMed Central

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

    1991-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  20. Hinge-Region O-Glycosylation of Human Immunoglobulin G3 (IgG3)*

    PubMed Central

    Plomp, Rosina; Dekkers, Gillian; Rombouts, Yoann; Visser, Remco; Koeleman, Carolien A.M.; Kammeijer, Guinevere S.M.; Jansen, Bas C.; Rispens, Theo; Hensbergen, Paul J.; Vidarsson, Gestur; Wuhrer, Manfred

    2015-01-01

    Immunoglobulin G (IgG) is one of the most abundant proteins present in human serum and a fundamental component of the immune system. IgG3 represents ∼8% of the total amount of IgG in human serum and stands out from the other IgG subclasses because of its elongated hinge region and enhanced effector functions. This study reports partial O-glycosylation of the IgG3 hinge region, observed with nanoLC-ESI-IT-MS(/MS) analysis after proteolytic digestion. The repeat regions within the IgG3 hinge were found to be in part O-glycosylated at the threonine in the triple repeat motif. Non-, mono- and disialylated core 1-type O-glycans were detected in various IgG3 samples, both poly- and monoclonal. NanoLC-ESI-IT-MS/MS with electron transfer dissociation fragmentation and CE-MS/MS with CID fragmentation were used to determine the site of IgG3 O-glycosylation. The O-glycosylation site was further confirmed by the recombinant production of mutant IgG3 in which potential O-glycosylation sites had been knocked out. For IgG3 samples from six donors we found similar O-glycan structures and site occupancies, whereas for the same samples the conserved N-glycosylation of the Fc CH2 domain showed considerable interindividual variation. The occupancy of each of the three O-glycosylation sites was found to be ∼10% in six serum-derived IgG3 samples and ∼13% in two monoclonal IgG3 allotypes. PMID:25759508

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

    PubMed

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

    2016-07-28

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

  2. Evaluation of IgG4 and total IgG antibodies against cysticerci and peptide antigens for the diagnosis of human neurocysticercosis by ELISA.

    PubMed

    Intapan, Pewpan M; Khotsri, Piyarat; Kanpittaya, Jaturat; Chotmongkol, Verajit; Maleewong, Wanchai; Morakote, Nimit

    2008-12-01

    To support the clinical diagnosis of human neurocysticercosis (NCC), we evaluated two peptides, HP6-3 and Ts45W-1, as well as crude saline extract (SE) of Tenia solium cysticerci as antigens for the detection of specific IgG4 subclass and total IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). The sera of definitive diagnosed NCC patients, patients infected with other parasitoses and healthy controls were examined. The diagnostic sensitivity for IgG4 and total IgG detection of the ELISA against SE antigen was 100% and 64.3% with a high amount of cross-reactions to taeniasis saginata at 88.9% (8/9) and 100% (9/9), respectively. The SE-based IgG4-ELISA showed the highest specificity (80.9%). Both peptide-based IgG4-ELISAs provided a superior sensitivity (78.6%) to the total IgG tests whereas their specificity was 66.7% for HP6-3 and 69.8% for Ts45W-1 only. The SE-based ELISA for the detection of specific IgG4 antibody can be used for the diagnosis of neurocysticercosis as well as for serological surveys of NCC endemic areas. The peptide-based IgG4 ELISAs potentially provide a reliable and cost effective alternative method independent from live parasite supply.

  3. Human IgG Subclasses against Enterovirus Type 71: Neutralization versus Antibody Dependent Enhancement of Infection

    PubMed Central

    Han, Jian-Feng; Wang, Guang-Chuan; Zhao, Hui; Li, Xiao-Feng; Deng, Yong-Qiang; Zhu, Shun-Ya; Wang, Xiao-Yu; Lin, Fang; Zhang, Fu-Jun; Chen, Wei; Qin, E-De; Qin, Cheng-Feng

    2013-01-01

    The emerging human enterovirus 71 (EV71) represents a growing threat to public health, and no vaccine or specific antiviral is currently available. Human intravenous immunoglobulin (IVIG) is clinical used in treating severe EV71 infections. However, the discovery of antibody dependent enhancement (ADE) of EV71 infection illustrates the complex roles of antibody in controlling EV71 infection. In this study, to identify the distinct role of each IgG subclass on neutralization and enhancement of EV71 infection, different lots of pharmaceutical IVIG preparations manufactured from Chinese donors were used for IgG subclass fractionation by pH gradient elution with the protein A-conjugated affinity column. The neutralization and ADE capacities on EV71 infection of each purified IgG subclass were then assayed, respectively. The neutralizing activity of human IVIG is mainly mediated by IgG1 subclass and to less extent by IgG2 subclass. Interestingly, IgG3 fraction did not have neutralizing activity but enhanced EV71 infection in vitro. These results revealed the different roles of human IgG subclasses on EV71 infection, which is of critical importance for the rational design of immunotherapy and vaccines against severe EV71 diseases. PMID:23700449

  4. IgG abzymes with peroxidase and oxidoreductase activities from the sera of healthy humans.

    PubMed

    Tolmacheva, Anna S; Blinova, Elena A; Ermakov, Evgeny A; Buneva, Valentina N; Vasilenko, Nataliya L; Nevinsky, Georgy A

    2015-09-01

    We present the evidence showing that small fractions of electrophoretically homogeneous immunoglobulin G (IgGs) from the sera of healthy humans and their Fab and F(ab)2 fragments oxidize 3,3'-diaminobenzidine through a peroxidase activity in the presence of H2 O2 and through an oxidoreductase activity in the absence of H2 O2 . During purification on protein G-Sepharose and gel filtration, the polyclonal IgGs partially lose the Me(2+) ions. After extensive dialysis of purified Abs against agents chelating metal ions, the relative peroxidase activity decreased dependently of IgG analyzed from 100 to ~10-85%, while oxidoreductase activity from 100 to 14-83%. Addition of external metal ions to dialyzed and non-dialyzed IgGs leads to a significant increase in their activity. Chromatography of the IgGs on Chelex non-charged with Cu(2+) ions results in the adsorption of a small IgG fraction bound with metal ions (~5%), while Chelex charged with Cu(2+) ions bind additionally ~38% of the total IgGs. Separation of Abs on both sorbents results in IgG separation to many different subfractions demonstrating various affinities to the chelating resin and different levels of the specific oxidoreductase and peroxidase activities. In the presence of external Cu(2+) ions, the specific peroxidase activity of several IgG subfractions achieves 20-27 % as compared with horseradish peroxidase (HRP, taken for 100%). The oxidoreductase activity of these fractions is ~4-6-fold higher than that for HRP. Antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases are known to represent critical defence mechanisms for preventing oxidative modifications of DNA, proteins, and lipids. Peroxidase and oxidoreductase activities of human IgGs could also play an important role in the protection of organisms from oxidative stress and toxic compounds.

  5. Lateral Flow Test Using Echinococcus granulosus Native Antigen B and Comparison of IgG and IgG4 Dipsticks for Detection of Human Cystic Echinococcosis

    PubMed Central

    Khalilpour, Akbar; Sadjjadi, Seyed Mahmoud; Moghadam, Zohreh Kazemi; Yunus, Muhammad Hafiznur; Zakaria, Nor Dyana; Osman, Sabariah; Noordin, Rahmah

    2014-01-01

    Cystic echinococcosis (CE) caused by infection with Echinococcus granulosus is of major concern for humans in many parts of the world. Antigen B was prepared from E. granulosus hydatid fluid, and Western blots confirmed eight batches showing a band corresponding to the 8-/12-kDa subunit with positive serum and no low-molecular mass band (< 15 kDa) with negative serum. The batches were pooled and used to prepare lateral flow immunoglobulin G4 (IgG4) and IgG dipsticks. Diagnostic sensitivity was determined using serum samples from 21 hydatidosis patients, and diagnostic specificity was established using sera from 17 individuals infected with other parasites and 15 healthy people. IgG4 dipstick had a diagnostic sensitivity of 95% (20 of 21) and a specificity of 100% (32 of 32). The IgG dipstick had a sensitivity of 100% (21 of 21) and a specificity of 87.5% (28 of 32). Thus, both IgG and IgG4 dipsticks had high sensitivities, but IgG4 had greater specificity for the diagnosis of human CE. PMID:25200268

  6. Intrachain disulfide bond in the core hinge region of human IgG4.

    PubMed Central

    Bloom, J. W.; Madanat, M. S.; Marriott, D.; Wong, T.; Chan, S. Y.

    1997-01-01

    IgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four-chain structure is maintained by strong noncovalent interactions between the amino-terminal half of pairs of heavy-light chains and between the carboxyl-terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy-light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor-alpha (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half-IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP571 (S229P), or with an IgG1 rather than IgG4 hinge region, CDP571(gamma 1), only trace amounts of nondisulfide bonded half-IgG tetramers were observed. Trypsin digest reversephase HPLC peptide mapping studies of CDP571 and CDP571(gamma 1) with on-line electrospray ionization mass spectroscopy supplemented with Edman sequencing identified the chemical factor preventing inter-heavy chain disulfide bond formation between half-IgG molecules: the two cysteines in the IgG4 and IgG1 core hinge region (CPSCP and CPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low-energy conformations of 31-33 kcal/mol and 40-42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP peptide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgG1 molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than

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

    PubMed

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

    2016-01-01

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

  8. 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

  9. 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

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

    PubMed

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

    2016-05-01

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

  11. IgG Avidity ELISA Test for Diagnosis of Acute Toxoplasmosis in Humans

    PubMed Central

    Rahbari, Amir Hossien; Keshavarz, Hossien; Mohebali, Mehdi; Rezaeian, Mostafa

    2012-01-01

    Serum samples, 100 in the total number, were collected from different laboratories in Tehran, Iran and tested for anti-Toxoplasma specific IgG and IgM antibodies using indirect immunofluorescent antibody test (IFAT). Using the IgG (chronic) and IgM (acute) positive samples, the IgG avidity test was performed by ELISA in duplicate rows of 96-well microtiter plates. One row was washed with 6 M urea and the other with PBS (pH 7.2), then the avidity index (AI) was calculated. Sixteen out of 18 (88.9%) sera with acute toxoplasmosis showed low avidity levels (AI≤50), and 76 out of 82 (92.7%) sera in chronic phase of infection showed high avidity index (AI>60). Six sera had borderline ranges of AI. The results showed that the IgG avidity test by ELISA could distinguish the acute and chronic stages of toxoplasmosis in humans. PMID:22711919

  12. Chimeric mouse human IgG3 antibodies with an IgG4-like hinge region induce complement-mediated lysis more efficiently than IgG3 with normal hinge.

    PubMed

    Norderhaug, L; Brekke, O H; Bremnes, B; Sandin, R; Aase, A; Michaelsen, T E; Sandlie, I

    1991-10-01

    We have altered the amino acid sequence of the hinge and the first constant domain (CH1) of mouse/human chimeric IgG3 antibodies by site-directed mutagenesis, so as to make the sequences identical to those of IgG4. All the mutant antibodies with altered hinge region were more active in complement activation and complement-mediated lysis than native IgG3. The mutations in CH1, however, did not alter the activity. This demonstrates the importance of the hinge region in modulating this effector function. The results show that the primary structure of neither CH1 nor the hinge of IgG4 is responsible for the lack of complement activation shown by this subclass.

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2016-03-01

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

  15. Engineered humanized dimeric forms of IgG are more effective antibodies

    PubMed Central

    1992-01-01

    Humanized IgG1 M195 (HuG1-M195), a complementarity determining region- grafted recombinant monoclonal antibody, is reactive with CD33, an antigen expressed on myelogenous leukemia cells. M195 is in use in trials for the therapy of acute myelogenous leukemia. Since biological activity of IgG may depend, in part, on multimeric Fab and Fc clustering, homodimeric forms of HuG1-M195 were constructed by introducing a mutation in the gamma 1 chain CH3 region gene to change a serine to a cysteine, allowing interchain disulfide bond formation at the COOH terminal of the IgG. Despite similar avidity, the homodimeric IgG showed a dramatic improvement in the ability to internalize and retain radioisotope in target leukemia cells. Moreover, homodimers were 100-fold more potent at complement-mediated leukemia cell killing and antibody-dependent cellular cytotoxicity using human effectors. Therefore, genetically engineered multimeric constructs of IgG may have advantages relative to those forms that are found naturally. PMID:1402660

  16. Directed evolution of Her2/neu-binding IgG1-Fc for improved stability and resistance to aggregation by using yeast surface display.

    PubMed

    Traxlmayr, Michael W; Lobner, Elisabeth; Antes, Bernhard; Kainer, Manuela; Wiederkum, Susanne; Hasenhindl, Christoph; Stadlmayr, Gerhard; Rüker, Florian; Woisetschläger, Max; Moulder, Kevin; Obinger, Christian

    2013-04-01

    An Fcab (Fc antigen binding) is a crystallizable fragment of IgG having C-terminal structural loops of CH3 domains engineered for antigen binding. Since introduction of novel binding sites might impair the immunoglobulin fold, repairing strategies are needed for improving the biophysical properties of promising binders without decreasing affinity to the antigen. Here, a directed evolution protocol was developed and applied for stabilization of a Her2/neu-binding Fcab. Distinct loop regions of the parental binder were softly randomized by parsimonious mutagenesis, followed by heat incubation of the yeast displayed protein library and selection for retained antigen binding. Selected Fcabs were expressed solubly in Pichia pastoris and human embryonic kidney 293 cells and characterized. Fcab clones that retained their affinity to Her2/neu but exhibited a significantly increased conformational stability and resistance to aggregation could be evolved. Moreover, we demonstrate that simultaneous selection for binding to the antigen and to structurally specific ligands (FcγRI and an antibody directed against the CH2 domain) yields even more stable Fcabs. To sum up, this study presents a very potent and generally applicable method for improving the fold and stability of antibodies, antibody fragments and alternative binding scaffolds. PMID:23267121

  17. Fully Human Monoclonal Antibody Inhibitors of the Neonatal Fc Receptor Reduce Circulating IgG in Non-Human Primates

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  19. IgG subclass antibodies to human cytomegalovirus (CMV) in normal human plasma samples and immune globulins and their neutralizing activities.

    PubMed

    Gupta, C K; Leszczynski, J; Gupta, R K; Siber, G R

    1996-06-01

    An enzyme-linked immunoabsorbent assay (ELISA) was developed for quantitation of IgG subclass antibodies to human cytomegalovirus (CMV) in human serum or plasma samples and in immune globulin (IG) preparations. The assay was based on the parallel titration of known concentrations of purified IgG subclass myeloma proteins and a specific CMV antiserum. The purified IgG subclass myeloma proteins were captured on an ELISA plate pre-coated with anti-human kappa, anti-human lambda or a mixture of anti-human kappa and lambda antibodies and the specific antiserum was titrated against CMV antigen coated on the plate. IgG subclass antibodies, captured or bound to antigen, were quantitated with IGG subclass heavy chain specific monoclonal antibodies. The method was highly reproducible, specific and sensitive. Using this method, 257 human plasma samples and 50 IG preparations were assayed for CMV specific IgG subclass and IgM antibodies. The major IgG subclass antibody to CMV was IgG1 which represented more than 96% of CMV IgG antibodies, followed by IgG3 (mean CMV IgG3 antibody content was 3% of IgG antibodies in IG preparations and 1.8% in plasma samples). A majority of the samples had low levels of IgG2 antibodies and a few samples exhibited low levels of IgG4 antibodies. IG preparations showed very low levels of CMV IgM antibodies whereas plasma samples had 14.2% of CMV antibodies (IgG and IgM) as IgM antibodies. Virus neutralizing (Nt) activity of these samples showed a significant correlation with CMV IgG1 antibodies. Nine samples of plasma and IGs were further evaluated for Nt activity of IgG1 and IgG3 antibodies by separating IgG3 from the rest of the antibodies with protein A agarose. IgG3 antibodies showed much higher Nt activity than IgG1 antibodies suggesting that enrichment of IgG3 antibodies in IG preparations may be useful in preparing CMV specific IG.

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

    PubMed

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

    2015-03-17

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

  1. Human intestinal alkaline phosphatase-binding IgG in patients with severe bacterial infections.

    PubMed Central

    Mäder, M; Kolbus, N; Meihorst, D; Köhn, A; Beuche, W; Felgenhauer, K

    1994-01-01

    Patterns of alkaline phosphatase (AP)-binding proteins were observed in the alkaline pH range of 6.5-9.5 upon isoelectric focusing and blotting of serum from patients with inflammatory diseases. After isolation using affinity chromatography on protein A or immunoaffinity chromatography on AP coupled to cyanogen bromide (CNBr)-activated Sepharose, the AP-binding protein was identified as IgG on Western blots and in ELISA using human IgG-specific antibodies. It was shown that this IgG binds to AP from both calf (bovine) and human intestine. However, it binds neither to the human liver-bone-kidney (LBK) isoform nor to bacterial AP. Moderate reaction was observed with human placental AP. Comparing patients with various diagnoses (n = 284), AP-binding antibodies were mainly found in severe bacterial infections. They were not detected in serum from healthy blood donors (n = 300). The presence of AP-binding IgG was independent of the infected organ and the bacterial species causing infection. This antibody may be useful for discriminating bacterial from viral infection and for indicating severe bacterial inflammation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8287614

  2. Purified human plasma kallikrein aggregates human blood neutrophils.

    PubMed Central

    Schapira, M; Despland, E; Scott, C F; Boxer, L A; Colman, R W

    1982-01-01

    Exposure of human blood polymorphonuclear leukocytes (PMN) to purified active plasma kallikrein resulted in PMN aggregation when kallikrein was present at concentrations ranging from 0.4 to 0.6 U/ml (0.18-0.27 microM). Kallikrein-induced PMN aggregation was not mediated through C5-derived peptides, because identical responses were observed whether or not kallikrein had been preincubated with an antibody to C5. Moreover, kallikrein was specific for aggregating PMN, because no aggregation was observed with Factor XII active fragments (23 nM), Factor XIa (0.6 U/ml or 15nM), thrombin (1.6 microM), plasmin (2 microM), porcine pancreatic elastase (2 microM), bovine pancreatic chymotrypsin (2 microM), or bradykinin (1 microM). Bovine pancreatic trypsin (2 microM) aggregated PMN, but to a lesser extent than kallikrein (0.18 microM). Kallikrein was a potent aggregant agent for PMN because similar responses were observed with kallikrein (0.5 U/ml or 0.23 microM) and an optimal dose (0.2 microM) of N-formyl-methionyl-leucyl-phenylalanine. In addition, PMN incubation with kallikrein resulted in stimulation of their oxidative metabolism as assessed by an increased oxygen uptake. Neutropenia and leukostasis observed in diseases associated with activation of the contact phase system may be the result of PMN aggregation by plasma kallikrein. PMID:6917855

  3. Prediction of reversible IgG1 aggregation occurring in a size exclusion chromatography column is enabled through a model based approach.

    PubMed

    Ojala, Frida; Sellberg, Anton; Hansen, Thomas Budde; Hansen, Ernst Broberg; Nilsson, Bernt

    2015-09-01

    One important aspect of antibody separation being studied today is aggregation, as this not only leads to a loss in yield, but aggregates can also be hazardous if injected into the body. The aim of this study was to determine whether the methodology applied in the previous study could be used to predict the aggregation of a different batch of IgG1, and to model the aggregation occurring in a SEC column. Aggregation was found to be reversible. The equilibrium parameter was found to be 272 M(-1) and the reaction kinetic parameter 1.33 × 10(-5) s(-1) , both within the 95% confidence interval of the results obtained in the previous work. The effective diffusivities were estimated to be 1.45 × 10(-13) and 1.90 10(-14) m(2) /s for the monomers and dimers, respectively. Good agreement was found between the new model and the chromatograms obtained in the SEC experiments. The model was also able to predict the decrease of dimers due to the dilution and separation in the SEC column during long retention times.

  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. 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.

  6. 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.

  7. Contribution of variable domains to the stability of humanized IgG1 monoclonal antibodies.

    PubMed

    Ionescu, Roxana M; Vlasak, Josef; Price, Colleen; Kirchmeier, Marc

    2008-04-01

    Temperature-induced unfolding of three humanized IgG1 monoclonal antibodies and their Fab and Fc fragments was monitored by differential scanning calorimetry at neutral pH. With some exceptions, the thermogram of the intact antibody presents two peaks and the transition with the larger experimental enthalpy contains the contribution from the Fab fragments. Although the measured enthalpy was similar for all three Fab fragments studied, the apparent melting temperatures were found to vary significantly, even for Fab fragments originating from the same human germline. Therefore, we propose to use the measured enthalpy of unfolding as the key parameter to recognize the unfolding events in the melting profile of an intact IgG1 antibody. If the variable domain sequences, resulting from complementarity determining regions (CDRs) grafting and humanization, destabilize the Fab fragment with respect to the CH3 domain, the first transition represents the unfolding of the Fab fragment and the CH2 domain, while the second transition represents CH3 domain unfolding. Otherwise, the first transition represents CH2 domain unfolding, and the second transition represents the unfolding of the Fab fragment and the CH3 domain. In some cases, the DSC profile may present three transitions, with the Fab unfolding occurring at distinct temperatures compared to the melting of the CH2 and CH3 domains. If the DSC profile of a humanized IgG1 monoclonal antibody cannot be described by the model above, the result may be an indication of significant structural heterogeneity and/or of disruption of the Fab cooperative unfolding. Low stability or heterogeneity of the Fab fragment may prove problematic for long-term storage or consistency of production. Therefore, understanding the features of a DSC profile is important for clone selection and process maturation in the early stages of development of therapeutic monoclonal antibodies.

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

    PubMed

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

    2015-06-01

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

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

    PubMed

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

    2014-08-15

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

  10. Anti-neutrophil cytoplasmic antibody-enriched IgG induces adhesion of human T lymphocytes to extracellular matrix proteins.

    PubMed

    Tomer, Y; Lider, O; Gilburd, B; Hershkoviz, R; Meroni, P L; Wiik, A; Shoenfeld, Y

    1997-06-01

    Recent studies have shown that anti-neutrophil cytoplasmic antibodies (ANCA) can activate neutrophils to adhere to endothelium, degranulate, and cause endothelial cell injury. These data have lead to the hypothesis that the T cell inflammatory response causing the vasculitis in Wegener's granulomatosis (WG) is secondary to stimulation of neutrophils by ANCA. So far there is no evidence for a direct effect of ANCA on lymphocytes. The present study was designed to examine whether lymphocytes can be directly stimulated by ANCA to adhere to endothelial extracellular matrix (ECM) proteins. Human and mouse ANCA-enriched IgG were tested for their ability to increase adhesion of human T lymphocytes to fibronectin, laminin, and intact ECM. Incubation of human T lymphocytes with human ANCA-enriched IgG increased adhesion of the lymphocytes in a dose-dependent manner to fibronectin, laminin, and intact ECM (the percentage adhesion to intact ECM was 55.7 +/- 3.1 and 45.0 +/- 1.0% for lymphocytes incubated with human IgG containing ANCA or control human IgG, respectively; P = 0.0045). The same induction of adhesion to fibronectin, laminin, and intact ECM was observed when the cells were incubated with the F(ab)2 fragment of ANCA-enriched IgG. Similarly, ANCA-enriched IgG produced in mice increased the adhesion of lymphocytes to fibronectin (the percentage adhesion to fibronectin was 29.7 +/- 4.3 and 16.6 +/- 1.9% for lymphocytes incubated with mouse IgG-ANCA or control mouse IgG, respectively; P = 0.0008). These results may suggest that ANCA can directly stimulate lymphocytes to adhere to endothelial ECM and to induce the vasculitic lesions of WG. It remains to be shown by which mechanisms ANCA stimulate lymphocytes to adhere to ECM. PMID:9175913

  11. 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.

  12. Self-aggregated deglycosylated IgA1 with or without IgG were associated with the development of IgA nephropathy.

    PubMed

    Yan, Y; Xu, L-X; Zhang, J-J; Zhang, Y; Zhao, M-H

    2006-04-01

    IgA nephropathy (IgAN) is the most common primary glomerulonephritis, with various pathological phenotypes. Our previous study suggested that aberrant glycosylation of serum IgA1 was associated with different pathological phenotypes of IgAN, and substantial evidence indicated that deglycosylated IgA1 had an increased tendency to form macromolecules. The aim of the current study was to investigate the composition of IgA1-containing macromolecules in different pathological phenotypes of IgAN. Sera from 10 patients with mild mesangial proliferative IgAN (mIgAN), 10 with focal proliferative sclerosing IgAN (psIgAN) and 10 healthy blood donors were collected. The sera were applied and IgA1 binding proteins (IgA1-BP) were eluted from the columns immobilized with desialylated IgA1 (DesIgA1/Sepharose) or desialylated/degalactosylated IgA1 (DesDeGalIgA1/Sepharose), respectively. The amounts of IgA1 and IgG and the glycoform of IgA1 in the IgA1-BP were detected by enzyme-linked immunosorbent assays (ELISAs) and were compared between patients with different pathological phenotypes and normal controls. The amount of IgA1 in IgA1-BP eluted from both columns was significantly higher in patients with both pathological phenotypes of IgAN than in normal controls. In IgA1-BP eluted from DesDeGalIgA1/Sepharose, the desialylation of IgA1 was much more pronounced in patients with both pathological phenotypes of IgAN than in normal controls, while the degalactosylation of IgA1 was much more pronounced only in patients with psIgAN than in normal controls. Furthermore, the amount of IgG in IgA1-BP eluted from DesDeGalIgA1/Sepharose was significantly higher in patients with psIgAN than in normal controls. In patients with psIgAN, the amount of IgG eluted from DesDeGalIgA1/Sepharose was much greater than from DesIgA1/Sepharose. In conclusion, self-aggregated deglycosylated IgA1 with or without IgG were associated with the development of IgAN.

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

    PubMed Central

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

    2015-01-01

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

  14. [Expression of human IL-35-IgG4 (Fc) fusion protein in CHO/DG44 cells].

    PubMed

    Tang, Jing; Gao, Wenda; Zhang, Qing; Zhang, Dawei; Chen, Yang; He, Bo; Liu, Quansheng

    2009-01-01

    We constructed the eukaryotic expression vector of human IL-35-IgG4 (Fc)-pOptiVEC-TOPO by gene recombination technique and expressed the fusion protein human IL-35-IgG4 (Fc) in CHO/DG44 cells. The two components of the newly discovered cytokine human IL-35, EBI3 and IL-12p35, were amplified by PCR from the cDNA library derived from the KG-I cells after LPS induction. The two PCR-amplified cDNA fragments of human IL-35 were linked by over-lapping PCR and then cloned into the IgG4 (Fc)-pOptiVEC-TOPO vector. The constructed plasmid with the recombinant cDNA IL-35-IgG4 (Fc) was verified by restriction enzyme digestion analysis, PCR and DNA sequencing. The verified plasmid with the recombinant cDNA was transfected into CHO/DG44 cells using Lipofectamine 2000. The success of the transfection was examined and confirmed by RT-PCR. After selection in alpha-MEM (-) medium, the IL-35-Ig G4 (Fc) positive CHO/DG44 clones were chosen and the media from these positive clones were collected to be used to purify the fusion protein. The positive CHO/DG44 clones were further cultured in increasing concentrations of MTX and the expression levels of the fusion protein IL-35-Ig G4 (Fc) were repetitively induced by MTX-induced gene amplification. The IL-35-IgG4 (Fc) fusion protein was purified from the media collected from the positive CHO/DG44 clones by protein G affinity chromatography and then identified by SDS-PAGE and Western blotting. The results showed that one protein band was found to match well with the predicted relative molecular mass of human IL-35-IgG4 (Fc) and this protein could specifically bind to anti-human IgG4 (Fc) monoclonal antibody. In conclusion, our study successfully established an IL-35-IgG4 (Fc) positive DG44 cell line which could stably express IL-35-IgG4 (Fc) fusion protein.

  15. 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

  16. Participation of nitric oxide signaling system in the cardiac muscarinic cholinergic effect of human chagasic IgG.

    PubMed

    Sterin-Borda, L; Leiros, C P; Goin, J C; Cremaschi, G; Genaro, A; Echagüe, A V; Borda, E

    1997-07-01

    The possible role of altered humoral immune response in the pathogenesis of the chronic chagasic cardioneuromyopathy was examined by analyzing the interaction of IgG from T. cruzi infected patients with cardiac muscarinic acetylcholine receptors (mAChR). Human chagasic IgG by activating cardiac M2 mAChR, simulated the agonist actions triggering negative inotropic effect, inositol phosphate accumulation, nitric oxide synthase stimulation and increased production of cyclic GMP. Inhibitors of phospholipase C, protein kinase C, calcium/calmodulin, nitric oxide synthase and guanylate cyclase activities; prevented chagasic IgG effects on signaling pathways involved in M2 mAChR activation. In addition, sodium nitroprusside or 8-bromo cyclic GMP, mimicked the chagasic IgG effect associated with cholinergic-mediated cellular transmembrane signals. Moreover, these chagasic IgG immunoprecipitated the mAChRs solubilized from cardiac membranes. By means of SDS-PAGE and immunoblotting analysis, chagasic sera recognized a band of 70-75 kDa. The major protein recognized by chagasic IgG had an Rf coincident with the peak of [3H] propylbenzilylcholine mustard with an apparent molecular weight similar to that of mAChRs, which disappeared in the presence of atropine. The specificity of this interaction was checked by immunoprecipitation of rat cardiac mAChR and immunoblotting of pure human M2 mAChRs. Chronic interaction of chagasic IgG with myocardial mAChRs, behaving as a muscarinic agonist, might lead to cell dysfunction or tissue damage. Also, these antibodies could produce desensitization, internalization or degradation of mAChRs; explaining the progressive blockade of mAChRs in myocardium with parasympathetic denervation, a phenomenon that has been described in the course of Chagas' cardioneuromyopathy. PMID:9236139

  17. Neutralizing IgG at the Portal of Infection Mediates Protection against Vaginal Simian/Human Immunodeficiency Virus Challenge

    PubMed Central

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

    2013-01-01

    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. PMID:23966410

  18. 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

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2015-12-01

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

  1. Variable region domain exchange in human IgGs promotes antibody complex formation with accompanying structural changes and altered effector functions.

    PubMed

    Chan, Lisa A; Phillips, Martin L; Wims, Letitia A; Trinh, K Ryan; Denham, Jerrod; Morrison, Sherie L

    2004-07-01

    Variable region domain exchanged IgG, or "inside-out (io)," molecules, were produced to investigate the effects of domain interactions on antibody structure and function. Studies using ultracentrifugation and electron microscopy showed that variable region domain exchange induces non-covalent multimerization through Fab domains. Surprisingly, variable region exchange also affected Fc-associated functions such as serum half-life and binding to protein G and FcgammaRI. These alterations were not merely a consequence of IgG aggregation. Both the extent of multimerization and alterations in Fc-associated properties depended on the IgG isotype.

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

    PubMed Central

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

    1984-01-01

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

  3. Evidence for trisulfide bonds in a recombinant variant of a human IgG2 monoclonal antibody.

    PubMed

    Pristatsky, Pavlo; Cohen, Steven L; Krantz, Debra; Acevedo, Jillian; Ionescu, Roxana; Vlasak, Josef

    2009-08-01

    The hinge region of human IgG2 contains four cysteine residues involved in disulfide linkages between the heavy chains, as well as the heavy and light chains. These linkages provide the fundamental framework of three distinct IgG2 disulfide isoforms recently described. Here, we detail another, disulfide-related post-translational modification in a recombinant variant of human IgG2. Heterogeneity associated with this antibody was separated into several fractions by anion-exchange chromatography (AEX), which is an important initial step that highlights the resolving power of surface charge-based HPLC techniques. Mass spectrometry of the intact antibody revealed weakly resolved discrete covalent additions of 25-35 Da in one of the two main AEX fractions. Digestion by endoproteinase Lys-C performed under nonreducing conditions, as well as tandem MS experiments, narrowed the modification to the peptide-containing disulfide-bridged hinge structure. High mass resolution and accuracy measurements of the peptide strongly suggested an addition of one or two S atoms. The modification could be eliminated by a mild reducing treatment of the intact antibody. Overall, these findings are consistent with the replacement of up to two disulfide bridges (S-S) with a like number of trisulfides (S-S-S) in the antibody hinge. The trisulfide modification is rather uncommon for proteins and its possible origins in the IgG2 variant are discussed.

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

    PubMed

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

    2004-01-01

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

  5. 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.

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

    PubMed

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

    2004-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

    Xu, Juan; Zhang, Chunhua

    2016-05-01

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

  9. 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

  10. Structural basis for cyclodextrins' suppression of human growth hormone aggregation

    PubMed Central

    Otzen, Daniel Erik; Knudsen, Benjamin Raerup; Aachmann, Finn; Larsen, Kim Lambertsen; Wimmer, Reinhard

    2002-01-01

    Many therapeutic proteins require storage at room temperature for extended periods of time. This can lead to aggregation and loss of function. Cyclodextrins (CDs) have been shown to function as aggregation suppressors for a wide range of proteins. Their potency is often ascribed to their affinity for aromatic amino acids, whose surface exposure would otherwise lead to protein association. However, no detailed structural studies are available. Here we investigate the interactions between human growth hormone (hGH) and different CDs at low pH. Although hGH aggregates readily at pH 2.5 in 1 M NaCl to form amorphous aggregates, the presence of 25 to 50 mM of various β-CD derivatives is sufficient to completely avoid this. α- and γ-CD are considerably less effective. Stopped-flow data on the aggregation reaction in the presence of β-CD are analyzed according to a minimalist association model to yield an apparent hGH-β-CD dissociation constant of ∼6 mM. This value is very similar to that obtained by simple fluorescence-based titration of hGH with β-CD. Nuclear magnetic resonance studies indicate that β-CD leads to a more unfolded conformation of hGH at low pH and predominantly binds to the aromatic side-chains. This indicates that aromatic amino acids are important components of regions of residual structure that may form nuclei for aggregation. PMID:12070330

  11. 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

  12. Serologic cross-reactivity of human IgM and IgG antibodies to five species of Ebola virus.

    PubMed

    Macneil, Adam; Reed, Zachary; Rollin, Pierre E

    2011-06-01

    Five species of Ebola virus (EBOV) have been identified, with nucleotide differences of 30-45% between species. Four of these species have been shown to cause Ebola hemorrhagic fever (EHF) in humans and a fifth species (Reston ebolavirus) is capable of causing a similar disease in non-human primates. While examining potential serologic cross-reactivity between EBOV species is important for diagnostic assays as well as putative vaccines, the nature of cross-reactive antibodies following EBOV infection has not been thoroughly characterized. In order to examine cross-reactivity of human serologic responses to EBOV, we developed antigen preparations for all five EBOV species, and compared serologic responses by IgM capture and IgG enzyme-linked immunosorbent assay (ELISA) in groups of convalescent diagnostic sera from outbreaks in Kikwit, Democratic Republic of Congo (n=24), Gulu, Uganda (n=20), Bundibugyo, Uganda (n=33), and the Philippines (n=18), which represent outbreaks due to four different EBOV species. For groups of samples from Kikwit, Gulu, and Bundibugyo, some limited IgM cross-reactivity was noted between heterologous sera-antigen pairs, however, IgM responses were largely stronger against autologous antigen. In some instances IgG responses were higher to autologous antigen than heterologous antigen, however, in contrast to IgM responses, we observed strong cross-reactive IgG antibody responses to heterologous antigens among all sets of samples. Finally, we examined autologous IgM and IgG antibody levels, relative to time following EHF onset, and observed early peaking and declining IgM antibody levels (by 80 days) and early development and persistence of IgG antibodies among all samples, implying a consistent pattern of antibody kinetics, regardless of EBOV species. Our findings demonstrate limited cross-reactivity of IgM antibodies to EBOV, however, the stronger tendency for cross-reactive IgG antibody responses can largely circumvent limitations in the

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

    PubMed

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

    2015-12-01

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

  14. Cross-clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG.

    PubMed Central

    Trkola, A; Pomales, A B; Yuan, H; Korber, B; Maddon, P J; Allaway, G P; Katinger, H; Barbas, C F; Burton, D R; Ho, D D

    1995-01-01

    We have tested three human monoclonal antibodies (MAbs) IgG1b12, 2G12, and 2F5) to the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1), and a tetrameric CD4-IgG molecule (CD4-IgG2), for the ability to neutralize primary HIV-1 isolates from the genetic clades A through F and from group O. Each of the reagents broadly and potently neutralized B-clade isolates. The 2F5 MAb and the CD4-IgG2 molecule also neutralized strains from outside the B clade, with the same breadth and potency that they showed against B-clade strains. The other two MAbs were able to neutralize a significant proportion of strains from outside the B clade, although there was a reduction in their efficacy compared with their activity against B-clade isolates. Neutralization of isolates by 2F5 correlated with their possession of the LDKW motif in a segment of gp41 near the membrane-spanning domain. The other two MAbs and CD4-IgG2 recognize discontinuous binding sites on gp120, and so no comparison between genetic sequence and virus neutralization was possible. Our data show that a vaccine based on the induction of humoral immunity that is broadly active across the genetic clades is not impossible if immunogens that express the epitopes for MAbs such as 2F5, 2G12, and IgG1b12 in immunogenic configurations can be created. Furthermore, if the three MAbs and CD4-IgG2 produce clinical benefit in immunotherapeutic trials in the United States or Europe, they may also do so elsewhere in the world. PMID:7474069

  15. Development of a Human IgG4 Bispecific Antibody for Dual Targeting of Interleukin-4 (IL-4) and Interleukin-13 (IL-13) Cytokines*

    PubMed Central

    Spiess, Christoph; Bevers, Jack; Jackman, Janet; Chiang, Nancy; Nakamura, Gerald; Dillon, Michael; Liu, Hongbin; Molina, Patricia; Elliott, J. Michael; Shatz, Whitney; Scheer, Justin M.; Giese, Glen; Persson, Josefine; Zhang, Yin; Dennis, Mark S.; Giulianotti, James; Gupta, Prateek; Reilly, Dorothea; Palma, Enzo; Wang, Jianyong; Stefanich, Eric; Scheerens, Heleen; Fuh, Germaine; Wu, Lawren C.

    2013-01-01

    Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes. PMID:23880771

  16. 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

  17. Characterization of the stability of a fully human monoclonal IgG after prolonged incubation at elevated temperature.

    PubMed

    Liu, Hongcheng; Gaza-Bulseco, Georgeen; Sun, Joanne

    2006-06-01

    The susceptible degradation sites of therapeutic proteins are routinely assessed under accelerated conditions such as exposure to chemicals or incubation at elevated temperature or a combination of both. A fully human monoclonal IgG(1) antibody was characterized after incubation at 40 degrees C for 6 months by employing mass spectrometry and chromatography analyses. It was found that deamidation, fragmentation and N-terminal glutamate cyclization to form pyroglutamate are the major degradation pathways. Three major deamidation sites were identified and one site in a small tryptic peptide accounted for more than 80% of the total. Peptide cleavage was observed at several positions between different pairs of amino acids. Most of the cleavage sites were located in the hinge or other flexible regions of the IgG molecule.

  18. A novel chemiluminescent immunoassay for detection of Toxoplasma gondii IgG in human sera.

    PubMed

    Holec-Gąsior, Lucyna; Ferra, Bartłomiej; Czechowska, Justyna; Serdiuk, Illia E; Krzymiński, Karol; Kur, Józef

    2016-08-01

    This study describes Toxoplasma gondii IgG chemiluminescent immunoassay (CLIA) based on the use of a novel immunochemical reagents in the form of the conjugates of original acridinium ester (AE) labels attached to antibodies and SAG2-GRA1-ROP1L chimeric antigen and shows that this test is useful for diagnostic purposes.

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

    PubMed Central

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

    2015-01-01

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

  20. Development of a Luciferase Immunoprecipitation System Assay To Detect IgG Antibodies against Human Respiratory Syncytial Virus Nucleoprotein

    PubMed Central

    Kumari, Sangeeta; Crim, Roberta Lynne; Kulkarni, Ashwin; Audet, Susette A.; Mdluli, Thembi; Murata, Haruhiko

    2014-01-01

    The nucleoprotein of respiratory syncytial virus (RSV-N) is immunogenic and elicits an IgG response following infection. The RSV-N gene was cloned into a mammalian expression vector, pREN2, and the expressed luciferase-tagged protein (Ruc-N) detected anti-RSV-N-specific IgG antibodies using a high-throughput immunoprecipitation method (the luciferase immunoprecipitation system [LIPS]-NRSV assay). The specificity of the assay was evaluated using monoclonal antibodies (MAbs) and monospecific pre- and postimmunization rabbit antisera. Blood serum samples from chimpanzees and humans with proven/probable RSV infection were also tested. The pre- and postimmunization serum samples from rabbits given human metapneumovirus (HMPV) or measles virus were negative when tested by the LIPS-NRSV assay, while antisera obtained after immunization with either the RSV-A or RSV-B strain gave positive signals in a dose-dependent manner. RSV-N MAb 858-3 gave a positive signal in the LIPS-NRSV assay, while MAbs against other paramyxovirus nucleoproteins or RSV-F or RSV-G did not. Serum samples from chimpanzees simultaneously immunized with vaccinia-RSV-F and vaccinia-RSV-G recombinant viruses were negative in the LIPS-NRSV assay; however, anti-RSV-N IgG responses were detected following subsequent RSV challenge. Seven of the 12 infants who were seronegative at 9 months of age had detectable anti-RSV-N antibodies when they were retested at 15 to 18 months of age. The LIPS-NRSV assay detects specific anti-RSV-N IgG responses that may be used as a biomarker of RSV infection. PMID:24403526

  1. Acidic-store depletion is required for human platelet aggregation.

    PubMed

    Amor, Nidhal Ben; Zbidi, Hanene; Bouaziz, Aicha; Jardin, Isaac; Isaac, Jardin; Hernández-Cruz, Juan M; Salido, Ginés M; Rosado, Juan A; Bartegi, Aghleb

    2009-10-01

    Platelet stimulation with thrombin induces an elevation in cytoplasmic free Ca(2+) concentration ([Ca(2+)]c) due to Ca(2+) release from intracellular stores and entry from the extracellular medium. Two different intracellular Ca(2+) stores have been described in human platelets: the dense tubular system and the lysosomal-like acidic stores. In the present study, we investigated the contribution of the acidic stores in thrombin-induced platelet aggregation. We have found that platelet aggregation induced by thrombin is reduced in a Ca(2+)-free medium. Discharge of the acidic Ca(2+) stores by treatment with the sarcoendoplasmic Ca(2+)-ATPase (SERCA)3 selective inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone reduced thrombin-evoked platelet aggregation. In the presence of 2,5-di-(tert-butyl)-1,4-hydroquinone, platelet aggregation induced by the protease-activated receptor (PAR)-1 and PAR-4 agonist peptides, SFLLRN and AYPGKF, respectively, was significantly reduced. In cells with depleted acidic stores, activation of GPIb-IX-V by thrombin resulted in reduced or no platelet aggregation in a medium containing 1 mmol/l Caor in a Ca(2+)-free medium, respectively. This finding suggests that Ca(2+) accumulation in the acidic Ca(2+) compartments is required for platelet aggregation induced by activation of the G-coupled PAR-1 and PAR-4 thrombin receptors and, by the occupation of the leucine-rich glycoprotein GPIb-IX-V and provide evidence supporting a functional role of the lysosomal-like acidic Ca(2+) stores in human platelets. PMID:19587585

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

    PubMed

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

    2010-10-01

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

  3. Inhibitory effects of magnolol and honokiol on human calcitonin aggregation

    PubMed Central

    Guo, Caiao; Ma, Liang; Zhao, Yudan; Peng, Anlin; Cheng, Biao; Zhou, Qiaoqiao; Zheng, Ling; Huang, Kun

    2015-01-01

    Amyloid formation is associated with multiple amyloidosis diseases. Human calcitonin (hCT) is a typical amyloidogenic peptide, its aggregation is associated with medullary carcinoma of the thyroid (MTC), and also limits its clinical application. Magnolia officinalis is a traditional Chinese herbal medicine; its two major polyphenol components, magnolol (Mag) and honokiol (Hon), have displayed multiple functions. Polyphenols like flavonoids and their derivatives have been extensively studied as amyloid inhibitors. However, the anti-amyloidogenic property of a biphenyl backbone containing polyphenols such as Mag and Hon has not been reported. In this study, these two compounds were tested for their effects on hCT aggregation. We found that Mag and Hon both inhibited the amyloid formation of hCT, whereas Mag showed a stronger inhibitory effect; moreover, they both dose-dependently disassembled preformed hCT aggregates. Further immuno-dot blot and dynamic light scattering studies suggested Mag and Hon suppressed the aggregation of hCT both at the oligomerization and the fibrillation stages, while MTT-based and dye-leakage assays demonstrated that Mag and Hon effectively reduced cytotoxicity caused by hCT aggregates. Furthermore, isothermal titration calorimetry indicated Mag and Hon both interact with hCT. Together, our study suggested a potential anti-amyloidogenic property of these two compounds and their structure related derivatives. PMID:26324190

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

    PubMed Central

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

    2015-01-01

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

  5. 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

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

    PubMed

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

    2016-04-01

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

  7. Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions.

    PubMed

    Grevys, Algirdas; Bern, Malin; Foss, Stian; Bratlie, Diane Bryant; Moen, Anders; Gunnarsen, Kristin Støen; Aase, Audun; Michaelsen, Terje Einar; Sandlie, Inger; Andersen, Jan Terje

    2015-06-01

    Engineering of the constant Fc part of monoclonal human IgG1 (hIgG1) Abs is an approach to improve effector functions and clinical efficacy of next-generation IgG1-based therapeutics. A main focus in such development is tailoring of in vivo half-life and transport properties by engineering the pH-dependent interaction between IgG and the neonatal Fc receptor (FcRn), as FcRn is the main homeostatic regulator of hIgG1 half-life. However, whether such engineering affects binding to other Fc-binding molecules, such as the classical FcγRs and complement factor C1q, has not been studied in detail. These effector molecules bind to IgG1 in the lower hinge-CH2 region, structurally distant from the binding site for FcRn at the CH2-CH3 elbow region. However, alterations of the structural composition of the Fc may have long-distance effects. Indeed, in this study we show that Fc engineering of hIgG1 for altered binding to FcRn also influences binding to both the classical FcγRs and complement factor C1q, which ultimately results in alterations of cellular mechanisms such as Ab-dependent cell-mediated cytotoxicity, Ab-dependent cellular phagocytosis, and Ab-dependent complement-mediated cell lysis. Thus, engineering of the FcRn-IgG1 interaction may greatly influence effector functions, which has implications for the therapeutic efficacy and use of Fc-engineered hIgG1 variants.

  8. Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions

    PubMed Central

    Grevys, Algirdas; Bern, Malin; Foss, Stian; Bratlie, Diane Bryant; Moen, Anders; Gunnarsen, Kristin Støen; Aase, Audun; Michaelsen, Terje Einar; Sandlie, Inger

    2015-01-01

    Engineering of the constant Fc part of monoclonal human IgG1 (hIgG1) Abs is an approach to improve effector functions and clinical efficacy of next-generation IgG1-based therapeutics. A main focus in such development is tailoring of in vivo half-life and transport properties by engineering the pH-dependent interaction between IgG and the neonatal Fc receptor (FcRn), as FcRn is the main homeostatic regulator of hIgG1 half-life. However, whether such engineering affects binding to other Fc-binding molecules, such as the classical FcγRs and complement factor C1q, has not been studied in detail. These effector molecules bind to IgG1 in the lower hinge–CH2 region, structurally distant from the binding site for FcRn at the CH2–CH3 elbow region. However, alterations of the structural composition of the Fc may have long-distance effects. Indeed, in this study we show that Fc engineering of hIgG1 for altered binding to FcRn also influences binding to both the classical FcγRs and complement factor C1q, which ultimately results in alterations of cellular mechanisms such as Ab-dependent cell-mediated cytotoxicity, Ab-dependent cellular phagocytosis, and Ab-dependent complement-mediated cell lysis. Thus, engineering of the FcRn–IgG1 interaction may greatly influence effector functions, which has implications for the therapeutic efficacy and use of Fc-engineered hIgG1 variants. PMID:25904551

  9. 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 Central

    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. PMID:25302805

  10. Variable domain-linked oligosaccharides of a human monoclonal IgG: structure and influence on antigen binding.

    PubMed Central

    Leibiger, H; Wüstner, D; Stigler, R D; Marx, U

    1999-01-01

    The variable-domain-attached oligosaccharide side chains of a human IgG produced by a human-human-mouse heterohybridoma were analysed. In addition to the conserved N-glycosylation site at Asn-297, an N-glycosylation consensus sequence (Asn-Asn-Ser) is located at position 75 in the variable region of its heavy chain. The antibody was cleaved into its antigen-binding (Fab) and crystallizing fragments. The oligosaccharides of the Fab fragment were released by digestion with various endo- and exoglycosidases and analysed by anion-exchange chromatography and fluorophore-assisted carbohydrate electrophoresis. The predominant components were disialyl- bi-antennary and tetra-sialyl tetra-antennary complex carbohydrates. Of note is the presence in this human IgG of oligosaccharides containing N-glycolylneuraminic acid and N-acetylneuraminic acid in the ratio of 94:6. Furthermore, we determined N-acetylgalactosamine in the Fab fragment of this antibody, suggesting the presence of O-linked carbohydrates. A three-dimensional structure of the glycosylated variable (Fv) fragment was suggested using computer-assisted modelling. In addition, the influence of the Fv-associated oligosaccharides of the CBGA1 antibody on antigen binding was tested in several ELISA systems. Deglycosylation resulted in a decreased antigen-binding activity. PMID:10024532

  11. 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

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

    PubMed

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

    2012-01-01

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

  13. 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.

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

    PubMed

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

    2014-01-01

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

  15. Engineering upper hinge improves stability and effector function of a human IgG1.

    PubMed

    Yan, Boxu; Boyd, Daniel; Kaschak, Timothy; Tsukuda, Joni; Shen, Amy; Lin, Yuwen; Chung, Shan; Gupta, Priyanka; Kamath, Amrita; Wong, Anne; Vernes, Jean-Michel; Meng, Gloria Y; Totpal, Klara; Schaefer, Gabriele; Jiang, Guoying; Nogal, Bartek; Emery, Craig; Vanderlaan, Martin; Carter, Paul; Harris, Reed; Amanullah, Ashraf

    2012-02-17

    Upper hinge is vulnerable to radical attacks that result in breakage of the heavy-light chain linkage and cleavage of the hinge of an IgG1. To further explore mechanisms responsible for the radical induced hinge degradation, nine mutants were designed to determine the roles that the upper hinge Asp and His play in the radical reactions. The observation that none of these substitutions could inhibit the breakage of the heavy-light chain linkage suggests that the breakage may result from electron transfer from Cys(231) directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His(229)-mediated hinge cleavage. On the other hand, the substitution of His(229) with Tyr showed promising advantages over the native antibody and other substitutions in improving the stability and function of the IgG1. This substitution inhibited the hinge cleavage by 98% and suggests that the redox active nature of Tyr did not enable it to replicate the ability of His to facilitate radical induced degradation. We propose that the lower redox potential of Tyr, a residue that may be the ultimate sink for oxidizing equivalents in proteins, is responsible for the inhibition. More importantly, the substitution increased the antibody's binding to FcγRIII receptors by 2-3-fold, and improved ADCC activity by 2-fold, while maintaining a similar pharmacokinetic profile with respect to the wild type. Implications of these observations for antibody engineering and development are discussed.

  16. Impact of methionine oxidation in human IgG1 Fc on serum half-life of monoclonal antibodies.

    PubMed

    Wang, Weirong; Vlasak, Josef; Li, Yunsong; Pristatsky, Pavlo; Fang, Yulin; Pittman, Tamara; Roman, Jeanette; Wang, Yang; Prueksaritanont, Thomayant; Ionescu, Roxana

    2011-03-01

    IgG monoclonal antibodies (mAbs) consist of two Fab fragments and one Fc fragment. The Fab fragments contain the variable regions and are responsible for drug specificity (via antigen binding); the Fc fragment contains constant regions and is responsible for effector functions (via interactions with Fcγ receptors) and extended serum half-life (via interaction with the neonatal Fc receptor, FcRn). There are two conserved methionine (Met) residues located in the FcRn binding site of the Fc fragment. It has been shown previously that oxidation of these two Met residues decreases the binding affinity to FcRn. We have further evaluated the impact of Met oxidation on serum half-lives of two humanized IgG1 mAbs in transgenic mice with human FcRn. Variable oxidation levels were obtained by several procedures: exposure to an oxidizing agent, accumulation during extended refrigerated storage, or chromatographic separation. Our results show that Met oxidation can result in a significant reduction of the serum circulation half-life and the magnitude of the change correlates well with the extent of Met oxidation and changes in FcRn binding affinities. The relatively low levels of Met oxidation accumulated during 3 years of refrigerated storage had minimal impact on FcRn binding and no detectable impact on the serum half-life.

  17. IgG antibodies against human papillomavirus type 16 E7 proteins in cervicovaginal washing fluid from patients with cervical neoplasia.

    PubMed

    Tjiong, M. Y.; Schegget, J. Ter; Tjiong-A-Hung, S. P.; Out, T. A.; Van Der Vange, N.; Burger, M. P. M.; Struyk, L.

    2000-07-01

    Little information is available about the cervicovaginal mucosal antibodies against human papillomavirus (HPV) proteins. In this study specific IgG antibodies against HPV 16 E7 protein were determined in paired samples of cervicovaginal washing fluid and serum from patients with cervical cancer (n = 22), cervical intraepithelial neoplasia (CIN) (n = 38), healthy individuals (n = 22), and serum from children (n = 41) by a radioactive immunoprecipitation assay (RIPA). HPV 16 E7 specific IgG antibodies were found in cervicovaginal washings (n = 8) and in sera (n = 8) of the patients with cervical cancer. About 60% of the patients with HPV 16 positive cervical cancer had HPV 16 E7 specific IgG antibodies. Titration studies showed that the IgG antibody reactivity in cervicovaginal washings was higher than in the paired serum samples of six patients with cervical cancer (P < 0.001). In the CIN group we found no IgG reactivity in the serum, but in five patients we found a low IgG reactivity in the cervicovaginal washings. No IgG reactivity was found in cervicovaginal washings and sera from healthy individuals and sera from children. HPV 16 E7 specific IgG antibodies seem to be locally produced in a number of patients with HPV 16 positive (pre)malignant cervical lesions. For more definitive evidence for the local production of these antibodies immunostaining should be performed to demonstrate the presence of specific anti-HPV 16 E7 IgG producing plasma cells in the cervical epithelium.

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

    PubMed Central

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

    2015-01-01

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

  19. Modulation of the murine immune response to human IgG by complexing with monoclonal antibodies. II. Antibody responses to idiotopes of the human IgG paraprotein and of the mouse monoclonal antibodies.

    PubMed

    Ling, N R; Elliott, D; Lowe, J

    1987-09-01

    Anti-idiotope antibodies produced by mice immunized with a human IgG paraprotein complexed with various mouse monoclonal antibodies (McAbs) have been measured. All animals receiving more than one injection of the paraprotein (free or complexed with a mouse McAb) produced antibodies to the idiotypes of the paraprotein. Complexing with a McAb, especially an anti-Fc-gamma McAb, enhanced the response. Antibodies to the idiotopes of mouse McAbs were more difficult to produce and their production was very dependent on the mode and schedule of the immunization. The best antisera were produced by mice receiving a course of injections of pre-formed complexes of the IgG paraprotein and McAbs. Four of five mice produced antibodies to the idiotopes of an anti-light chain McAb (C4) after a course of immunization (one primary plus four boosts) of an IgG-C4 complex. Two of the six mice receiving a similar course of injections of the paraprotein complexed with an anti-gamma McAb (A55) produced high titres of antibodies to A55 idiotypes. Responses were enhanced when complexes were prepared with a pool of McAbs. It is probable that the formation of large multi-cross-linked complexes containing the McAb under study is important in generating the response. Once a response is initiated, very high titres may be achieved.

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

    PubMed Central

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

    2013-01-01

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

  1. Immuno-column for on-line quantification of human serum IgG antibodies to Helicobacter pylori in human serum samples.

    PubMed

    Molina, Luis; Messina, Germán A; Stege, Patrícia W; Salinas, Eloy; Raba, Julio

    2008-09-15

    This study report an human serum IgG antibodies to Helicobacter pylori quantitation procedure based on the multiple use of an immobilized H. pylori antigen on an immuno-column incorporated into an a flow-injection (FI) analytical system. The immuno-adsorbent column was prepared by packing 3-aminopropyl-modified controlled-pore glass (APCPG) covalently linking H. pylori antigens in a 3-cm of Teflon tubing (0.5 i.d.). Antibodies in the serum sample are allowed to react immunologically with the immobilized H. pylori antigen, and the bound antibodies are quantified by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. p-Aminophenyl phosphate (pAPP) was converted to p-aminophenol (pAP) by AP and an electroactive product was quantified on glassy carbon electrode (GCE) modified with multiwall carbon nanotubes (MWCNT) (GCE-CNTs) at 0.30 V. The total assay time was 25 min. The calculated detection limits for amperometric detection and the ELISA procedure are 0.62 and 1.8 UmL(-1), respectively. Reproducibility assays were made using repetitive standards of H. pylori-specific antibody and the intra- and inter-assay coefficients of variation were below 5%. The immuno-affinity method showed higher sensitivity and lower time-consumed, demonstrate its potential usefulness for early assessment of human serum immunoglobulin G (IgG) antibodies to H. pylori. PMID:18761158

  2. 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

  3. 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.

  4. Enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to human cytomegalovirus.

    PubMed

    Sarov, I; Andersen, P; Andersen, H K

    1980-02-01

    A solid-phase enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to cytomegalovirus (CMV) is described. The assay used purified CMV and extracts of CMV infected cells as antigen. Antigens were desiccated onto the bottom surface of polystyrene microcuvettes. The antibodies bound to the antigens were assayed by anti-IgG-alkaline phosphate conjugate followed by addition of the enzyme substrate. Titration curves have been obtained from the sera of 35 blood donors and of 23 patients. Comparison of results obtained by ELISA with those obtained by complement fixation (CF) shows that there is agreement between the tests. Both purified CMV and extracts of CMV infected cells were found to be suitable antigens. Purified CMV was of value particularly in those sera which show high reactivity against control antigen. The ELISA technique described is approximately 412 to 548 times more sensitive than the CF test when purified CMV or extracts of CMV infected cells, respectively, are used as antigens. No significant heterotypic rise to CMV was observed by ELISA in three sets of sera with seroconversion to herpes simplex virus. The ELISA technique gives objective results, is easily performed, and may be adaptable as a routine test both for serological diagnosis of CMV infection and for screening of the general population.

  5. Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody.

    PubMed

    Vlasak, Josef; Bussat, Marie C; Wang, Shiyi; Wagner-Rousset, Elsa; Schaefer, Mark; Klinguer-Hamour, Christine; Kirchmeier, Marc; Corvaïa, Nathalie; Ionescu, Roxana; Beck, Alain

    2009-09-15

    Despite technological advances, detection of deamidation in large proteins remains a challenge and the use of orthogonal methods is needed for unequivocal assignment. By a combination of cation-exchange separation, papain digestion, and a panel of mass spectrometry techniques we identified asparagine deamidation in light chain complementarity determining region 1 (CDR1) of a humanized IgG1 monoclonal antibody. The reaction yields both Asp and isoAsp, which were assigned by Edman degradation and by isoAsp detection using protein isoaspartate methyltransferase. The deamidated antibody variants were less potent in antigen binding compared to the nondegraded antibody. Changes in near-UV CD spectra, susceptibility to papain cleavage in an adjacent CDR2 loop, and the tendency of the newly formed isoAsp to undergo isomerization suggest local perturbations in the structure of the isoAsp-containing antibody.

  6. The uptake of IgG by human placental chorionic villi: a correlated autoradiographic and wide aperture counting study.

    PubMed

    Ockleford, C D; Clint, J M

    1980-01-01

    The uptake of 3H-IgG into human placental chorionic villi in vitro takes place for at least 1 h at 37 degrees C and at 4 degrees C. The rate of uptake is lower at the latter temperature, but still about 20 per cent of the 37 degrees C total. Measurement of cell-associated redioactivity at 4 degrees C cannot therefore be used as a measure of binding: genuine uptake, probably as a result of endocytosis, appears to occur at this temperature. Some proportion of the uptake of IgG at 37 degrees C can be inhibited by colchicine and by cytochalasin B, but some is refractory to these treatments. A coated vesicle-enriched fraction isolated from placenta previously incubated with 3H-IgG was found to be associated with radioisotope.

  7. Study of the aggregation of human insulin Langmuir monolayer.

    PubMed

    Liu, Wei; Johnson, Sheba; Micic, Miodrag; Orbulescu, Jhony; Whyte, Jeffrey; Garcia, Andrew R; Leblanc, Roger M

    2012-02-21

    The human insulin (HI) Langmuir monolayer at the air-water interface was systematically investigated in the presence and absence of Zn(II) ions in the subphase. HI samples were dissolved in acidic (pH 2) and basic (pH 9) aqueous solutions and then spread at the air-water interface. Spectroscopic data of aqueous solutions of HI show a difference in HI conformation at different pH values. Moreover, the dynamics of the insulin protein showed a dependence on the concentration of Zn(II) ions. In the absence of Zn(II) ions in the subphase, the acidic and basic solutions showed similar behavior at the air-water interface. In the presence of Zn(II) ions in the subphase, the surface pressure-area and surface potential-area isotherms suggest that HI may aggregate at the air-water interface. It was observed that increasing the concentration of Zn(II) ions in the acidic (pH 2) aqueous solution of HI led to an increase of the area at a specific surface pressure. It was also seen that the conformation of HI in the basic (pH 9) medium had a reverse effect (decrease in the surface area) with the increase of the concentration of Zn(II) ions in solution. From the compression-decompression cycles we can conclude that the aggregated HI film at air-water interface is not stable and tends to restore a monolayer of monomers. These results were confirmed from UV-vis and fluorescence spectroscopy analysis. Infrared reflection-absorption and circular dichroism spectroscopy techniques were used to determine the secondary structure and orientation changes of HI by zinc ions. Generally, the aggregation process leads to a conformation change from α-helix to β-strand and β-turn, and at the air-water interface, the aggregation process was likewise seen to induce specific orientations for HI in the acidic and basic media. A proposed surface orientation model is presented here as an explanation to the experimental data, shedding light for further research on the behavior of insulin as a Langmuir

  8. 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.

  9. 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.

  10. Aggregation behavior of cationic nanohydrogel particles in human blood serum.

    PubMed

    Nuhn, Lutz; Gietzen, Sabine; Mohr, Kristin; Fischer, Karl; Toh, Kazuko; Miyata, Kanjiro; Matsumoto, Yu; Kataoka, Kazunori; Schmidt, Manfred; Zentel, Rudolf

    2014-04-14

    For systemic siRNA delivery applications, well-defined drug carriers are required that guarantee stability for both carrier and cargo. Among various concepts progressing in market or final development, cationic nanohydrogel particles may serve as novel transport media especially designed for siRNA-in vivo experiments. In this work, the interaction of nanohydrogel particles with proteins and serum components was studied via dynamic light scattering in human blood serum as novel screening method prior to applications in vivo. The formation of larger aggregates mostly caused by charge interaction with albumin could be suppressed by nanogel loading with siRNA affording a neutral zeta potential for the complex. Preliminary in vivo studies confirmed the results inside the light-scattering cuvette. Although both carrier and cargo may have limited stability on their own under physiological relevant conditions, they can form safe and stable complexes at a charge neutralized ratio and thus making them applicable to systemic siRNA delivery.

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

    PubMed

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

    2015-06-01

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

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

    EPA Science Inventory

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

  13. 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

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

    PubMed

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

    2016-01-01

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

  15. Uncovering the Mechanism of Aggregation of Human Transthyretin*

    PubMed Central

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

    2015-01-01

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

  16. Interactions between DMPC liposomes and the serum blood proteins HSA and IgG.

    PubMed

    Sabín, Juan; Prieto, Gerardo; Ruso, Juan M; Messina, Paula V; Salgado, Francisco J; Nogueira, Montserrat; Costas, Miguel; Sarmiento, Félix

    2009-02-12

    The interaction between two serum blood proteins, namely human serum albumin (HSA) and human immunoglobulin G (IgG), with 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) liposomes has been studied in detail using dynamic light scattering, flow cytometry, enzyme-linked immunosorbent assay (ELISA), electrophoretic mobility, differential scanning calorimetry (DSC), and surface tension measurements. HSA and IgG interact with liposomes forming molecular aggregates that remain stable at protein concentrations beyond those of total liposome coverage. Both HSA and IgG penetrate into the liposome bilayer. An ELISA assay indicates that the Fc region of IgG is the one that is immersed in the DMPC membrane. The liposome-protein interaction is mainly of electrostatic nature, but an important hydrophobic contribution is also present.

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

    PubMed

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

    2016-04-01

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

  18. 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.

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

    PubMed

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

    2012-12-01

    Fetal bovine serum (FBS) is the most frequently used serum for the cultivation of mammalian cells. However, since animal-derived materials might not be appropriate due to safety issues, allogeneic human serum (HS) has been used to replace FBS, particularly for the culture of human cells. While there has been a debate about the advantages of HS, its precise effect on human adult stem cells have not been clarified. The present study aimed to investigate the effect of HS on the human eyelid adipose stem cells (HEACs) in vitro. When HEACs were cultivated in a medium containing 10% HS, many cells moved into several spots and aggregated there. The phenomenon was observed as early as 9 days following 10% HS treatment, and 12 days following 5% HS plus 5% FBS treatment. However, the aggregation was never observed when the same cells were cultivated with 10% FBS or bovine serum albumin. To examine whether cell density might affect the aggregation, cells were seeded with different densities on 12-well dish. Until the beginning of aggregation, cells seeded at low densities exhibited the longest culture period of 16 days whereas cells seeded at high densities showed the shortest period of 9 days to form aggregation. The number of cells was 15.1±0.2×10(4) as the least for the low density group, and 29.3±2.8×10(4) as the greatest for the high density group. When human cord blood serum or normal bovine serum was examined for the same effect on HEACs, interestingly, cord blood serum induced the aggregation of cells whereas bovine serum treatment has never induced. When cells were cultivated with 10% HS for 9 days, they were obtained and analyzed by RT-PCR. Compared to FBS-cultivated HEACs, HS-cultivated HEACs did not express VIM, and less expressed GATA4, PALLD. On the other hand, HS-cultivated HEACs expressed MAP2 more than FBS-cultivated HEACs. In conclusion, human adult stem cells could move and form aggregates by the treatment with human body fluids.

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

    PubMed Central

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

    2012-01-01

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

  1. 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.

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

    PubMed

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

    2016-08-01

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

  3. Monomeric IgG in intravenous Ig preparations is a functional antagonist of FcgammaRII and FcgammaRIIIb.

    PubMed

    van Mirre, Edwin; Teeling, Jessica L; van der Meer, Jos W M; Bleeker, Wim K; Hack, C Erik

    2004-07-01

    Intravenous Ig preparations (IVIg), originally developed as a substitution therapy for patients with low plasma IgG, are nowadays frequently used in the treatment of various immune diseases. However, the mechanism of action of IVIg in these diseases remains elusive and is often referred to as "immunomodulatory." We hypothesized that monomeric IgG may act as a low-affinity FcgammaR antagonist and sought experimental evidence for this hypothesis. Human neutrophils as well FcgammaRIIa-transfected IIA1.6 cells were used as FcgammaR-positive cells and aggregated IgG (aIgG) or stable dimeric IgG as FcgammaR-specific agonists for these cells. We found that monomeric IgG purified from IVIg at concentrations similar to that of IgG in plasma, diminished the binding of stable dimeric IgG to FcgammaRIIa transfectants, reduced aIgG-induced influx of Ca(2+) ions into the cytosol of neutrophils, and attenuated the aIgG-induced release of elastase. Notably, monomeric IgG by itself did not elicit these responses, nor did it affect these processes in response to fMLP. Absorption of IgG from normal plasma revealed that plasma IgG exerted similar effects as monomeric IgG in IVIg. In addition, adding monomeric IgG to blood of healthy volunteers showed a dose-dependent decrease of aIgG-induced elastase release. Finally, we observed decreased aIgG-induced polymorphonuclear neutrophil responses in two hypogammaglobulinemic patients upon treatment with IVIg. We conclude that monomeric IgG at physiological levels acts as a low-affinity FcgammaR antagonist. Moreover, FcgammaR antagonism constitutes an immunomodulatory effect of IVIg.

  4. 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

  5. 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. PMID:26038982

  6. 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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  9. 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

  10. The influence of local antichlamydial antibody on the acquisition and persistence of human ocular chlamydial infection: IgG antibodies are not protective.

    PubMed Central

    Bailey, R. L.; Kajbaf, M.; Whittle, H. C.; Ward, M. E.; Mabey, D. C.

    1993-01-01

    In order to study the effect of antichlamydial antibodies in ocular secretions on resistance to ocular chlamydial infection and clearance of this infection, we have performed linked longitudinal studies in a Gambian village in which trachoma is endemic. We have measured IgG and IgA antibody levels to a local serotype B isolate of Chlamydia trachomatis by amplified enzyme immunoassay, and chlamydial antigen levels in conjunctival swabs using a commercially available immunoassay which detects chlamydial glycolipid. Having previously demonstrated that sharing a bedroom with a case of active trachoma is a risk factor for acquisition of the disease, we have analyzed the effect of IgG and IgA antibody on the acquisition and persistence of clinical trachoma after controlling for age, sex, exposure to infection and for the presence of chlamydial antigen using a Poisson regression model. We have found that the presence of antichlamydial IgG in ocular secretions of disease-free subjects is associated with an increased incidence of trachoma. IgA antibody shows an opposite trend, but this is not statistically significant. One possible explanation of these findings is that antichlamydial IgG antibodies enhance the infectivity of C. trachomatis for the human eye; this could have major implications for the development of a chlamydial vaccine. PMID:8405158

  11. The IgG detected in the C1q solid-phase immune-complex assay is not always of immune-complex nature.

    PubMed

    Hack, C E; Belmer, A J

    1986-01-01

    The properties of the solid-phase C1q immune-complex assay as well as the nature of the IgG detected by this assay in patients' sera were investigated. Aggregated IgG was used as a model for immune complexes. Aggregated IgG bound to solid-phase C1q was detected by 125I-anti-IgG. Fluid-phase C1q (either in normal human serum or purified) neither inhibited the binding of aggregated IgG to solid-phase C1q nor dissociated bound aggregated IgG from the solid-phase C1q. Therefore, we concluded that the solid-phase C1q has a higher affinity for aggregated IgG than the fluid-phase C1q, probably because of the polymerization of the solid-phase C1q. To get more insight into the nature of the IgG detected by the C1q solid-phase assay in patients' sera, we investigated whether C4 and/or C3 were present on it. With the use of 125I-anti-C4 and 125I-anti-C3 instead of 125I-anti-IgG, C4 and C3, respectively, were easily detected on the aggregated IgG that had bound to the solid-phase C1q. The lower limit of detection of these assays was 30 micrograms aggregated IgG/ml of normal human serum. Sera of patients suffering from rheumatoid arthritis and systemic lupus erythematosus were tested with these assays and, despite positive results with 125I-anti-IgG, no positive results were obtained with either 125I-anti-C4 or 125I-anti-C3. So, on the IgG detected by the C1q solid-phase assay in patients' sera, neither C4 nor C3 are present. Furthermore, in five of the six sera tested, this IgG sedimented as monomeric IgG. Therefore, it seems unjustified to refer to this IgG as circulating immune complexes.

  12. Acid-induced aggregation propensity of nivolumab is dependent on the Fc.

    PubMed

    Liu, Boning; Guo, Huaizu; Xu, Jin; Qin, Ting; Xu, Lu; Zhang, Junjie; Guo, Qingcheng; Zhang, Dapeng; Qian, Weizhu; Li, Bohua; Dai, Jianxin; Hou, Sheng; Guo, Yajun; Wang, Hao

    2016-01-01

    Nivolumab, an anti-programmed death (PD)1 IgG4 antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG1), panitumumab (IgG2) and atezolizumab (aglyco-IgG1) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG1 < aglyco-IgG1 < IgG2 < IgG4. This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG4 backbone. PMID:27310175

  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. PMID:9020315

  14. 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

  15. 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.

  16. Antibody germline characterization of cross-neutralizing human IgGs against 4 serotypes of dengue virus.

    PubMed

    Pitaksajjakul, Pannamthip; Benjathummarak, Surachet; Pipattanaboon, Chonlatip; Wongwit, Waranya; Okabayashi, Tamaki; Kuhara, Motoki; Misaki, Ryo; Fujiyama, Kazuhito; Ramasoota, Pongrama

    2014-04-01

    Dengue virus (DENV), a re-emerging virus, constitutes the largest vector-borne disease virus, with 50-100 million cases reported every year. Although DENV infection induces lifelong immunity against viruses of the same serotypes, the subsequent infection with the heterologous serotypes can cause more severe form of the disease, such as Dengue Haemorrhagic Fever (DHF) or Dengue Shock Syndrome (DSS). However, there is neither approved vaccine nor specific drugs available to treat this disease. In this study, previously developed 19 human monoclonal antibodies (HuMAbs) showing strong to moderate cross neutralizing activity were selected. Most of them (13/19) were targeted to domain II of envelop glycoprotein. To understand and clarify the recognition properties, the maturation mechanisms comprising Variable/Diversity/Joining (VDJ) recombination, Variable Heavy (VH)/Variable Light (VL) chain pairing, variability at junctional site, and somatic hypermutation (SHM) of those antibodies were studied and compared with their predecessor germline sequences. IMGT/V-QUEST database was applied to analyze the isolated VH and VL sequences. To confirm the correction of isolated VH/VL, 3 HuMAbs (1A10H7, 1B3B9, 1G7C2) was transiently expressed in HEK293T cell. All three clones of the expressed recombinant IgG (rIgG) showed the same binding and neutralizing activity as same as those from hybridomas. The data obtained in this study will elucidate the properties of those HuMAbs for further genetic modification, and its binding epitopes. PMID:24637211

  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. 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.

  19. Construction of a stability landscape of the CH3 domain of human IgG1 by combining directed evolution with high throughput sequencing.

    PubMed

    Traxlmayr, Michael W; Hasenhindl, Christoph; Hackl, Matthias; Stadlmayr, Gerhard; Rybka, Jakub D; Borth, Nicole; Grillari, Johannes; Rüker, Florian; Obinger, Christian

    2012-10-26

    One of the most important but still poorly understood issues in protein chemistry is the relationship between sequence and stability of proteins. Here, we present a method for analyzing the influence of each individual residue on the foldability and stability of an entire protein. A randomly mutated library of the crystallizable fragment of human immunoglobulin G class 1 (IgG1-Fc) was expressed on the surface of yeast, followed by heat incubation at 79°C and selection of stable variants that still bound to structurally specific ligands. High throughput sequencing allowed comparison of the mutation rate between the starting and selected library pools, enabling the generation of a stability landscape for the entire CH3 domain of human IgG1 at single residue resolution. Its quality was analyzed with respect to (i) the structure of IgG1-Fc, (ii) evolutionarily conserved positions and (iii) in silico calculations of the energy of unfolding of all variants in comparison with the wild-type protein. In addition, this new experimental approach allowed the assignment of functional epitopes of structurally specific ligands used for selection [Fc γ-receptor I (CD64) and anti-human CH2 domain antibody] to distinct binding regions in the CH2 domain. PMID:22846908

  20. 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.

  1. Cataract-related changes in protein aggregates of human lens studied by ultracentrifugation.

    PubMed

    Twardowski, J; Hoja, D

    1990-01-01

    In human lens, cataract development causes an increase in the amount of protein aggregates. Their specific density of 1.25 g/ml is much higher than that of 1.14 g/ml and 1.06 g/ml obtained for water-soluble proteins. During the formation of water-insoluble aggregates their size varies up to some micrometers. Infrared spectra of water-soluble protein and in the aggregates confirm that the content of bound water in aggregates decreases. PMID:2279587

  2. A single amino acid in the second Ig-like domain of the human Fc gamma receptor II is critical for human IgG2 binding.

    PubMed

    Warmerdam, P A; van de Winkel, J G; Vlug, A; Westerdaal, N A; Capel, P J

    1991-08-15

    The low-affinity human Fc gamma RIIa is encoded by a single gene with allelic variation, defined by low-responder and high-responder alleles (LR and HR). The HR Fc gamma RIIa transcript interacts strongly with murine (m) IgG1 complexes, in contrast to the LR Fc gamma RIIa. Furthermore, the transcripts can be discriminated by mAb 41H16, which recognizes an epitope expressed on the HR Fc gamma RIIa molecule. We report that this receptor is also polymorphic in its reactivity with human (h) IgG2. Binding studies using well-defined hIgG dimers revealed that LR Fc gamma RIIa molecules can efficiently bind hIgG2, in contrast to HR Fc gamma RIIa. Previous work of others showed one amino acid difference between the allelic forms of Fc gamma RII. We, however, found a second amino acid difference between both allelic forms. In this study, hybrid Fc gamma RIIa molecules were constructed to determine the epitope for mAb 41H16 and the binding domain for mIgG1 and hIgG2 complexes. Our data point to the importance of the amino acid at position 131, located in the second Ig-like domain of Fc gamma RIIa. When an arginine residue is present at amino acid position 131, the receptor is recognized by mAb 41H16. Furthermore, the receptor can bind mIgG1-sensitized indicator E, but binds hIgG2 dimers only weakly. When a histidine residue is present at this amino acid position, hIgG2 dimers do bind efficiently to Fc gamma RII, whereas mIgG1-sensitized E and mAb 41H16 exhibit a strongly diminished binding.

  3. [Use of anti-D (Rh) IgG or intramuscular polyvalent human immunoglobulin in the treatment of chronic autoimmune thrombocytopenic purpura].

    PubMed

    Vizcaíno, G; Diez-Ewald, M; Arteaga-Vizcaíno, M; Torres, E

    1992-01-01

    The present study compares the effect of the intramuscular injection of low doses of IgG anti-D or human polyvalent immunoglobulin (Ig) on the platelet count of patients with CATP. Forty patients (14 children, 26 adults), 11 who had undergone splenectomy, were divided in the following groups of treatment: 20 patients received a single injection of 300 micrograms of IgG anti-D, 6 patients received the same dose as above plus 0.5 mg/kg daily of prednisone v.o and 14 patients received 640 mg of polyvalent Ig. Each patient was sequentially studied by measuring peripheral blood parameters, reticulocyte index, direct Coombs' test and C3-C4 determinations. Their blood group and Rh factor had been previously determined. The platelet response was evaluated as refractory (no response) and favorable (platelet increment over 50,000/microliters compared with initial platelet count). Patients with a favorable response over a month were considered as a prolonged remission. The results showed a favorable platelet response in 74% of the patients that received a single injection of IgG anti-D alone (one of the patients was Rh negative) or associated to prednisone, and 42.8% of the cases when polyvalent Ig was used. The patients who had not undergone splenectomy obtained better results than the group with splenectomy (62% vs 45%) and children showed a better response than adults (78.5% vs 46.1%). Forty five percent of prolonged remissions (including the Rh negative patient) were obtained with both schemes of IgG anti-D administration and only 28.5% when polyvalent Ig was used. The remissions were significantly longer with IgG anti-D (p < 0.01). The hematological and serological parameters did not show any significant modifications in all the cases and there was no adverse effects with the treatment. In conclusion, the intramuscular injection of immunoglobulins, especially IgG anti-D, produce an increase in the platelet count in some patients with CATP, several of them can obtain

  4. 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

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

    PubMed Central

    Buxbaum, Laurence U.

    2013-01-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  10. Inhibitors of ex vivo aggregation of human platelets induced by decompression, during reduced barometric pressure.

    PubMed

    Murayama, M; Kumaroo, K K

    1986-05-15

    It has been shown experimentally ex vivo that human platelet aggregation is induced by decompression (reduced pressure) produced by various means, i.e., reduced barometric pressure, reduced hydrostatic pressure, and reduced hydrodynamic pressure due to Bernoulli's principle. We report here that the spontaneous platelet aggregation induced by reduced barometric pressure (253 torr for three hours) is inhibited by 1:10(7) diluted Japanese herbal plant oil (JHP) and also by two of its major constituents, menthone and menthol with the median inhibitory concentration (IC50) in the millimolar range. These drugs gave essentially similar results when collagen and ADP were used as aggregating agents. Inhibitor concentrations were determined by microscopic examination of platelets in wet preparations when the aggregating stimulus was reduced pressure and by optical aggregometry when collagen and ADP were the aggregating agents. Potential usefulness of these compounds in the prevention of decompression syndrome (DCS) and acute mountain sickness (AMS) are discussed.

  11. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells.

    PubMed

    Li, Tong; Paudel, Hemant K

    2016-01-01

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

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

    PubMed Central

    Li, Tong; Paudel, Hemant K.

    2016-01-01

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

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

    SciTech Connect

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

    1994-05-01

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

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

    PubMed

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

    2016-05-01

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

  15. Positive and negative aggregation responses to cultured human tumor cell lines among different normal individuals.

    PubMed

    Bastida, E; Ordinas, A; Jamieson, G A

    1982-01-01

    Platelets from approximately 50% (7/16) of normal individuals have been shown to have greater sensitivity to aggregation induced by critical threshold concentrations of three human tumor cell lines. These results may have implications for the genetics and epidemiology of human neoplastic disease.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  18. Purification of granulosa cells from human ovarian follicular fluid using granulosa cell aggregates.

    PubMed

    Quinn, M C J; McGregor, S B; Stanton, J L; Hessian, P A; Gillett, W R; Green, D P L

    2006-01-01

    Human follicular fluid can provide a source of human granulosa cells for scientific study. However, removing potentially contaminating cells, such as white and red blood cells, is important for molecular and in vitro studies. We have developed a purification technique for human granulosa cells based on the selection of cellular aggregates. Human granulosa cells from 21 IVF patients were collected. A 50% Percoll gradient was used to remove red blood cells, and granulosa cell aggregates were collected, washed and processed for histology, electron microscopy, flow cytometry analysis, cell culture and RNA extraction. Granulosa cell aggregates were found to be homogeneous and free of white blood cells after histological and electron microscopic analysis. White blood cell contamination, measured by flow cytometry, was found to be between 2 and 4%. Polymerase chain reaction analysis revealed expression of known human granulosa cell genes and a white blood cell marker. Human granulosa cells grown in vitro showed flattened fibroblast-like morphology with lipid droplets consistent with previous reports. Cultured cells expressed the FSH receptor. Selection of human granulosa cell aggregates following centrifugation through a Percoll gradient provides an efficient method of selecting granulosa cells, suitable for both molecular and in vitro studies.

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

    PubMed

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

    2003-12-01

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

  20. 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

  1. An improved Protein G with higher affinity for human/rabbit IgG Fc domains exploiting a computationally designed polar network

    PubMed Central

    Jha, Ramesh K.; Gaiotto, Tiziano; Bradbury, Andrew R.M.; Strauss, Charlie E.M.

    2014-01-01

    Protein G is an IgG binding protein that has been widely exploited for biotechnological purposes. Rosetta protein modeling identified a set of favorable polar mutations in Protein G, at its binding interface with the Fc domain of Immunoglobulin G, that were predicted to increase the stability and tighten the binding relative to native Protein G, with only a minor perturbation of the binding mode seen in the crystal structure. This triple mutant was synthesized and evaluated experimentally. Relative to the native protein G, the mutant showed a 3.5-fold enhancement in display level on the surface of yeast and a 5-fold tighter molar affinity for rabbit and human IgG. We attribute the improved affinity to a network of hydrogen bonds exploiting specific polar groups on human and rabbit Fc. The relative specificity increased as well since there was little affinity enhancement for goat and mouse Fc, while the affinity for rat Fc was poorer by half. This designed Protein G will be useful in biotechnological applications as a recombinant protein, where its improved affinity, display and specificity will increase antibody capture sensitivity and capacity. Furthermore, the display of this protein on the surface of yeast introduces the concept of the use of yeast as an affinity matrix. PMID:24632761

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

    SciTech Connect

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

    2015-10-31

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

  3. 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.

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

    PubMed

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

    1999-08-01

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

  5. A critical role for the regulation of Syk from agglutination to aggregation in human platelets.

    PubMed

    Shih, Chun-Ho; Chiang, Tin-Bin; Wang, Wen-Jeng

    2014-01-10

    Agglucetin, a tetrameric glycoprotein (GP) Ibα agonist from Formosan Agkistrodon acutus venom, has been characterized as an agglutination inducer in human washed platelets (WPs). In platelet-rich plasma (PRP), agglucetin dramatically elicits a biphasic response of agglutination and subsequent aggregation. For clarifying the intracellular signaling events from agglutination to aggregation in human platelets, we examined the essential signaling molecules involved through the detection of protein tyrosine phosphorylation (PTP). In WPs, an anti-GPIbα monoclonal antibody (mAb) AP1, but not a Src kinase inhibitor PP1, completely inhibited agglucetin-induced agglutination. However, PP1 but not AP1 had a potent suppression on platelet aggregation by a GPVI activator convulxin. The PTP analyses showed agglucetin alone can cause a weak pattern involving sequential phosphorylation of Lyn/Fyn, Syk, SLP-76 and phospholipase Cγ2 (PLCγ2). Furthermore, a Syk-selective kinase inhibitor, piceatannol, significantly suppressed the aggregating response in agglucetin-activated PRP. Analyzed by flow cytometry, the binding capacity of fluorophore-conjugated PAC-1, a mAb recognizing activated integrin αIIbβ3, was shown to increase in agglucetin-stimulated platelets. Again, piceatannol but not PP1 had a concentration-dependent suppression on agglucetin-induced αIIbβ3 exposure. Moreover, the formation of signalosome, including Syk, SLP-76, VAV, adhesion and degranulation promoting adapter protein (ADAP) and PLCγ2, are required for platelet aggregation in agglucetin/fibrinogen-activated platelets. In addition, GPIbα-ligation via agglucetin can substantially promote the interactions between αIIbβ3 and fibrinogen. Therefore, the signal pathway of Lyn/Fyn/Syk/SLP-76/ADAP/VAV/PLCγ2/PKC is sufficient to trigger platelet aggregation in agglucetin/fibrinogen-pretreated platelets. Importantly, Syk may function as a major regulator for the response from GPIbα-initiated agglutination to

  6. A critical role for the regulation of Syk from agglutination to aggregation in human platelets.

    PubMed

    Shih, Chun-Ho; Chiang, Tin-Bin; Wang, Wen-Jeng

    2014-01-10

    Agglucetin, a tetrameric glycoprotein (GP) Ibα agonist from Formosan Agkistrodon acutus venom, has been characterized as an agglutination inducer in human washed platelets (WPs). In platelet-rich plasma (PRP), agglucetin dramatically elicits a biphasic response of agglutination and subsequent aggregation. For clarifying the intracellular signaling events from agglutination to aggregation in human platelets, we examined the essential signaling molecules involved through the detection of protein tyrosine phosphorylation (PTP). In WPs, an anti-GPIbα monoclonal antibody (mAb) AP1, but not a Src kinase inhibitor PP1, completely inhibited agglucetin-induced agglutination. However, PP1 but not AP1 had a potent suppression on platelet aggregation by a GPVI activator convulxin. The PTP analyses showed agglucetin alone can cause a weak pattern involving sequential phosphorylation of Lyn/Fyn, Syk, SLP-76 and phospholipase Cγ2 (PLCγ2). Furthermore, a Syk-selective kinase inhibitor, piceatannol, significantly suppressed the aggregating response in agglucetin-activated PRP. Analyzed by flow cytometry, the binding capacity of fluorophore-conjugated PAC-1, a mAb recognizing activated integrin αIIbβ3, was shown to increase in agglucetin-stimulated platelets. Again, piceatannol but not PP1 had a concentration-dependent suppression on agglucetin-induced αIIbβ3 exposure. Moreover, the formation of signalosome, including Syk, SLP-76, VAV, adhesion and degranulation promoting adapter protein (ADAP) and PLCγ2, are required for platelet aggregation in agglucetin/fibrinogen-activated platelets. In addition, GPIbα-ligation via agglucetin can substantially promote the interactions between αIIbβ3 and fibrinogen. Therefore, the signal pathway of Lyn/Fyn/Syk/SLP-76/ADAP/VAV/PLCγ2/PKC is sufficient to trigger platelet aggregation in agglucetin/fibrinogen-pretreated platelets. Importantly, Syk may function as a major regulator for the response from GPIbα-initiated agglutination to

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

    PubMed Central

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

    2015-01-01

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

  8. 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.

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

    PubMed Central

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

    1969-01-01

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

  10. Aggregation of human immunodeficiency virus type 1 by human salivary secretions.

    PubMed

    Bergey, E J; Cho, M I; Hammarskjöld, M L; Rekosh, D; Levine, M J; Blumberg, B M; Epstein, L G

    1993-01-01

    Human immunodeficiency virus (HIV-1) is generally transmitted by parenteral contact with infected body secretions. Although extensive epidemiological data and familial studies have failed to provide any conclusive data that saliva may act as a vehicle for transmission of AIDS, both professional and public anxieties remain. The present study, as well as others, suggests that salivary secretions may act as inhibitors of HIV-1 replication in vitro. In our study, the inhibitory activity was determined to be associated mainly with secretions obtained from the human submandibular-sublingual glands. Human submandibular-sublingual (HSMSL) and parotid (HPS) salivas were collected and tested for their ability to modulate the replication of HIV-1, using a plaque assay on HeLa/CD4+ cell monolayers. Initial results examining freshly collected salivary samples from ten individuals confirmed the results previously obtained by Fox et al. (1988, 1989). An average plaque reduction of approximately 66% was obtained with HSMSL, in contrast to 34% reduction obtained with HPS. Titration of the inhibitory activity in HSMSL showed detectable levels at a 1:500 dilution. Comparison of inhibitory activity of dialyzed and lyophilized saliva to fresh saliva indicated little difference between the two samples when filtration occurred after the addition of HIV-1. However, the effect of filtration was significantly diminished in the lyophilized samples. Electron microscopic examination of the saliva-HIV incubates revealed the aggregation/entrapment of virus particles by salivary components. These results suggest that human salivary secretions (with HSMSL > HPS) may have a role in modulating the infectivity of HIV-1.

  11. Effect of cocoa products and flavanols on platelet aggregation in humans: a systematic review.

    PubMed

    Peluso, Ilaria; Palmery, Maura; Serafini, Mauro

    2015-07-01

    Previous evidence suggested an active role of cocoa products and flavanols in modulating platelet aggregation. However, cocoa flavanols are characterized by a low bioavailability that can deeply affect their presence in biological fluids and raise questions on their biological effect in humans. We performed a systematic search on Medline, Embase, Cochrane and ProQuest databases, until April 2015, on the effect of cocoa products on platelet aggregation in human intervention studies. We identified 13 interventions, of which only five involved repeated administration. Different effects were observed on the basis of the platelet aggregation test used, whereas neither a longer duration of treatment nor a higher dose was associated with a higher inhibition of platelet aggregation. In conclusion, the reviewed results suggest that consumption of cocoa products in bolus administration positively affects platelet aggregation in both healthy subjects and diseased patients. On the other hand, more evidence is required in order to assess the effect of long-term cocoa product ingestion and to identify the bioactive components involved.

  12. 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...

  13. 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 ...

  14. Trypsin and thrombin accelerate aggregation of human endocrine pancreas precursor cells.

    PubMed

    Wei, Chiju; Geras-Raaka, Elizabeth; Marcus-Samuels, Bernice; Oron, Yoram; Gershengorn, Marvin C

    2006-02-01

    Human islet-derived precursor cells (hIPCs) and human pancreatic ductal carcinoma (PANC-1) cells can be induced to form aggregates that subsequently differentiate into hormone-expressing islet-like cell aggregates (ICAs). We show that challenge of hIPCs or PANC-1 cells with thrombin or trypsin resulted in stimulation of signaling via the inositol-tris-phosphate second messenger pathway leading to rapid, transient increases in cytosolic calcium ion concentration in the majority of the cells. Because we found that hIPCs, PANC-1 cells, human fetal pancreas, and human adult islets express two protease-activated receptors (PARs), PAR-1 and PAR-2, we tested whether the effects of thrombin and trypsin were mediated, at least in part, by these receptors. Peptide agonists that are relatively specific for PAR-1 (SFLLRN-amide) or PAR-2 (SLIGRL-amide) stimulated increases in inositol phosphates and cytosolic calcium ion concentration, and increased the phosphorylation of Rho, a small G-protein associated with cytoskeletal changes affecting cellular morphology and migration. Most importantly, we show that these agonists increased the rate of hIPC aggregation leading to the formation of more viable, smaller ICAs. Our data show that thrombin and trypsin accelerate aggregation, an early stage of hIPC differentiation in vitro, and imply that pancreatic trypsin and thrombin may be involved in islet development in vivo. PMID:16021635

  15. An enzyme immunoassay based micro-neutralization test for titration of antibodies to human cytomegalovirus (CMV) and its correlation with direct ELISA measuring CMV IgG antibodies.

    PubMed

    Gupta, C K; Leszczynski, J; Gupta, R K; Siber, G R

    1996-03-01

    An ELISA-based micro-neutralization (Nt) test in MRC-5 cells for titration of neutralizing antibodies against human cytomegalovirus (CMV) in human plasma and preparations of immune globulins was developed to eliminate microscopic reading of cytopathic effect (CPE), a process that is subjective and time consuming. Un-neutralized CMV from the Nt reaction and grown in MRC-5 cells as per the standard micro-Nt test was coated in the same plates by various methods and CMV antigen was quantified by polyclonal or monoclonal CMV antibodies. Optimal coating of plates with CMV antigen (100 TCID50 of virus grown on MRC-5 cells for 7 days) was obtained by freezing/thawing of virus infected MRC-5 cells in phosphate buffered saline, ph 7.2. The CMV antigen treated sequentially with CMV monoclonal antibody to late nuclear protein antigen, goat anti-mouse IgG3 alkaline phosphatase conjugate and phosphatase substrate gave an absorbance of 1 at 410 nm wavelength whereas uninfected MRC-5 cells treated under similar conditions did not show any absorbance. The optimal Nt reaction occurred at 37 degrees C for 1-2 h and was unaffected by complement. At 4 degrees C, CMV was inactivated in 1-2 h. The antibody titres were affected by the virus dose used in the Nt test over a range of 20 to 798 TCID50. When the titre was determined against a reference serum, the effect of virus dose on the Nt titre was reduced. Complete neutralization virus read microscopically correlated with ELISA absorbance of < 0.1. CPE produced by approximately 1 TCID50 of CMV showed an absorbance of 0.1 or more. The correlation coefficient (r) between Nt titres and CMV IgG antibodies determined by ELISA was 0.69 (P < 0.001) for 257 human plasma samples and 0.85 (P < 0.001) for 50 immune globulin preparations.

  16. Aggregation of Streptococcus pneumoniae by a pneumococcal capsular polysaccharide-specific human monoclonal IgM correlates with antibody efficacy in vivo.

    PubMed

    Fabrizio, Kevin; Manix, Catherine; Guimaraes, Allan J; Nosanchuk, Joshua D; Pirofski, Liise-Anne

    2010-05-01

    Acquired antibody immunity to Streptococcus pneumoniae (pneumococcus) has been linked to serotype (ST)-specific opsonic antibodies to the relevant pneumococcal capsular polysaccharide (PPS) that mediate protection by enhancing the bactericidal effect of host phagocytes. Despite the well-recognized role of opsonic IgG in host defense against pneumococcus, PPS-specific monoclonal antibodies (MAbs) that mediate protection against lethal challenge with ST3 pneumococcus in mice but do not promote phagocytic killing in vitro (nonopsonic antibodies) have been described. In this study, we sought to determine the biological activity of one such MAb, A7 (a human PPS3-specific IgM), and the mechanism by which it mediates protection. In vitro studies demonstrated that coincubation of A7 with ST3 in the absence of phagocytes or a complement source resulted in a reduction in CFU on blood agar plates that was largely reversible by sonication. A chromogenic cellular proliferation assay demonstrated that A7 did not affect replication of ST3 in liquid culture. The ability of A7 to induce aggregation of ST3 was confirmed by fluorescence microscopy and flow cytometry: A7 induced aggregation of ST3, and in the presence of a complement source, A7 promoted deposition of complement component 3 (C3) on aggregated bacteria in a dose-dependent fashion. Similarly, administration of preincubated mixtures of A7 and ST3 intraperitoneally to mice protected them from the lethality of ST3 in a dose-dependent fashion. These findings suggest that A7-mediated aggregation enhances resistance to ST3, most likely by enhancing C3 deposition on the ST3 capsule, thereby promoting host antipneumococcal activity in vivo.

  17. 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.

  18. Possible role of free radical altered IgG in the etiopathogenesis of rheumatoid arthritis.

    PubMed

    Swaak, A J; Kleinveld, H A; Kloster, J F; Hack, C E

    1989-01-01

    Alteration of IgG by oxygen-derived free radicals has been implicated in an in vivo process which renders IgG autoantigenic and leads to the production of rheumatoid factor (RF) and the perpetuation of inflammation, as in rheumatoid arthritis (RA). In this study the impact of UV irradiation on IgG was investigated as well as the ability of RF to bind to UV-altered gamma globulin. Inhibition studies of the binding of 125I aggregated human gamma globulin (AHG) to RF-coated sepharose beads show that UV-irradiated IgG is able to bind RF to the same extent as AHG. Binding studies to 125I-C1q proved that UV-irradiated IgG could bind the first complement component, but also that the complement system could be activated as illustrated by the C3a generation. These results support the hypothesis that free radical damage to gamma globulins plays a role in the chronicity of the inflammatory reaction in RA.

  19. [Effects of human disturbance on soil aggregates content and their organic C stability in Karst regions].

    PubMed

    Wei, Ya-wei; Su, Yi-rong; Chen, Xiang-bi; He, Xun-yang; Qin, Wen-geng; Wei, Guo-fu

    2011-04-01

    Taking the primary forest land (PF), natural restoration land (NR), grazing grassland burned annually in winter (GB), and maize-sweet potato cropland (MS) in Karst regions of Northwest Guangxi as test objects, this paper studied the soil aggregates content and their organic C stability in the four ecosystems under different human disturbance patterns. The soil water-stable aggregates (>0.25 mm) content in PF, NR, and GB accounted for more than 70%, while that in MS was only 37%. The destruction rate of soil aggregates structure in the four ecosystems decreased in the sequence of MS (54.9%) > GB (23.2%) > NR (9.8%) and PF (9.6%), with significant differences among them (P<0.05). With increasing incubation time, the mineralization rate of soil aggregate organic C decreased after an initial increase and kept stable after 20 days, and increased with decreasing aggregate size. In the same size aggregates, the mineralization rate of organic C in the four ecosystems increased in the sequence of MS < GB and NR < PF. In PF, the mineralization ratio of soil organic C was 1.7% - 3.8%, being significantly higher than that in NR, GB, and MS. The cumulative mineralization amount of soil organic C had the same change trend with the mineralization rate. The contents of soil organic C and aggregate organic C were significantly positively correlated with the mineralization rate and cumulative mineralization amount of organic C, respectively, and significantly negatively correlated with the mineralization ratio of organic C.

  20. Interaction of Human Serum Album and C60 Aggregates in Solution

    PubMed Central

    Song, Maoyong; Liu, Shufang; Yin, Junfa; Wang, Hailin

    2011-01-01

    An important property of C60 in aquatic ecotoxicology is that it can form stable aggregates with nanoscale dimensions, namely nC60. Aggregation allows fullerenes to remain suspended for a long time, and the reactivity of individual C60 is substantially altered in this aggregate form. Herein, we investigated the interaction of nC60 and human serum album (HSA) using the methods of fluorescence, fluorescence dynamics, circular dichroism (CD), and site marker competitive experiments. We proposed a binding model consistent with the available experimental results for the interactions of nC60 with HSA. During the interaction process, the structure and conformation of HSA were affected, leading to functional changes of drug binding sites of HSA. PMID:21954338

  1. Inhibitory effect of compounds from Zingiberaceae species on human platelet aggregation.

    PubMed

    Jantan, I; Raweh, S M; Sirat, H M; Jamil, S; Mohd Yasin, Y H; Jalil, J; Jamal, J A

    2008-04-01

    Twelve compounds isolated from Alpinia mutica Roxb., Kaempferia rotunda Linn., Curcuma xanthorhiza Roxb., Curcuma aromatica Valeton and Zingiber zerumbet Smith (Family: Zingiberaceae) and three synthesized derivatives of xanthorrhizol were evaluated for their ability to inhibit arachidonic acid- (AA), collagen- and ADP-induced platelet aggregation in human whole blood. Antiplatelet activity of the compounds was measured in vitro by the Chrono Log whole blood aggregometer using an electrical impedance method. Among the compounds tested, curcumin from C. aromatica, cardamonin, pinocembrine and 5,6-dehydrokawain from A. mutica and 3-deacetylcrotepoxide from K. rotunda showed strong inhibition on platelet aggregation induced by AA with IC(50) values of less than 84 microM. Curcumin was the most effective antiplatelet compound as it inhibited AA-, collagen- and ADP-induced platelet aggregation with IC(50) values of 37.5, 60.9 and 45.7 microM, respectively.

  2. Aggregation analysis of Con A binding proteins of human seminal plasma: a dynamic light scattering study.

    PubMed

    Tomar, Anil Kumar; Sooch, Balwinder Singh; Singh, Sarman; Yadav, Savita

    2013-02-01

    Concanavalin A (Con A) binding fraction of human seminal plasma is vital as it shows decapacitating activity and contains proteins which have critical roles in fertility related processes. Con A binding proteins were isolated by lectin affinity chromatography. These proteins form high molecular weight aggregates at near physiological pH (7.0) as inferred by gel filtration. Aggregation analysis was performed by dynamic light scattering (DLS). DLS analysis was also performed at different pH values and in presence of various additives including NaCl, EDTA, cholesterol and sugars, such as d-glucose, d-fructose and d-mannose to identify their effect on aggregation size. The results indicate that degree of aggregation was highly reduced in presence of d-fructose, EDTA and at lower and higher pH values as depicted by lowering of hydrodynamic radii. This aggregation behaviour might be decisive for fertility related events with a suggestive role towards inhibition of premature capacitation.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2016-05-15

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

  5. IgG subclass distributions of anti-horse serum antibodies and natural venom-antibodies produced in response to antivenom injection or snake bite in humans.

    PubMed

    Ameno, S; Ameno, K; Fuke, C; Kiryu, T; Ijiri, I

    1990-01-01

    The Japanese Mamushi (Agkistrodon halys blomhoffi, BOIE) is the most common snake in Japan. Bite victims treated with antivenom (horse serum) can produce antibodies against the horse serum and the snake venom. We studied distributions of the IgG subclasses of both these antibodies produced in response to antivenom injection and snake bite. We found that IgG1 and IgG4 of each antibody in the victims' serum were present for a long period of time. PMID:2343468

  6. Subcutaneous IgG in neurologic diseases.

    PubMed

    Berger, Melvin

    2014-01-01

    Subcutaneous administration of IgG (SCIG) has become widely used in primary immune deficiency diseases but it has only recently been studied for maintenance therapy in autoimmune peripheral neuropathies, such as chronic idiopathic demyelinating polyneuropathy and multifocal motor neuropathy. Weekly self-administration of SCIG is safe and well-tolerated, and results in steady-state serum IgG levels, as contrasted with the peaks and troughs of monthly immune globulin (human) for intravenous use. Freedom from the need for venous access or medical personnel for infusions, flexibility in scheduling, convenience of home therapy, and improved clinical stability due to the steady-state IgG levels, lead many patients to prefer SCIG to immune globulin (human) for intravenous use. Long-term studies are needed to determine if the constant IgG levels and clinical stability translate into better long-term outcomes.

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

    PubMed

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

    2014-05-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    1992-01-01

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

  12. 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

  13. 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.

  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. 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.

  16. 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

  17. Neutralizing Polyclonal IgG Present during Acute Infection Prevents Rapid Disease Onset in Simian-Human Immunodeficiency Virus SHIVSF162P3-Infected Infant Rhesus Macaques

    PubMed Central

    Jaworski, J. Pablo; Kobie, James; Brower, Zachary; Malherbe, Delphine C.; Landucci, Gary; Sutton, William F.; Guo, Biwei; Reed, Jason S.; Leon, Enrique J.; Engelmann, Flora; Zheng, Bo; Legasse, Al; Park, Byung; Dickerson, Mary; Lewis, Anne D.; Colgin, Lois M. A.; Axthelm, Michael; Messaoudi, Ilhem; Sacha, Jonah B.; Burton, Dennis R.; Forthal, Donald N.; Hessell, Ann J.

    2013-01-01

    Simian-human immunodeficiency virus (SHIV) models for human immunodeficiency virus (HIV) infection have been widely used in passive studies with HIV neutralizing antibodies (NAbs) to test for protection against infection. However, because SHIV-infected adult macaques often rapidly control plasma viremia and any resulting pathogenesis is minor, the model has been unsuitable for studying the impact of antibodies on pathogenesis in infected animals. We found that SHIVSF162P3 infection in 1-month-old rhesus macaques not only results in high persistent plasma viremia but also leads to very rapid disease progression within 12 to 16 weeks. In this model, passive transfer of high doses of neutralizing IgG (SHIVIG) prevents infection. Here, we show that at lower doses, SHIVIG reduces both plasma and peripheral blood mononuclear cell (PBMC)-associated viremia and mitigates pathogenesis in infected animals. Moreover, production of endogenous NAbs correlated with lower set-point viremia and 100% survival of infected animals. New SHIV models are needed to investigate whether passively transferred antibodies or antibodies elicited by vaccination that fall short of providing sterilizing immunity impact disease progression or influence immune responses. The 1-month-old rhesus macaque SHIV model of infection provides a new tool to investigate the effects of antibodies on viral replication and clearance, mechanisms of B cell maintenance, and the induction of adaptive immunity in disease progression. PMID:23885083

  18. 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.

  19. IMC-A12, a human IgG1 monoclonal antibody to the insulin-like growth factor I receptor.

    PubMed

    Rowinsky, Eric K; Youssoufian, Hagop; Tonra, James R; Solomon, Phillip; Burtrum, Douglas; Ludwig, Dale L

    2007-09-15

    Targeted monoclonal antibody therapy is an important strategy in cancer therapeutics. Among the most promising characteristics of therapeutic targets are those that modulate the growth and survival of malignant neoplasms and their sensitivity to anticancer therapies. The insulin-like growth factor-I receptor (IGF-IR) is overexpressed in many types of solid and hematopoietic malignancies, and has been implicated as a principal cause of heightened proliferative and survival signaling. IGF-IR has also been shown to confer resistance to cytotoxic, hormonal, and targeted therapies, suggesting that therapeutics targeting IGF-IR may be effective against a broad range of malignancies. IMC-A12 (ImClone Systems Incorporated), a fully human monoclonal IgG1 antibody that binds with high affinity to the IGF-IR, inhibits ligand-dependent receptor activation and downstream signaling. IMC-A12 also mediates robust internalization and degradation of the IGF-IR. In human tumor xenograft models, IGF-IR blockade by IMC-A12 results in rapid and profound growth inhibition of cancers of the breast, lung, colon, and pancreas, and many other neoplasms. Although promising single-agent activity has been observed, the most impressive effects of targeting the IGF-IR with IMC-A12 have been noted when this agent was combined with cytotoxic agents or other targeted therapeutics. The results with IMC-A12 to date suggest that it may be an effective therapeutic in a diverse array of oncologic indications.

  20. Ex vivo human platelet aggregation induced by decompression during reduced barometric pressure, hydrostatic, and hydrodynamic (Bernoulli) effect.

    PubMed

    Murayama, M

    1984-03-01

    Decompression of human platelet-rich plasma (PRP) in siliconized glass or plastic to 380 mm Hg for 3 hours at 38 degrees C produced platelet aggregation independent of pO2. Aggregation also took place when PRP was compressed to 8,000 PSI and then decompressed slowly to one atmosphere (14.7 PSI) without gas bubble formation. Platelets also aggregated when plasma was decompressed hydrodynamically (Bernoulli effect) at room temperature. It was also found that the drugs piracetam (2-oxypyrolidine acetamide) and pentoxifylline (1-(5-oxohexyl)-theobromine) at 0.5 and 1.0 mM prevent thrombocyte aggregation. Implications for mountain sickness are discussed.

  1. 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

  2. Aggregation of complement receptors on human neutrophils in the absence of ligand

    PubMed Central

    1987-01-01

    C3bi receptors (CR3) on human polymorphonuclear leukocytes (PMN) bind ligand-coated particles and promote their ingestion. The binding activity of CR3 is not constitutive but is transiently enabled by phorbol esters (Wright, S. D., and B. D. Meyer, 1986, J. Immunol. 136:1759-1764). Our observations indicate that the capacity of CR3 to bind ligand is tightly correlated with the degree of ligand-independent aggregation of the receptor in the plane of the membrane. Fixed PMN were labeled with anti-CR3 monoclonal antibodies and streptavidin colloidal gold before viewing in the electron microscope either en face or in thin section. On unstimulated PMN, gold particles marking CR3 were dispersed randomly. Stimulation of PMN for 25 min with phorbol myristate acetate (PMA) dramatically enhances binding of C3bi-coated particles, and the CR3 on such stimulated cells was observed in clusters containing more than six gold particles. CR3 was not aggregated over coated pits. After 50 min in PMA, the binding activity of CR3 falls, and the distribution of CR3 was again observed to be disperse. If a hydrophilic phorbol ester was washed away after a 20-min stimulation, binding activity remains elevated for at least 50 min, and CR3 remained aggregated. Thus, clustering of CR3 was temporally correlated with its ability to bind ligand and initiate phagocytosis. Unlike CR3, Fc receptors and HLA did not exhibit changes in their aggregation state in response to PMA. Treating PMN with formyl- methionyl-leucyl-phenylalanine, which enhances expression of CR3 but not its function, did not lead to aggregation of CR3. These observations suggest that a clustered configuration is a precondition necessary for binding ligand and signaling phagocytosis. PMID:2958480

  3. 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.

  4. Humanizing murine IgG3 anti-GD2 antibody m3F8 substantially improves antibody-dependent cell-mediated cytotoxicity while retaining targeting in vivo

    PubMed Central

    Cheung, Nai-Kong V.; Guo, Hongfen; Hu, Jian; Tassev, Dimiter V.; Cheung, Irene Y.

    2012-01-01

    Murine IgG3 anti-GD2 antibody m3F8 has shown anti-neuroblastoma activity in Phase I/II studies, where antibody-dependent cell-mediated cytotoxicity (ADCC) played a key role. Humanization of m3F8 should circumvent human anti-mouse antibody (HAMA) response and enhance its ADCC properties to reduce dosing and pain side effect. Chimeric 3F8 (ch3F8) and humanized 3F8 (hu3F8-IgG1 and hu3F8-IgG4) were produced and purified by protein A affinity chromatography. In vitro comparison was made with m3F8 and other anti-GD2 antibodies in binding, cytotoxicity, and cross-reactivity assays. In GD2 binding studies by SPR, ch3F8 and hu3F8 maintained KD comparable to m3F8. Unlike other anti-GD2 antibodies, m3F8, ch3F8 and hu3F8 had substantially slower koff.. Similar to m3F8, both ch3F8 and hu3F8 inhibited tumor cell growth in vitro, while cross-reactivity with other gangliosides was comparable to that of m3F8. Both peripheral blood mononuclear cell (PBMC)-ADCC and polymorphonuclear leukocytes (PMN)-ADCC of ch3F8 and hu3F8-IgG1 were more potent than m3F8. This superiority was consistently observed in ADCC assays, irrespective of donors or NK-92MI-transfected human CD16 or CD32, whereas complement mediated cytotoxicity (CMC) was reduced. As expected, hu3F8-IgG4 had near absent PBMC-ADCC and CMC. Hu3F8 and m3F8 had similar tumor-to-non tumor ratios in biodistribution studies. Anti-tumor effect against neuroblastoma xenografts was better with hu3F8-IgG1 than m3F8. In conclusion, humanizing m3F8 produced next generation anti-GD2 antibodies with substantially more potent ADCC in vitro and anti-tumor activity in vivo. By leveraging ADCC over CMC, they may be clinically more effective, while minimizing pain and HAMA side effects. A Phase I trial using hu3F8-IgG1 is ongoing. PMID:22754766

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

    PubMed

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

    1999-03-01

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

  6. 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.

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

    PubMed Central

    2015-01-01

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

  8. 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

  9. Neutralization activity and kinetics of two broad-range human monoclonal IgG1 derived from recombinant Fab fragments and directed against Hepatitis C virus E2 glycoprotein.

    PubMed

    Diotti, Roberta Antonia; Sautto, Giuseppe Andrea; Solforosi, Laura; Mancini, Nicasio; Clementi, Massimo; Burioni, Roberto

    2012-10-01

    Hepatitis C virus (HCV) is the major cause of chronic liver disease worldwide. There is evidence that neutralizing anti-HCV antibodies may find potential applications in novel prophylactic and therapeutic strategies. This paper describes the very high neutralization activity and unique biological features of two broadly cross-reactive and cross-neutralizing anti-HCV human monoclonal IgG1 derived from human monoclonal recombinant Fab fragments.

  10. Human tonsillar IgE biosynthesis in vitro. I. Enhancement of IgE and IgG synthesis in the presence of pokeweed mitogen by T-cell irradiation

    SciTech Connect

    Ohta, K.; Manzara, T.; Harbeck, R.J.; Kirkpatrick, C.H.

    1982-02-01

    A study of the events regulating human IgE biosynthesis in vitro was undertaken with tonsillar lymphocytes. IgG synthesis was also studied to evaluate the specificity of our observations. T-cell irradiation significantly enhanced synthesis of IgE by pokeweed mitogen (PWM)-stimulated B cells from 12 of 18 donors and IgG in all 18 donors. This enhancement was the result of de novo immunoglobulin synthesis, since the amount of IgE and IgG spontaneously released from lysed and lysed-and-cultured mononuclear cells was significantly less than that detected in the cell cultures, and the augmentation was completely ablated by the treatment of the cells with cycloheximide or mitomycin C. Enhancement was also dependent on the presence of PWM; T-cell irradiation did not enhance IgE synthesis in unstimulated cultures. Moreover, this enhancement was also observed in the co-cultures of B cells and allogeneic irradiated T cells. These observations suggest that radiosensitive T cells exert a suppressive activity that contributes to regulation of human IgE and IgG synthesis and that the suppressor function as well as the helper function can overcome allogeneic disparities.

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

    SciTech Connect

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

    1986-03-01

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

  12. 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.

  13. Expression and gene transcript of Fc receptors for IgG, HLA class II antigens and Langerhans cells in human cervico-vaginal epithelium.

    PubMed Central

    Hussain, L A; Kelly, C G; Fellowes, R; Hecht, E M; Wilson, J; Chapman, M; Lehner, T

    1992-01-01

    The mechanism of transmission of HIV from the male to the female genital tract or in the reverse order is not clear. CD4 glycoprotein is the receptor for HIV and Langerhans cells and the related dendritic cells could play a role in the initial transmission of HIV. Fc receptors (FcR) for IgG might be involved in antibody-mediated binding of HIV. We carried out an immunohistological study of normal human cervical and vaginal epithelia for the presence of CD4 glycoprotein, Langerhans cells and FcR to IgG. CD4+ glycoprotein was not found in the vaginal or cervical epithelium, with the exception of a few endocervical epithelial cells. A small number of CD4+ mononuclear cells were found in the endocervical epithelium of a third of the specimens but a large number of CD4+ cells was found in the submucosa of most of the cervical and vaginal specimens. Langerhans cells expressing CD4, HLA class II, Fc gamma R2 and Fc gamma R3 were detected in most vaginal, ectocervical and transformation zone epithelia and in 9/14 endocervical tissues. Fc gamma R3 was detected in about two-thirds of the columnar endocervical epithelium and the transformation zone. A smaller number of specimens expressed Fc gamma R2 in these epithelia, but Fc gamma R1 was not detected. We then demonstrated mRNA for Fc gamma R3 in the columnar endocervical epithelial cells and transformation zone by in situ hybridization, using a CD16-RNA probe. Fc gamma R3 and Fc gamma R2 gene transcripts were also found in fetal cervical tissue by applying the polymerase chain reaction to amplify portions of the Fc gamma R3 and Fc gamma R2 coding sequences in cDNA prepared from fetal RNA. HLA-DR was found in the endocervical cells, transformation zone and in Langerhans cells of all specimens. The presence of Langerhans cells, Fc gamma receptors and HLA class II antigen offers three potential mechanisms for cervico-vaginal HIV transmission: (i) direct HIV infection of Langerhans cells, (ii) binding of HIV antibody complexes

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

    PubMed

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

    2015-06-01

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

  15. 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

  16. Spatial and temporal control of cell aggregation efficiently directs human pluripotent stem cells towards neural commitment.

    PubMed

    Miranda, Cláudia C; Fernandes, Tiago G; Pascoal, Jorge F; Haupt, Simone; Brüstle, Oliver; Cabral, Joaquim M S; Diogo, Maria Margarida

    2015-10-01

    3D suspension culture is generally considered a promising method to achieve efficient expansion and controlled differentiation of human pluripotent stem cells (hPSCs). In this work, we focused on developing an integrated culture platform for expansion and neural commitment of hPSCs into neural precursors using 3D suspension conditions and chemically-defined culture media. We evaluated different inoculation methodologies for hPSC expansion as 3D aggregates and characterized the resulting cultures in terms of aggregate size distribution. It was demonstrated that upon single-cell inoculation, after four days of culture, 3D aggregates were composed of homogenous populations of hPSC and were characterized by an average diameter of 139 ± 26 μm, which was determined to be the optimal size to initiate neural commitment. Temporal analysis revealed that upon neural specification it is possible to maximize the percentage of neural precursor cells expressing the neural markers Sox1 and Pax6 after nine days of culture. These results highlight our ability to define a robust method for production of hPSC-derived neural precursors that minimizes processing steps and that constitutes a promising alternative to the traditional planar adherent culture system due to a high potential for scaling-up. PMID:25866360

  17. Conglutinin-like factors in human saliva--relation to other salivary aggregating factors--.

    PubMed

    Murai, Y

    1980-12-01

    This study was conducted to examine the relation between conglutinin-like factors and other bacterial aggregating factors in human saliva. Human and guinea pig complement intermediate cells (EAC4b,3b) were prepared by using and anticomplementary agent K-76 COONa. Conglutinin-like factors and agglutinins for sensitized sheep erythrocytes in parotid and whole saliva from seven subjects were examined. Whole saliva from the subjects with a periodontal disease showed a lower activity than that from the subjects with a clinically normal gingiva. It seems, therefore, that some strum component from the gingival crevice inhibit the aggregation of sensitized sheep erthrocytes by saliva as in the case of the conglutination of EAC4b, 3b cells. Conglutinin-like factors appeard over a wide region including both the void volume and the secretory IgA region in gel filtration of human whole saliva on Sepharose 4B. The void volume fractions contained a high conglutinin-like factor activity but no Iga activity. These data suggest that conglutinin-like factors are not polymers of IgA but complexes of glycoproteins or those on which IgA is bound furthermore. PMID:6936093

  18. Glyoxylate lowers metabolic ATP in human platelets without altering adenylate energy charge or aggregation.

    PubMed

    Dangelmaier, Carol A; Holmsen, Holm

    2014-01-01

    Human blood platelets adhere to exposed collagen at the site of vascular injury, initiating a signaling cascade leading to fibrinogen activation, secretion of granules and aggregation, thus producing a stable thrombus. All these steps require metabolic ATP. In this study we have labeled the metabolic pool of ATP with nucleotides, treated platelets with various inhibitors and have monitored their ability to be activated. Incubating platelets with glyoxylate dramatically reduced the ATP level without a change in the adenylate energy charge (AEC). This reduction of ATP did not affect ADP-induced primary or secondary aggregation, whereas glyoxal, methyl glyoxal, or the combination of antimycin plus deoxyglucose reduced both ATP and AEC and inhibited aggregation. The reduction of ATP by glyoxylate was almost quantitatively matched by an increase in hypoxanthine without elevation of ADP. AMP, IMP or inosine, acetoacetate, aspartate, or glutamate had no effect on glyoxylate-induced breakdown of ATP, while pyruvate stopped the ATP reduction fast and efficiently. Glyoxylate also lowered the citrate content. The glyoxylate-induced breakdown of ATP coincided with an increase in fructose-1,6-bisphosphate, indicating that the phosphofructokinase reaction was the main ATP-consuming step. Glyoxylate was a substrate for lactate dehydrogenase although with a Km almost 100 times higher than pyruvate. We suggest that glyoxylate primarily competes with pyruvate in the pyruvate dehydrogenase reaction, thus lowering the citrate concentration, which in turn activates phosphofructokinase. Clearly, lowering of ATP in the cytosol by more than 50% does not affect platelet aggregation provided that the AEC is not reduced.

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

    PubMed Central

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

    2016-01-01

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

  20. Analysis of lysine clipping of a humanized Lewis-Y specific IgG antibody and its relation to Fc-mediated effector function.

    PubMed

    Antes, Bernhard; Amon, Sabine; Rizzi, Andreas; Wiederkum, Susi; Kainer, Manuela; Szolar, Oliver; Fido, Markus; Kircheis, Ralf; Nechansky, Andreas

    2007-06-01

    During the analytical characterization of the humanized Lewis-Y specific monoclonal antibody IGN311 (IgG1/kappa) used for passive anti-cancer therapy in humans, isoelectric focusing (IEF) experiments revealed that IGN311 batches produced in serum-containing and serum-free medium, respectively, displayed different banding patterns. The additional bands in the IEF pattern correlated with additional peaks observed by subsequent cation exchange (CEX)-HPLC analysis. Since the IEF pattern is one of the specification criteria in the quality control of monoclonal antibodies and a non-matching pattern may be indicative for lot-to-lot inconsistency, this phenomenon was investigated in detail. First, we investigated whether a difference in antibody glycosylation was the cause for the observed charge heterogeneity. De-N-glycosylation experiments demonstrated that charge heterogeneity observed in the IEF pattern is not a consequence of glycosylation. In contrast, sample treatment by carboxypeptidase B, removing the carboxy-terminal lysine residues from the two heavy chains of the antibody, resulted in reduced charge heterogeneity eliminating the two most basic bands observed in IEF. These data were supported by reversed phase HPLC-MALDI-TOF-MS analysis of enzymatically cleaved peptides of the antibody as well as by carboxy-terminal sequencing of the heavy chains. It was demonstrated that the differences in the IEF banding pattern were due to lysine clipping occurring during the production of the antibody. The antibody batch produced under serum-free conditions was less affected by lysine clipping. Both antibody variants--clipped and unclipped--elicited the same potency in a complement dependent cytotoxicity (CDC) assay demonstrating that lysine clipping of IGN311 does not impair Fc-mediated effector functions.

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

    PubMed

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

    2016-05-01

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

  2. Sequence-dependent abnormal aggregation of human Tau fragment in an inducible cell model.

    PubMed

    Liu, Xiao-Ling; Hu, Ji-Ying; Hu, Meng-Yun; Zhang, Yi; Hong, Zheng-Yuan; Cheng, Xiao-Qing; Chen, Jie; Pang, Dai-Wen; Liang, Yi

    2015-08-01

    A pathological hallmark of Alzheimer disease (AD) is the accumulation of misfolded hyperphosphorylated microtubule-associated protein Tau within neurons, forming neurofibrillary tangles and leading to synaptic dysfunction and neuronal death. Here we study sequence-dependent abnormal aggregation of human fragment Tau244-372 in an inducible cell model. As evidenced by confocal laser scanning microscopy, Western blot, and immunogold electron microscopy, fibril-forming motifs are essential and sufficient for abnormal aggregation of Tau244-372 in SH-SY5Y neuroblastoma cells induced by Congo red: when its two fibril-forming segments PHF6 and PHF6* are deleted, Tau244-372 does lose its ability to form fibrils in SH-SY5Y cells, and the replacement of PHF6 and PHF6* with an unrelated amyloidogenic sequence IFQINS from human lysozyme does rescue the fibril-forming ability of Tau244-372 in SH-SY5Y cells. By contrast, insertion of a non-fibril forming peptide GGGGGG does not drive the disabled Tau244-372 to misfold in SH-SY5Y cells. Furthermore, as revealed by quantum dots based probes combined with annexin V staining, annexin V-FITC apoptosis detection assay, and immunofluorescence, fibril-forming motifs are essential and sufficient for early apoptosis of living SH-SY5Y cells induced by abnormal aggregation of Tau244-372. Our results suggest that fibril-forming motifs could be the determinants of Tau protein tending to misfold in living cells, thereby inducing neuronal apoptosis and causing the initiation and development of AD.

  3. 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.

  4. Comparison of Three Commercially Available Serologic Assays Used To Detect Human Parvovirus B19-Specific Immunoglobulin M (IgM) and IgG Antibodies in Sera of Pregnant Women

    PubMed Central

    Butchko, Allyson R.; Jordan, Jeanne A.

    2004-01-01

    A split-sample study was conducted to evaluate the performances of three enzyme immunoassays (EIAs) utilizing one or more conformational antigens to detect human parvovirus B19 (B19V)-specific immunoglobulin M (IgM) or IgG in the sera of 198 pregnant women. We compared EIAs available from Biotrin International, Inc. (Dublin, Ireland), Medac Diagnostika (Wedel, Germany), and Mikrogen (Martinsried, Germany). Specimens with discordant results were analyzed further using an immunofluorescence assay (Biotrin). Equivocal data accounted for close to half of all the discrepant results for both IgM and IgG, with 7 of 15 discrepant results from the Medac and Mikrogen kits involving equivocal data and the Biotrin kit giving a single equivocal result. For each specimen, a consensus was established from the four test results if agreement occurred among at least three of four results. Overall, the highest percentage of agreement with the consensus results was seen when Biotrin kits were used; 194 (100%) of 194 and 194 (99.5%) of 195 results for IgM and IgG, respectively, agreed with the consensus results. When Medac kits were used, 189 (97.4%) of 194 and 191 (97.9%) of 195 results for IgM and IgG, respectively, agreed with the consensus, and when Mikrogen kits were used, 179 (92.3%) of 194 and 193 (99%) of 195 results for IgM and IgG, respectively, agreed with the consensus. Given the consensus results, the Medac EIA appeared to generate presumed false-positive results for IgM and the Mikrogen EIA appeared to generate presumed false-positive results for IgG and IgM. In summary, the Biotrin EIAs produced far fewer equivocal results than the other assays and results of the Biotrin EIAs agreed more often with the consensus results than did those of the other commercially available EIAs for detecting B19V-specific IgM and IgG antibodies. PMID:15243081

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

    PubMed Central

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

    2014-01-01

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

  6. Partial characterization of the shift from IgG to IgA synthesis in the clonal differentiation of human leukemic bone marrow-derived lymphocytes

    PubMed Central

    1975-01-01

    An unusual B-cell proliferation was noted in an individual (Tun) which was characterized by the presence of two separate populations of chronic lymphocytic leukemia (CLL) cell staining on the surface and in the cytoplasm for either IgG(k) or IgA(k). Utilizing an idiotypic antiserum prepared from the associated serum monoclonal IgG(k) protein the idiotype was detected on the surface and in the cytoplasm of both the IgG- and IgA-bearing cell populations. These observations are consistent with a common clonal origin and a switch mechanism involving IgG and IgA synthesis. Sequential-labeling of Surface Ig and intracellular Ig with antisera conjugated to opposite fluorochromes documented the progressive maturation of the terminal differentiation of the IgA-bearing cell population at a level before morphologically distinct plasma cells. The distribution and pattern of surface and cytoplasmic IgG and IgA staining in individual cells suggest that the direction of switching is from IgG to IgA synthesis. The demonstration of shared idiotypic specificity between the IgG- and IgA-bearing populations is consistent with a transition in Ig heavy chain synthesis resulting from an alternation in the CH gene. It is concluded that certain CLL clones may manifest a switch from IgG to IgA synthesis at a level of B-cell differentiation which encompasses both the B lymphocyte and the Ig-synthesizing plasma cell. PMID:809529

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

    PubMed

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

    2016-02-01

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

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

    PubMed

    Luo, Shen; Zhang, Baolin

    2015-01-01

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

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

    PubMed

    Luo, Shen; Zhang, Baolin

    2015-01-01

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

  10. IgG4-related disease of the rectum

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-08-01

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

  12. 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.

  13. 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. PMID:26579725

  14. 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. PMID:26742760

  15. 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

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

    PubMed Central

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

    2014-01-01

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

  17. Effect of prenylated flavonoids and chalcones isolated from Artocarpus species on platelet aggregation in human whole blood.

    PubMed

    Jantan, Ibrahim; Mohd Yasin, Yusyila H; Jamil, Shajarahtunnur; Sirat, Hasnah; Basar, Norazah

    2010-07-01

    Five prenylflavonoids and two prenylchalcones from Artocarpus lowii King, A. scortechinii King and A. teysmanii Miq., and acetylated derivatives of cycloheterophyllin and artonin E were investigated for their ability to inhibit arachidonic acid (AA), collagen and adenosine diphosphate (ADP)-induced platelet aggregation in human whole blood by using an electrical impedance method. Among the tested compounds, only cycloheterophyllin inhibited AA-induced platelet aggregation with an IC(50) value of 100.9 microM. It also showed strong inhibition against ADP-induced aggregation, with an IC(50) value of 57.1 microM. Isobavachalcone, 2',4'-dihydroxy-4-methoxy-3'-prenyldihydrochalcone, cycloartobiloxanthone, artonin E and artonin E triacetate showed selective inhibition against ADP-induced aggregation, with IC(50) values ranging from 55.3 to 192.0 microM, but did not show such effect against other inducers.

  18. Covalent cross-links in oxygen free radical altered human immunoglobulin G.

    PubMed

    Kleinveld, H A; Hack, C E; Swaak, A J; van Noort, W L; van Eijk, H G; Koster, J F

    1988-01-01

    The damaging effect of an oxygen free radical generating system, i.e. ultraviolet irradiation, on human immunoglobulin G (IgG) was studied. The free radical altered IgG was analysed by a high performance liquid chromatograph equipped with a TSK G 3000 SW-column. Gel filtration of 120 min UV-irradiated IgG resulted in three clearly distinguished peaks corresponding to polymer IgG (MW greater than 500 kD), dimer IgG (MW 300 kD) and monomer IgG (MW 150 kD). Analysis of oxygen free radical altered and aggregated IgG by SDS-PAGE and subsequent silver-staining revealed inter- and intra-molecular reduction (by beta-mercaptoethanol)-resistant cross-links between IgG-molecules were formed. Comparison of amino acid analyses of native IgG with oxygen free radical aggregated polymer IgG showed significant reductions in tyrosine- (7.0%) and histidine- (6.5%) content. These findings suggest that tyrosine and histidine are involved in covalent cross-linking between IgG-molecules caused by oxygen free radicals. These alterations on IgG induced by free radical-activity might render it antigenic, and could initiate the production of rheumatoid factors (RF).

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

    PubMed Central

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

    2009-01-01

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

  20. Expression of Tight Junction Molecule In The Human Serum-Induced Aggregation of Human Abdominal Adipose-Derived Stem Cells In Vitro

    PubMed Central

    Yoon, A Young; Yun, Sujin; Yang, HyeJin; Lim, Yoon Hwa; Kim, Haekwon

    2014-01-01

    Previously we have shown that human abdominal adipose derived-stem cells (ADSCs) could aggregate during the high-density culture in the presence of human serum (HS). In the present study, we observed that human cord blood serum (CBS) and follicular fluid (HFF) also induced aggregation. Similarly, porcine serum could induce aggregation whereas bovine and sheep sera induced little aggregation. qRT-PCR analyses demonstrated that, compared to FBS-cultured ADSCs, HScultured cells exhibited higher level of mRNA expression of CLDN3, -6, -7, -15, and -16 genes among the tight junction proteins. ADSCs examined at the time of aggregation by culture with HS, BSA, HFF, CBS, or porcine serum showed significantly higher level of mRNA expression of JAM2 among JAM family members. In contrast, cells cultured in FBS, bovine serum or sheep serum, showed lower level of JAM2 expression. Immunocytochemical analyses demonstrated that the aggregates of HS-cultured cells (HS-Agg) showed intense staining against the anti-JAM2 antibody whereas neither non-aggregated cells (HS-Ex) nor FBS-cultured cells exhibited weak staining. Western blot results showed that HS-Agg expressed JAM2 protein more prominently than HS-Ex and FBS-cultured cells, both of latter reveled weaker intensity. These results suggest that the aggregation property of ADSCs during high-density culture would be dependent on the specific components of serum, and that JAM2 molecule could play a role in the animal sera-induced aggregation in vitro. PMID:25949191

  1. Adenosine diphosphate-induced aggregation of human platelets in flow through tubes: III. Shear and extrinsic fibrinogen-dependent effects.

    PubMed

    Goldsmith, H L; Frojmovic, M M; Braovac, S; McIntosh, F; Wong, T

    1994-01-01

    The effect of shear rate and fibrinogen concentration on adenosine diphosphate-induced aggregation of suspensions of washed human platelets in Poiseuille flow at 23 degrees C was studied using a previously described double infusion technique and resistive particle counter size analysis. Using suspensions of multiple-centrifuged and -washed cells in Tyrodes-albumin [3 x 10(5) microliters-1; (17)] with [fibrinogen] from 0 to 1.2 microM, the rate and extent of aggregation with 0.7 microM ADP in Tyrodes-albumin were measured over a range of mean transit times from 0.2 to 43 s, and at mean tube shear rates, G, = 41.9, 335 and 1,335 s-1. As measured by the decrease in singlet concentration, aggregation at 1.2 microM fibrinogen increased with increasing G up to 1,335 s-1, in contrast to that previously reported in citrated plasma, in which aggregation reached a maximum at G = 335 s-1. Without added fibrinogen, there was no aggregation at G = 41.9 s-1; at G = 335 s-1, there was significant aggregation but with an initial lag time, aggregation increasing further at G = 1,335 s-1. Without added fibrinogen, aggregation was abolished at all G upon incubation with the hexapeptide GRGDSP, but was almost unaffected by addition of an F(ab')2 fragment of an antibody to human fibrinogen. Aggregation in the absence of added fibrinogen was also observed at 37 degrees C. The activation of the multiple-washed platelets was tested using flow cytometry with the fluorescently labelled monoclonal antibodies FITC-PAC1 and FITC-9F9. It was shown that 57% of single cells in unactivated PRT expressed maximal GPIIb-IIIa fibrinogen receptors (MoAb PAC1) and 54% expressed pre-bound fibrinogen (MoAb 9F9), with further increases on ADP activation. However, incubation with GRGDSP and the F(ab')2 fragment did not inhibit the prebound fibrinogen. Moreover, relatively unactivated cells (8% expressing receptor, 14% prebound fibrinogen), prepared from acidified cPRP by single centrifugation with 50 nM of

  2. 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

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

    SciTech Connect

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

    1990-07-01

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

  4. 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.

  5. Liquid–liquid diffusion crystallization improves the X-ray diffraction of EndoS, an endo-β-N-acetyl­glucosaminidase from Streptococcus pyogenes with activity on human IgG

    PubMed Central

    Trastoy, Beatriz; Lomino, Joseph V.; Wang, Lai-Xi; Sundberg, Eric J.

    2013-01-01

    Endoglycosidase S (EndoS) is an enzyme secreted by Streptococcus pyogenes that specifically hydrolyzes the β-1,4-di-N-acetylchitobiose core glycan on immunoglobulin G (IgG) antibodies. One of the most common human pathogens and the cause of group A streptococcal infections, S. pyogenes secretes EndoS in order to evade the host immune system by rendering IgG effector mechanisms dysfunctional. On account of its specificity for IgG, EndoS has also been used extensively for chemoenzymatic synthesis of homogeneous IgG glycoprotein preparations and is being developed as a novel therapeutic for a wide range of autoimmune diseases. The structural basis of its enzymatic activity and substrate specificity, however, remains unknown. Here, the purification and crystallization of EndoS are reported. Using traditional hanging-drop and sitting-drop vapor-diffusion crystallization, crystals of EndoS were grown that diffracted to a maximum of 3.5 Å resolution but suffered from severe anisotropy, the data from which could only be reasonably processed to 7.5 Å resolution. When EndoS was crystallized by liquid–liquid diffusion, it was possible to grow crystals with a different space group to those obtained by vapor diffusion. Crystals of wild-type endoglycosidase and glycosynthase constructs of EndoS grown by liquid–liquid diffusion diffracted to 2.6 and 1.9 Å resolution, respectively, with a greatly diminished anisotropy. Despite extensive efforts, the failure to reproduce these liquid–liquid diffusion-grown crystals by vapor diffusion suggests that these crystallization methods each sample a distinct crystallization space. PMID:24316841

  6. Liquid-liquid diffusion crystallization improves the X-ray diffraction of EndoS, an endo-β-N-acetylglucosaminidase from Streptococcus pyogenes with activity on human IgG.

    PubMed

    Trastoy, Beatriz; Lomino, Joseph V; Wang, Lai Xi; Sundberg, Eric J

    2013-12-01

    Endoglycosidase S (EndoS) is an enzyme secreted by Streptococcus pyogenes that specifically hydrolyzes the β-1,4-di-N-acetylchitobiose core glycan on immunoglobulin G (IgG) antibodies. One of the most common human pathogens and the cause of group A streptococcal infections, S. pyogenes secretes EndoS in order to evade the host immune system by rendering IgG effector mechanisms dysfunctional. On account of its specificity for IgG, EndoS has also been used extensively for chemoenzymatic synthesis of homogeneous IgG glycoprotein preparations and is being developed as a novel therapeutic for a wide range of autoimmune diseases. The structural basis of its enzymatic activity and substrate specificity, however, remains unknown. Here, the purification and crystallization of EndoS are reported. Using traditional hanging-drop and sitting-drop vapor-diffusion crystallization, crystals of EndoS were grown that diffracted to a maximum of 3.5 Å resolution but suffered from severe anisotropy, the data from which could only be reasonably processed to 7.5 Å resolution. When EndoS was crystallized by liquid-liquid diffusion, it was possible to grow crystals with a different space group to those obtained by vapor diffusion. Crystals of wild-type endoglycosidase and glycosynthase constructs of EndoS grown by liquid-liquid diffusion diffracted to 2.6 and 1.9 Å resolution, respectively, with a greatly diminished anisotropy. Despite extensive efforts, the failure to reproduce these liquid-liquid diffusion-grown crystals by vapor diffusion suggests that these crystallization methods each sample a distinct crystallization space.

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

    PubMed Central

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

    2014-01-01

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

  8. 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.

  9. Orally given gastroprotective capsaicin does not modify aspirin-induced platelet aggregation in healthy male volunteers (human phase I examination).

    PubMed

    Sandor, B; Papp, J; Mozsik, Gy; Szolcsanyi, J; Keszthelyi, Zs; Juricskay, I; Toth, K; Habon, Tamas

    2014-12-01

    Capsaicin is a well-known component of red pepper. Recent studies have shown that capsaicin could prevent gastric ulcer provoked by various NSAID-s like acetylsalicylic acid (ASA). Primary objective of this human clinical phase I trial was to investigate whether two different doses of capsaicin co-administered with ASA could alter the inhibitory effect of ASA on platelet aggregation. 15 healthy male subjects were involved in the study and treated orally with 400 μg capsaicin, 800 μg capsaicin, 500 mg ASA, 400 μg capsaicin+500 mg ASA and 800 μg capsaicin+500 mg ASA. Blood was drawn before and 1, 2, 6 and 24 hours after the drug administration. After that epinephrine induced platelet aggregation was measured by optical aggregometry. Between treatments, volunteers had a 6-day wash-out period. Our results showed that capsaicin had no effect on platelet aggregation, while as expected, ASA monotherapy resulted in a significant and clinically effective platelet aggregation inhibition (p ≤ 0.001). The combined ASA-capsaicin therapies reached equivalent effectiveness in platelet aggregation inhibition as ASA monotherapy. Our investigation proved that capsaicin did not influence the inhibitory effect of ASA on platelet aggregation, thus the capsaicin-ASA treatment would combine the antiplatelet effect of ASA with the possible gastroprotection of capsaicin.

  10. 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).

  11. Induction of aggregation of Raji human B-lymphoblastic cells by vasoactive intestinal peptide.

    PubMed Central

    Robichon, A; Sreedharan, S P; Yang, J; Shames, R S; Gronroos, E C; Cheng, P P; Goetzl, E J

    1993-01-01

    Subsets of neurons in the thymic cortex, Peyer's patches and lymphoid tissues of the respiratory system deliver vasoactive intestinal peptide (VIP) at nanomolar concentrations. The possible effects of VIP on B-cell adhesiveness in these tissues were examined in studies of the homotypic aggregation (HA) of human B-lymphoblastoid cells of the Raji line, which express a mean of 27,950 VIP receptors/cell with a mean Kd of 0.8 nM. Mean HA, assessed microscopically, attained a maximum of 54% after 8 hr with 0.1 microgram/ml of phorbol 12-myristate 13-acetate (PMA) (P < 0.01) and 31% after 24 hr with 10(-8) M VIP (P < 0.05), as contrasted with 13% and 20% at the respective times in medium alone, and both stimuli also increased the mean size of aggregates. The presence of the phosphodiesterase inhibitor Ro 20-1724 permitted 10(-9) M VIP, which had no effect alone, to raise the mean cyclic AMP content of Raji cells by more than 10-fold and concurrently to elevate mean HA from 55% in medium alone at 48 hr to 70% and from 55% at 72 hr to 68% (P < 0.05 for both). Monoclonal antibodies to lymphocyte function-associated (LFA-1) adhesive protein and to intercellular adherence molecule-1 (ICAM-1) suppressed significantly the HA of Raji cells induced by VIP and PMA. The effects of VIP on compartmental immunity in the lungs and intestines thus may be mediated in part by increases in lymphocyte adhesiveness, which could contribute to the regional accumulation of specifically immunocompetent cells. Images Figure 2 PMID:8104888

  12. Upregulation of NRG-1 and VAMP-1 in Human Brain Aggregates Exposed to Clozapine

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2009-09-01

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

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

    PubMed

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

    2016-09-01

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

  15. MotionExplorer: exploratory search in human motion capture data based on hierarchical aggregation.

    PubMed

    Bernard, Jürgen; Wilhelm, Nils; Krüger, Björn; May, Thorsten; Schreck, Tobias; Kohlhammer, Jörn

    2013-12-01

    We present MotionExplorer, an exploratory search and analysis system for sequences of human motion in large motion capture data collections. This special type of multivariate time series data is relevant in many research fields including medicine, sports and animation. Key tasks in working with motion data include analysis of motion states and transitions, and synthesis of motion vectors by interpolation and combination. In the practice of research and application of human motion data, challenges exist in providing visual summaries and drill-down functionality for handling large motion data collections. We find that this domain can benefit from appropriate visual retrieval and analysis support to handle these tasks in presence of large motion data. To address this need, we developed MotionExplorer together with domain experts as an exploratory search system based on interactive aggregation and visualization of motion states as a basis for data navigation, exploration, and search. Based on an overview-first type visualization, users are able to search for interesting sub-sequences of motion based on a query-by-example metaphor, and explore search results by details on demand. We developed MotionExplorer in close collaboration with the targeted users who are researchers working on human motion synthesis and analysis, including a summative field study. Additionally, we conducted a laboratory design study to substantially improve MotionExplorer towards an intuitive, usable and robust design. MotionExplorer enables the search in human motion capture data with only a few mouse clicks. The researchers unanimously confirm that the system can efficiently support their work. PMID:24051792

  16. 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.

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    1992-01-01

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

  20. Enhanced apoptotic effects of dihydroartemisinin-aggregated gelatin and hyaluronan nanoparticles on human lung cancer cells.

    PubMed

    Sun, Qian; Teong, Benjamin; Chen, I-Fen; Chang, Shwu Jen; Gao, Jimin; Kuo, Shyh-Ming

    2014-04-01

    Recent studies suggest that dihydroartemisinin (DHA), a derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua L., has anticancer properties. Due to poor water solubility, poor oral activity, and a short plasma half-life, large doses of DHA have to be injected to achieve the necessary bioavailability. This study examined increasing DHA bioavailability by encapsulating DHA within gelatin (GEL) or hyaluronan (HA) nanoparticles via an electrostatic field system. Observations from transmission electron microscopy show that DHA in GEL and HA nanoparticles formed GEL/DHA and HA/DHA aggregates that were approximately 30-40 nm in diameter. The entrapment efficiencies for DHA were approximately 13 and 35% for the GEL/DHA and HA/DHA aggregates, respectively. The proliferation of A549 cells was inhibited by the GEL/DHA and HA/DHA aggregates. Fluorescent annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining displayed low background staining with annexin V-FITC or PI on DHA-untreated cells. In contrast, annexin V-FITC and PI stains dramatically increased when the cells were incubated with GEL/DHA and HA/DHA aggregates. These results suggest that DHA-aggregated GEL and HA nanoparticles exhibit higher anticancer proliferation activities than DHA alone in A549 cells most likely due to the greater aqueous dispersion after hydrophilic GEL or HA nanoparticles aggregation. These results demonstrate that DHA can aggregate with nanoparticles in an electrostatic field environment to form DHA nanosized aggregates. PMID:24039154

  1. In vitro proliferation and production of cytokine and IgG by human PBMCs stimulated with polysaccharide extract from plants endemic to Gabon.

    PubMed

    Mengome, Line Edwige; Voxeur, Aline; Akue, Jean Paul; Lerouge, Patrice

    2014-11-13

    Polysaccharides were extracted from seven plants endemic to Gabon to study their potential immunological activities. Peripheral blood mononuclear cell (PBMC) (5×10⁵ cells/mL) proliferation, cytokine and immunoglobulin G (IgG) assays were performed after stimulation with different concentrations of polysaccharide fractions compared with lipopolysaccharides (LPS) and concanavalin A (ConA) from healthy volunteers. The culture supernatants were used for cytokine and IgG detection by enzyme-linked immunosorbent assay (ELISA). The results show that pectin and hemicellulose extracts from Uvaria klainei, Petersianthus macrocarpus, Trichoscypha addonii, Aphanocalyx microphyllus, Librevillea klaineana, Neochevalierodendron stephanii and Scorodophloeus zenkeri induced production levels that were variable from one individual to another for IL-12 (3-40 pg/mL), IL-10 (6-443 pg/mL), IL-6 (7-370 pg/mL), GM-CSF (3-170 pg/mL) and IFN-γ (5-80 pg/mL). Only hemicelluloses from Aphanocalyx microphyllus produce a small amount of IgG (OD=0.034), while the proliferation of cells stimulated with these polysaccharides increased up to 318% above the proliferation of unstimulated cells. However, this proliferation of PBMCs was abolished when the pectin of some of these plants was treated with endopolygalacturonase (p<0.05), but the trend of cytokine synthesis remained the same, both before and after enzymatic treatment or saponification. This study suggests that these polysaccharides stimulate cells in a structure-dependent manner. The rhamnogalacturonan-I (RGI) fragment alone was not able to induce the proliferation of PBMC.

  2. Comparative characterization of pulmonary surfactant aggregates and alkaline phosphatase isozymes in human lung carcinoma tissue.

    PubMed

    Iino, Nozomi; Matsunaga, Toshiyuki; Harada, Tsuyoshi; Igarashi, Seiji; Koyama, Iwao; Komoda, Tsugikazu

    2007-05-01

    Alkaline phosphatase (AP) isozymes are surfactant-associated proteins (SPs). Since several different AP isozymes have been detected in the pneumocytes of lung cancer patients, we attempted to identify the relationship between pulmonary surfactant aggregate subtypes and AP isozymes. Pulmonary surfactant aggregates were isolated from carcinoma and non-carcinoma tissues of patients with non-small cell carcinoma of the lung. Upon analysis, ultraheavy, heavy, and light surfactant aggregates were detected in the non-carcinoma tissues, but no ultraheavy surfactant aggregates were found in the carcinoma tissues. Surfactant-associated protein A (SP-A) was detected as two bands (a 27-kDa band and a 54-kDa band) in the ultraheavy, heavy, and light surfactant aggregates found in the non-carcinoma tissues. Although both SP-A bands were detected in the heavy and light surfactant aggregates from adenocarcinoma tissues, the 54-kDa band was not detected in squamous cell carcinoma tissues. Liver AP (LAP) was detected in the heavy and light surfactant aggregates from both non-carcinoma and squamous carcinoma tissues, but not in heavy surfactant aggregates from adenocarcinoma tissues. A larger amount of bone type AP (BAP) was found in light surfactant aggregate fractions from squamous cell carcinomas than those from adenocarcinoma tissues or non-carcinoma tissues from patients with either type of cancer. LAP, BAP, and SP-A were identified immunohistochemically in type II pneumocytes from non-carcinoma tissues and adenocarcinoma cells, but no distinct SP-A staining was observed in squamous cell carcinoma tissues. The present study has thus revealed several differences in pulmonary surfactant aggregates and AP isozymes between adenocarcinoma tissue and squamous cell carcinoma tissue.

  3. Stimulation of human neutrophils by soluble and insoluble immunoglobulin aggregates. Secretion of granule constituents and increased oxidation of glucose.

    PubMed Central

    Henson, P M; Oades, Z G

    1975-01-01

    Reaction of human neutrophils with aggregated immunoglobulin on nonphagocytosable surfaces results in secretion of granule enzymes (exocytosis of granules) and stimulation of glucose oxidation by the nexose monophosphate pathway (HMP). The role of HMP stimulation in the enzyme secretion and some requirements for the two neutrophil activities have been examined. It was found that (a) HMP stimulation could be selectively inhibited under conditions where release of granule enzymes remained unchanged or was enhanced, for example, by reduced glucose concentration or by 2-deoxyglucose. (b) Removal of Ca++ and addition of agents which increased the intracellular levels of cyclic AMP (cAMP), however, prevented both activities, while colchicine had greater inhibitory activity on HMP stimulation than upon secretion. (c) Neutrophils incubated in suspension with particulate aggregated gamma-globulin phagocytosed the particles and exhibited a stimulated HMP and released granule enzymes. In contrast, incubation in suspension with soluble aggregated gamma-globulin resulted in the stimulated HMP only. Granule evzymes were not liberated. 300-fold less soluble aggregates bound to a surface, however readily induced exocytosis of granules from adherent neutrophils. This demonstrates the importance of surface effects in the induction of secretion from neutrophils. Aggregated immunoglobulin reacting with neutrophil Fc receptors thus induces both degranulation (exocytosis) and increased HMP activity. The pathways leading to these events are separable although apparently sharing some common steps, including the initiating events. Images PMID:51031

  4. 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.

  5. Cysteine Racemization on IgG Heavy and Light Chains

    PubMed Central

    Zhang, Qingchun; Flynn, Gregory C.

    2013-01-01

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

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

    PubMed

    Walker, W S

    1976-04-01

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

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

    PubMed

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

    2006-08-01

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

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

    PubMed

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

    2006-08-01

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

  9. An enzyme-linked immunosorbent assay (ELISA) for IgG and IgA antibodies to respiratory syncytial virus in low dilutions of secretions of human serum and secretions.

    PubMed

    Nandapalan, N; Routledge, E; Toms, G L

    1984-01-01

    An enzyme-linked immunosorbent assay (ELISA) has been developed for titration of IgG and IgA antibodies to respiratory syncytial (RS) virus in low dilutions of human serum, colostrum, and nasopharyngeal secretions. Previously the sensitivity of RS virus ELISA on such specimens has been limited by nonspecific absorption of antibody, particularly IgA, to crude antigen preparations. For IgG antibody estimation in infant sera, this unwanted binding was reduced to workable levels by increasing the serum, salt, and detergent concentration of the diluent. Residual nonspecific binding of IgA in colostra appeared mainly due to antigen lipids or to lipoproteins. This was markedly reduced by partitioning Triton X-100-treated infected cell lysate antigens in Arklone. Using the modified ELISA technique for anti-RS virus IgA, good correlations were found with unfixed cell membrane immunofluorescence (MIF) for colostra (r = 0.81, P less than 0.001) and nasal secretions from adult volunteers. In several samples nonspecific absorption of antibody precluded MIF assay, but did not affect the ELISA. Although there was an overall correlation between ELISA for anti-RS IgG antibody in sera, the complement fixation test (r = 0.75, P less than 0.001), and MIF test (r = 0.82, P less than 0.001), the sensitivity of ELISA for antibody responses in convalescent sera of infants from 3 months to 2 years was poor. Conversely, the sensitivity of ELISA for antibody in the sera of older children and for transplacentally acquired antibody in very young infants was higher than that for the other two tests. ELISA was thus less reliable than either CF or MIF for detecting antibody rises in paired infant sera, particularly where maternally acquired antibody remained in the acute serum. The reasons for this apparent disparity are discussed.

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

    PubMed

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

    2015-10-01

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

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

    SciTech Connect

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

    2009-02-20

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

  12. Zeta Potential and Aggregation of Virus-Like Particle of Human Norovirus and Feline Calicivirus Under Different Physicochemical Conditions.

    PubMed

    Samandoulgou, Idrissa; Fliss, Ismaïl; Jean, Julie

    2015-09-01

    Although the spread of human norovirus reportedly depends on its ability to bind to food materials, the mechanism of the phenomenon remains unknown. Since protein size and electrical charge are reportedly important parameters in their adsorption, the current work is focused on determining human noroviruses isoelectric point (IEP), electrical charge and aggregate size at different pH, ionic strength (IS), and temperature. Using the baculovirus expression vector system, we produced and purified virus-like particles (VLPs) of GI.1 and GII.4 noroviruses and feline calicivirus, determined their IEP, and examined their size and electrical charge using a Zetasizer Nano ZS apparatus. Shape and size were also visualized using transmission electron microscopy. IEPs were found close to pH 4. Net charge increased as the pH deviated from the IEP. VLPs were negatively charged at all IS tested and showed a gradual decrease in charge with increasing IS. At low temperature, VLPs were 20-45 nm in diameter at pH far from their IEP and under almost all IS conditions, while aggregates appeared at or near the IEP. At increased temperatures, aggregates appeared at or near the IEP and at high IS. Aggregation at the IEP was also confirmed by microscopy. This suggests that electrostatic interactions would be the predominant factor in VLPs adhesion at pH far from 4 and at low ionic strength. In contrast, non-electrostatic interactions would prevail at around pH 4 and would be reinforced by aggregates, since size generally favors multiple bonding with sorbents. PMID:26001534

  13. 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.

  14. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

    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.

  16. Ultrastructural mapping of methyldopa and anti-D IgG erythrocyte antigen receptors.

    PubMed Central

    Masouredis, S P; Sudora, E

    1975-01-01

    The ultrastructural distribution pattern and site density of alpha-methyldopa immunoglobin G (alpha-MD IgG) on the red cell membrane was observed and compared with that of anti-D IgG, with ferritin-conjugated rabbit anti-human IgG and [125I]anti-D. alpha-MD IgG binds to all common types of human red cells, both Rho (D) positive and negative, to give a random, aperiodic distribution pattern grossly indistinguishable from the red cell D receptor site pattern. alpha-MD IgG inhibits the binding of [125I]anti-D to D-positive red cells when the reaction is controlled with respect to total reaction volume, ionic strength, and the appropriate concentrations of the two IgG reactants. To determine if a alpha-MD IgG binds to the D-antigen receptor, D-positive red cells were sensitized with alpha-MD and [125I]anti-D IgG spearately and with both IgG preparations. The cell-bound radioactivity served to identify what proportion of the total ferritin-labeled IgG sites were due to anti-D. With nonsaturating concentrations of anti-D the number of IgG sites observed was equal to the sum of the sites found when the red cell was sensitized separately with alpha-MD and anti-D IgG. With saturating concentrations of anti-D there was a reduction in the expected number of IgG sites, indicating that alpha-MD IgG was excluded from binding. There was no comparable interaction of alpha-MD IgG and anti-D IgG when D-negative red cells were used. The results obtained indicate that alpha-MD IgG does not bind to the D antigen. The interaction between alpha-MD IgG and anti-D IgG for binding sites on the red cell membrane may be due to the close physical proximity of the two receptors, so as to produce steric hindrance in binding of the two IgG preparations when both are present. The alpha-MD IgG receptor appears to be a part of the Rh antigen complex that occurs in both D-positive and D-negative red cells and probably contains receptors for other types of warm-antibody immune hemolytic anemias

  17. 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

  18. 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 ...

  19. 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.

  20. 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.

  1. Conformational stability and aggregation of therapeutic monoclonal antibodies studied with ANS and Thioflavin T binding.

    PubMed

    Kayser, Veysel; Chennamsetty, Naresh; Voynov, Vladimir; Helk, Bernhard; Trout, Bernhardt L

    2011-01-01

    Characterization of aggregation profiles of monoclonal antibodies (mAb) is gaining importance because an increasing number of mAb-based therapeutics are entering clinical studies and gaining marketing approval. To develop a successful formulation, it is imperative to identify the critical biochemical properties of each potential mAb drug candidate. We investigated the conformational change and aggregation of a human IgG1 using external dye-binding experiments with fluorescence spectroscopy and compared the aggregation profiles obtained to the results of size-exclusion chromatography. We show that using an appropriate dye at selected mAb concentration, unfolding or aggregation can be studied. In addition, dye-binding experiments may be used as conventional assays to study therapeutic mAb stability. PMID:21540645

  2. 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.

  3. Structural and pathway complexity of beta-strand reorganization within aggregates of human transthyretin(105-115) peptide.

    PubMed

    Li, Da-Wei; Han, Li; Huo, Shuanghong

    2007-05-17

    Interstrand conformational rearrangements of human transthyretin peptide (TTR(105-115)) within dimeric aggregates were simulated by means of molecular dynamics (MD) with implicit solvation model for a total length of 48 micros. The conformations sampled in the MD simulations were clustered to identify free energy minima without any projections of free energy surface. A connected graph was constructed with nodes (=clusters) and edges corresponding to free energy minima and transitions between nodes, respectively. This connected graph which reflects the complexity of the free energy surface was used to extract the transition disconnectivity graph, which reflects the overall free energy barriers between pairs of free energy minima but does not contain information on transition paths. The routes of transitions between important free energy minima were obtained by further processing the original graph and the MD data. We have found that both parallel and antiparallel aggregates are populated. The parallel aggregates with different alignment patterns are separated by nonnegligible free energy barriers. Multiroutes exist in the interstrand conformational reorganization. Most visited routes do not dominant the kinetics, while less visited routes contribute a little each but they are numerous and their total contributions are actually dominant. There are various kinds of reptation motions, including those through a beta-bulge, side-chain aided reptation, and flipping or rotation of a hairpin formed by one strand.

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

    PubMed

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

    1989-01-01

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

  5. 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.

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

    PubMed Central

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

    2015-01-01

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

  7. Human α-Defensins Inhibit BK Virus Infection by Aggregating Virions and Blocking Binding to Host Cells*

    PubMed Central

    Dugan, Aisling S.; Maginnis, Melissa S.; Jordan, Joslynn A.; Gasparovic, Megan L.; Manley, Kate; Page, Rebecca; Williams, Geoffrey; Porter, Edith; O'Hara, Bethany A.; Atwood, Walter J.

    2008-01-01

    BK virus (BKV) is a polyomavirus that establishes a lifelong persistence in most humans and is a major impediment to success of kidney grafts. The function of the innate immune system in BKV infection and pathology has not been investigated. Here we examine the role of antimicrobial defensins in BKV infection of Vero cells. Our data show that α-defensin human neutrophil protein 1 (HNP1) and human α-defensin 5 (HD5) inhibit BKV infection by targeting an early event in the viral lifecycle. HD5 treatment of BKV reduced viral attachment to cells, whereas cellular treatment with HD5 did not. Colocalization studies indicated that HD5 interacts directly with BKV. Ultrastructural analysis revealed HD5-induced aggregation of virions. HD5 also inhibited infection of cells by other related polyomaviruses. This is the first study to demonstrate polyomavirus sensitivity to defensins. We also show a novel mechanism whereby HD5 binds to BKV leading to aggregation of virion particles preventing normal virus binding to the cell surface and uptake into cells. PMID:18782756

  8. Cell aggregation optimizes the differentiation of human ESCs and iPSCs into pancreatic bud-like progenitor cells.

    PubMed

    Toyoda, Taro; Mae, Shin-Ichi; Tanaka, Hiromi; Kondo, Yasushi; Funato, Michinori; Hosokawa, Yoshiya; Sudo, Tomomi; Kawaguchi, Yoshiya; Osafune, Kenji

    2015-03-01

    Embryonic pancreatic bud cells, the earliest pancreas-committed cells, generated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into mature pancreatic β-cells in vivo, indicating the feasibility of hESC/iPSC-based cell therapy for diabetes. However, the key factors required for the differentiation of these cells into pancreatic bud cells are incompletely understood. The purpose of this study was to establish culture conditions that efficiently induce PDX1(+)NKX6.1(+) pancreatic bud cells from hESCs/iPSCs. We differentiated a hESC line, KhES-3, into pancreatic lineages with a stepwise protocol recapitulating developmental process. The induction rate of PDX1(+)NKX6.1(+) cells was correlated with cell density in adherent cultures, and markedly improved with cell aggregation cultures. The positive effects of cell aggregation cultures on the differentiation of pancreatic bud cells were reproduced in multiple hESC/iPSC lines. The human PDX1(+)NKX6.1(+) cells developed into pancreatic epithelia after implantation into immunocompromised mice. Moreover, human C-peptide secretion into mouse bloodstream was stimulated by glucose challenges after in vivo maturation. Taken together, these results suggest that cultures with high cell density are crucial for the differentiation of pancreas-committed progenitor cells from hESCs/iPSCs. Our findings may be applicable for the development of hESC/iPSC-based cell therapy for diabetes.

  9. HHV-6 IgG4 isotype response following measles infection.

    PubMed

    Ferreyra, Leonardo; Bustos, Dolores; Biganzoli, Patricia; Isa, Maria Beatriz; Don, Paola Sicilia; Ribechini, Eliana; Nates, Silvia Viviana; Pavan, Jorge Victorio

    2010-03-01

    Human herpesvirus 6 (HHV-6) is widespread in the human population by infecting most individuals in early childhood. After primary infection, HHV-6 establishes a latent infection by remaining in circulating mononuclear cells of healthy individuals. The HHV-6 antibody titer increases after primary infection with measles virus. The present study was undertaken to determine the specific antiviral IgG1, IgG2, IgG3, and IgG4 subclass response patterns to HHV-6 in HHV-6-seropositive individuals with natural measles virus infection, measles vaccination, and rubella virus infection. The purpose of this study was to examine HHV-6-specific IgG isotype response in patients with acute virus coinfection. Serum samples were obtained from individuals who were seropositive for HHV-6 after natural primary infection with measles virus during an outbreak, measles vaccination, or rubella virus infection, and from healthy individuals. Sera were examined by indirect immunofluorescence assays for detection of HHV-6-specific IgG1, IgG2, IgG3, and IgG4 antibodies. A high percentage (69%) of those infected with measles virus had an HHV-6 IgG1 and IgG4 response (P < 0.001, chi(2) test), whereas persons vaccinated against measles, those infected with rubella, and healthy individuals showed an HHV-6 IgG1 response. These results demonstrate that natural measles virus infection induces an HHV-6 IgG isotype response, which suggests a shift in immune activity from a Th1 to a Th2 response. J. Med. Virol. 82:396-399, 2010. (c) 2010 Wiley-Liss, Inc.

  10. Serum total IgG and IgG4 levels in thyroid eye disease

    PubMed Central

    Sy, Aileen; Silkiss, Rona Z

    2016-01-01

    Purpose To investigate the relationship between immunoglobulin G (IgG)4-related disease (IgG4-RD) and thyroid eye disease (TED) with respect to IgG levels. Patients and methods A retrospective review of total IgG, IgG subclass, and thyroid stimulating immunoglobulin (TSI) levels in 24 patients with TED. Results Five patients (20.8%) demonstrated serum IgG4 levels consistent with IgG4-RD without any additional systemic disease. Total IgG and IgG subclass levels were found to be an inadequate proxy for TSI elevation. Conclusion There may be a subtype of TED patients with elevated IgG4 in the absence of IgG4-RD systemic findings. PMID:27799828

  11. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-02

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

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

    PubMed Central

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

    2015-01-01

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

  15. Aggregate Risk of Cardiovascular Disease among Adolescents Perinatally Infected with the Human Immunodeficiency Virus

    PubMed Central

    Patel, Kunjal; Wang, Jiajia; Jacobson, Denise L.; Lipshultz, Steven E.; Landy, David C.; Geffner, Mitchell E.; DiMeglio, Linda A.; Seage, George R.; Williams, Paige L.; Van Dyke, Russell B.; Siberry, George K.; Shearer, William T.; Young, Luciana; Scott, Gwendolyn B.; Wilkinson, James D.; Fisher, Stacy D.; Starc, Thomas J.; Miller, Tracie L.

    2014-01-01

    Background Perinatally HIV-infected adolescents may be susceptible to aggregate atherosclerotic cardiovascular disease (CVD) risk, as measured by the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) coronary arteries (CA) and abdominal aorta (AA) risk scores, due to prolonged exposure to HIV and antiretroviral therapy. Methods and Results CA and AA PDAY scores were calculated for 165 perinatally HIV-infected adolescents, using a weighted combination of modifiable risk factors: dyslipidemia, cigarette smoking, hypertension, obesity, and hyperglycemia. Demographic and HIV-specific predictors of scores ≥ 1 were identified and trends in scores over time were assessed. 48% and 24% of the perinatally HIV-infected adolescents had CA and AA scores ≥ 1, representing increased CVD risk factor burden. Significant predictors of CA scores ≥ 1 included male sex, history of an AIDS-defining condition, longer duration of use of a ritonavir-boosted protease inhibitor, and no prior use of tenofovir. Significant predictors of AA scores ≥ 1 included suppressed viral load, history of an AIDS-defining condition, and longer duration of boosted protease inhibitor use. No significant changes in CA and AA risk scores were observed over the 4-year study period. Conclusions A substantial proportion of perinatally HIV-infected youth have high PDAY scores reflecting increased aggregate atherosclerotic CVD risk factor burden. High scores were predicted by HIV disease severity and boosted protease inhibitor use. PDAY scores may be useful in identifying high-risk youth who may benefit from early lifestyle or clinical interventions. PMID:24366631

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2016-08-15

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

  18. 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.

  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. PMID:25174509

  20. [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.

  1. [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. PMID:25328251

  2. Physical instability, aggregation and conformational changes of recombinant human bone morphogenetic protein-2 (rhBMP-2).

    PubMed

    Luca, Ludmila; Capelle, Martinus A H; Machaidze, Gia; Arvinte, Tudor; Jordan, Olivier; Gurny, Robert

    2010-05-31

    The influence of two different pH values on the physical stability of recombinant human bone morphogenetic protein-2 (rhBMP-2) in aqueous solution was evaluated in the present work. RhBMP-2 in solution at pH 4.5 or 6.5 was characterized by intrinsic and extrinsic (Nile Red and 1,8-ANS) fluorescence spectroscopy, 90 degrees light-scattering and transmission electron microscopy (TEM). Compared to the pH 4.5 solution, rhBMP-2 at pH 6.5 had (i) a stronger intrinsic fluorescence intensity, (ii) a longer fluorescence lifetime, (iii) a stronger 90 degrees light-scattering intensity, (iv) a stronger Nile Red fluorescence intensity, (v) a higher Nile Red fluorescence anisotropy, (vi) a lower 1,8-ANS fluorescence intensity, (vii) a higher 1,8-ANS fluorescence anisotropy and (viii) a longer 1,8-ANS fluorescence lifetime. Electron microscopy showed that rhBMP-2 at pH 4.5 contained aggregates of about 100 nm in diameter. More and larger protein aggregates (0.1-2 microm) were observed in solution at pH 6.5. Taken together, these results indicate conformational changes and increased aggregation of rhBMP-2 at pH 6.5 compared to pH 4.5, demonstrating a strong influence of pH on rhBMP-2 physical stability. These observations must be considered when developing a delivery system for rhBMP-2.

  3. 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.

  4. Intrinsic differences in the initiation of B cell receptor signaling favor responses of human IgG(+) memory B cells over IgM(+) naive B cells.

    PubMed

    Davey, Angel M; Pierce, Susan K

    2012-04-01

    The acquisition of long-lived memory B cells (MBCs) is critical for the defense against many infectious diseases. Despite their importance, little is known about how Ags trigger human MBCs, even though our understanding of the molecular basis of Ag activation of B cells in model systems has advanced considerably. In this study, we use quantitative, high-resolution, live-cell imaging at the single-cell and single-molecule levels to describe the earliest Ag-driven events in human isotype-switched, IgG-expressing MBCs and compare them with those in IgM-expressing naive B cells. We show that human MBCs are more robust than naive B cells at each step in the initiation of BCR signaling, including interrogation of Ag-containing membranes, formation of submicroscopic BCR oligomers, and recruitment and activation of signaling-associated kinases. Despite their robust response to Ag, MBCs remain highly sensitive to FcγRIIB-mediated inhibition. We also demonstrate that in the absence of Ag, a portion of MBC receptors spontaneously oligomerized, and phosphorylated kinases accumulated at the membrane and speculate that heightened constitutive signaling may play a role in maintaining MBC longevity. Using high-resolution imaging, we have provided a description of the earliest events in the Ag activation of MBCs and evidence for acquired cell-intrinsic differences in the initiation of BCR signaling in human naive and MBCs.

  5. The Epstein-Barr virus-binding site on CD21 is involved in CD23 binding and interleukin-4-induced IgE and IgG4 production by human B cells.

    PubMed Central

    Henchoz-Lecoanet, S; Jeannin, P; Aubry, J P; Graber, P; Bradshaw, C G; Pochon, S; Bonnefoy, J Y

    1996-01-01

    Human CD21 has previously been described as a receptor for the C3d,g and iC3b proteins of complement, as a receptor for the gp350/220 envelope glycoprotein of the Epstein-Barr virus (EBV) and also as a receptor for inerferon-alpha (IFN-alpha). Structurally, CD21 consists of 15 to 16 short consensus repeats (SCR) of 60 to 75 amino acids followed by a transmembrane domain and an intracytoplasmic region. We reported that CD23, a low-affinity receptor for IgE (Fc epsilon R2), is a new functional ligand for CD21. We recently found that the sites of interaction of CD23 on CD21 are on SCR 5 to 8 and 1-2. The first site is a lectin-sugar type of interaction and the second site is a protein-protein interaction. We report here that amongst the other ligands for CD21 (EBV, C3d,g and IFN-alpha), only EBV is able to inhibit the binding of CD23 to CD21. Furthermore, even a peptide from gp350/220 of EBV known to bind to CD21 is able to decrease CD23 binding to CD21. Since CD23/CD21 pairing is important in the control of IgE production, we tested the effect of the EBV-derived peptide on immunoglobulin production from peripheral blood mononuclear cells and purified tonsillar B cells. Interestingly, the EBV-peptide inhibited IgE and IgG4 production induced by interleukin-4, in a dose-dependent manner. The same results were obtained using either peripheral blood mononuclear cells or purified tonsillar B cells. Another CD21 ligand, C3, did not affect binding of CD23 to CD21 nor the production of IgE and IgG4. This study indicates that blocking CD23 binding to CD21 SCR 2 on human B cells selectively modulates immunoglobulin production. PMID:8707347

  6. 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

  7. 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

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

    PubMed

    Bergström, Joakim J E; Heyman, Birgitta

    2015-01-01

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

  9. 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

  10. Engineering the variable region of therapeutic IgG antibodies

    PubMed Central

    Tsunoda, Hiroyuki; Kuramochi, Taichi; Sampei, Zenjiro; Ishii, Shinya; Hattori, Kunihiro

    2011-01-01

    Since the first generation of humanized IgG1 antibodies reached the market in the late 1990s, IgG antibody molecules have been extensively engineered. The success of antibody therapeutics has introduced severe competition in developing novel therapeutic monoclonal antibodies, especially for promising or clinically validated targets. Such competition has led researchers to generate so-called second or third generation antibodies with clinical differentiation utilizing various engineering and optimization technologies. Parent IgG antibodies can be engineered to have improved antigen binding properties, effector functions, pharmacokinetics, pharmaceutical properties and safety issues. Although the primary role of the antibody variable region is to bind to the antigen, it is also the main source of antibody diversity and its sequence affects various properties important for developing antibody therapeutics. Here we review recent research activity in variable region engineering to generate superior antibody therapeutics. PMID:21406966

  11. Engineering new bone via a minimally invasive route using human bone marrow-derived stromal cell aggregates, microceramic particles, and human platelet-rich plasma gel.

    PubMed

    Chatterjea, Anindita; Yuan, Huipin; Fennema, Eelco; Burer, Ruben; Chatterjea, Supriyo; Garritsen, Henk; Renard, Auke; van Blitterswijk, Clemens A; de Boer, Jan

    2013-02-01

    There is a rise in the popularity of arthroscopic procedures in orthopedics. However, the majority of cell-based bone tissue-engineered constructs (TECs) rely on solid preformed scaffolding materials, which require large incisions and extensive dissections for placement at the defect site. Thus, they are not suitable for minimally invasive techniques. The aim of this study was to develop a clinically relevant, easily moldable, bone TEC, amenable to minimally invasive techniques, using human mesenchymal stromal cells (hMSCs) and calcium phosphate microparticles in combination with an in situ forming platelet-rich plasma gel obtained from human platelets. Most conventional TECs rely on seeding and culturing single-cell suspensions of hMSCs on scaffolds. However, for generating TECs amenable to the minimally invasive approach, it was essential to aggregate the hMSCs in vitro before seeding them on the scaffolds as unaggregated MSCs did not generate any bone. Twenty four hours of in vitro aggregation was determined to be optimal for maintaining cell viability in vitro and bone formation in vivo. Moreover, no statistically significant difference was observed in the amount of bone formed when the TECs were implanted via an open approach or a minimally invasive route. TECs generated using MSCs from three different human donors generated new bone through the minimally invasive route in a reproducible manner, suggesting that these TECs could be a viable alternative to preformed scaffolds employed through an open surgery for treating bone defects.

  12. 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.

  13. Characterization of the promoter of the human gene encoding the high-affinity IgG receptor: Transcriptional induction by. gamma. -interferon is mediated through common DNA response elements

    SciTech Connect

    Pearse, R.N.; Feinman, R.; Ravetch, J.V. )

    1991-12-15

    Expression of the high-affinity receptor for IgG (Fc{sub {gamma}}RI) is restricted to cells of myeloid lineage and is induced by {gamma}-interferon (IFN-{gamma}) but not by IFN-{alpha}/{beta}. The organization of the human Fc{sub {gamma}}RI gene has been determined and the DNA elements governing its cell type-restricted transcription and IFN-{gamma} induction are reported here. A 39-nucleotide sequence (IFN-{gamma} response region, or GRR) is defined that is both necessary and sufficient for IFN-{gamma} inducibility. Sequence analysis of the GRR reveals the presence of promoter elements initially defined for the major histocompatibility complex class 2 genes: i.e., X, H, and {gamma}-IRE sequences. Comparison of a number of genes whose expression is induced selectively by IFN-{gamma} indicated that the presence of these elements is a general feature of IFN-{gamma}-responsive genes. The studies suggest that the combination of X, H, and {gamma}-IRE elements is a common motif in the pathway of transcriptional induction by this lymphokine.

  14. Development of a valve-based cell printer for the formation of human embryonic stem cell spheroid aggregates.

    PubMed

    Faulkner-Jones, Alan; Greenhough, Sebastian; King, Jason A; Gardner, John; Courtney, Aidan; Shu, Wenmiao

    2013-03-01

    In recent years, the use of a simple inkjet technology for cell printing has triggered tremendous interest and established the field of biofabrication. A key challenge has been the development of printing processes which are both controllable and less harmful, in order to preserve cell and tissue viability and functions. Here, we report on the development of a valve-based cell printer that has been validated to print highly viable cells in programmable patterns from two different bio-inks with independent control of the volume of each droplet (with a lower limit of 2 nL or fewer than five cells per droplet). Human ESCs were used to make spheroids by overprinting two opposing gradients of bio-ink; one of hESCs in medium and the other of medium alone. The resulting array of uniform sized droplets with a gradient of cell concentrations was inverted to allow cells to aggregate and form spheroids via gravity. The resulting aggregates have controllable and repeatable sizes, and consequently they can be made to order for specific applications. Spheroids with between 5 and 140 dissociated cells resulted in spheroids of 0.25-0.6 mm diameter. This work demonstrates that the valve-based printing process is gentle enough to maintain stem cell viability, accurate enough to produce spheroids of uniform size, and that printed cells maintain their pluripotency. This study includes the first analysis of the response of human embryonic stem cells to the printing process using this valve-based printing setup.

  15. 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

  16. 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

  17. Effect of methylglyoxal modification on stress-induced aggregation of client proteins and their chaperoning by human alphaA-crystallin.

    PubMed

    Biswas, Ashis; Wang, Benlian; Miyagi, Masaru; Nagaraj, Ram H

    2008-02-01

    alpha-Crystallin prevents protein aggregation under various stress conditions through its chaperone-like properties. Previously, we demonstrated that MGO (methylglyoxal) modification of alphaA-crystallin enhances its chaperone function and thus may affect transparency of the lens. During aging of the lens, not only alphaA-crystallin, but its client proteins are also likely to be modified by MGO. We have investigated the role of MGO modification of four model client proteins (insulin, alpha-lactalbumin, alcohol dehydrogenase and gamma-crystallin) in their aggregation and structure and the ability of human alphaA-crystallin to chaperone them. We found that MGO modification (10-1000 microM) decreased the chemical aggregation of insulin and alpha-lactalbumin and thermal aggregation of alcohol dehydrogenase and gamma-crystallin. Surface hydrophobicity in MGO-modified proteins decreased slightly relative to unmodified proteins. HPLC and MS analyses revealed argpyrimidine and hydroimidazolone in MGO-modified client proteins. The degree of chaperoning by alphaA-crystallin towards MGO-modified and unmodified client proteins was similar. Co-modification of client proteins and alphaA-crystallin by MGO completely inhibited stress-induced aggregation of client proteins. Our results indicate that minor modifications of client proteins and alphaA-crystallin by MGO might prevent protein aggregation and thus help maintain transparency of the aging lens. PMID:17941823

  18. Intermediate conformation between native β-sheet and non-native α-helix is a precursor of trifluoroethanol-induced aggregation of Human Carbonic Anhydrase-II

    SciTech Connect

    Gupta, Preeti; Deep, Shashank

    2014-06-20

    Highlights: • HCAII forms amyloid-like aggregates at moderate concentration of trifluoroethanol. • Protein adopts a state between β-sheet and α-helix at moderate % of TFE. • Hydrophobic surface(s) of partially structured conformation forms amyloid. • High % of TFE induces stable α-helical state preventing aggregation. - Abstract: In the present work, we examined the correlation between 2,2,2-trifluoroethanol (TFE)-induced conformational transitions of human carbonic anhydrase II (HCAII) and its aggregation propensity. Circular dichroism data indicates that protein undergoes a transition from β-sheet to α-helix on addition of TFE. The protein was found to aggregate maximally at moderate concentration of TFE at which it exists somewhere between β-sheet and α-helix, probably in extended non-native β-sheet conformation. Thioflavin-T (ThT) and Congo-Red (CR) assays along with fluorescence microscopy and transmission electron microscopy (TEM) data suggest that the protein aggregates induced by TFE possess amyloid-like features. Anilino-8-naphthalene sulfonate (ANS) binding studies reveal that the exposure of hydrophobic surface(s) was maximum in intermediate conformation. Our study suggests that the exposed hydrophobic surface and/or the disruption of the structural features protecting a β-sheet protein might be the major reason(s) for the high aggregation propensity of non-native intermediate conformation of HCAII.

  19. 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.

  20. 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.

  1. Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity

    PubMed Central

    Quast, Isaak; Keller, Christian W.; Maurer, Michael A.; Giddens, John P.; Tackenberg, Björn; Wang, Lai-Xi; Münz, Christian; Nimmerjahn, Falk; Dalakas, Marinos C.; Lünemann, Jan D.

    2015-01-01

    IgG molecules exert both pro- and antiinflammatory effector functions based on the composition of the fragment crystallizable (Fc) domain glycan. Sialylated IgG Fc domains have antiinflammatory properties that are attributed to their ability to increase the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we report that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-mediated cytotoxicity (CDC). Fc sialylation of a CD20-targeting antibody had no impact on antibody-dependent cellular cytotoxicity and did not change the affinity of the antibody for activating Fcγ receptors. In contrast, the presence of sialic acid abrogated the increased binding of C1q to Fc-galactosylated IgG1 and resulted in decreased levels of C3b deposition on the cell surface. Similar to monoclonal antibodies, sialic acid inhibited the increased C1q binding to galactosylated Fc fragments in human polyclonal IgG. In sera derived from patients with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system in which humoral immune responses mediate tissue damage, induction of IgG Fc sialylation was associated with clinical disease remission. Thus, impairment of CDC represents an FcγR-independent mechanism by which Fc-sialylated glycovariants might limit proinflammatory IgG effector functions. PMID:26436649

  2. Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity.

    PubMed

    Quast, Isaak; Keller, Christian W; Maurer, Michael A; Giddens, John P; Tackenberg, Björn; Wang, Lai-Xi; Münz, Christian; Nimmerjahn, Falk; Dalakas, Marinos C; Lünemann, Jan D

    2015-11-01

    IgG molecules exert both pro- and antiinflammatory effector functions based on the composition of the fragment crystallizable (Fc) domain glycan. Sialylated IgG Fc domains have antiinflammatory properties that are attributed to their ability to increase the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we report that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-mediated cytotoxicity (CDC). Fc sialylation of a CD20-targeting antibody had no impact on antibody-dependent cellular cytotoxicity and did not change the affinity of the antibody for activating Fcγ receptors. In contrast, the presence of sialic acid abrogated the increased binding of C1q to Fc-galactosylated IgG1 and resulted in decreased levels of C3b deposition on the cell surface. Similar to monoclonal antibodies, sialic acid inhibited the increased C1q binding to galactosylated Fc fragments in human polyclonal IgG. In sera derived from patients with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system in which humoral immune responses mediate tissue damage, induction of IgG Fc sialylation was associated with clinical disease remission. Thus, impairment of CDC represents an FcγR-independent mechanism by which Fc-sialylated glycovariants might limit proinflammatory IgG effector functions.

  3. CD23 molecule acts as a galactose-binding lectin in the cell aggregation of EBV-transformed human B-cell lines.

    PubMed

    Kijimoto-Ochiai, S; Uede, T

    1995-06-01

    Epstein-Barr virus (EBV)-transformed human B-cell lines, L-KT9 and DH3 cells express CD23 antigen, and grow in a mixture of single and aggregated cells. The CD23 molecule has high amino acid sequence homology with C-type lectin and recently we have shown that the solubilized CD23 molecule can really interact with galactose residues on glycoproteins. In this study, therefore, we tested whether CD23 antigen on the cell surface really acts as a galactose-binding lectin in the aggregation of these cells. The EBV-transformed cells (L-KT9) were separated into an aggregated-cell-rich fraction and a single-cell-rich fraction. Aggregated cells disaggregated after removal of galactose by beta-galactosidase treatment, whereas single cells made large aggregation on sialidase treatment, and this aggregation was inhibited in the presence of asialo-fetuin. On the other hand, naturally aggregated cells become single cells with anti-CD23 monoclonal antibody (mAB) as well as the soluble form of CD23, but not with anti-CD21 mAB. In addition, L-KT9 and DH3 cells bound to asialo-fetuin-coupled Sepharose (ASF-Sepharose) and this binding was significantly inhibited by pre-treatment of cells with anti-CD23, but not with anti-CD21 or other anti-adhesion molecules. From these results, we conclude that the naturally aggregated state of EBV-transformed cells occurs mainly through the interaction of CD23 as a lectin molecule and galactose residues as its ligand.

  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. Microarray analysis of cultured human brain aggregates following cortisol exposure: implications for cellular functions relevant to mood disorders.

    PubMed

    Salaria, S; Chana, G; Caldara, F; Feltrin, E; Altieri, M; Faggioni, F; Domenici, E; Merlo-Pich, E; Everall, I P

    2006-09-01

    Increased cortisol levels in humans are often observed in patients suffering from mood disorders. In this study human fetal brain aggregates were exposed to cortisol at 500 nM for 3 weeks, as an in-vitro model of chronic cortisol exposure. Microarray analysis on extracted mRNA using the Affymetrix U133A platform was then performed. Our results demonstrated a significant effect of cortisol on 1648 transcripts; 736 up-regulated and 912 down-regulated genes. The most differentially regulated biological categories were: RNA processing, protein metabolism, and cell growth. Within these categories we observed a down-regulation of fibroblast growth factor 2 (FGF2) (-1.5-fold) and aquaporin4 (AQP4) (-1.7-fold), alongside an up-regulation of fibroblast growth factor 9 (FGF9) (+1.7-fold) and vesicle associated membrane protein2 (VAMP2) (+1.7-fold). FGF2, FGF9, AQP4 and VAMP2 changes were confirmed at the protein level by immunohistochemistry. Alterations in FGF transcripts are in keeping with recent literature demonstrating such effects in patients with mood disorders. PMID:16844382

  6. 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. PMID:23669544

  7. 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.

  8. 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.

  9. 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.

  10. X-ray diffraction from intact tau aggregates in human brain tissue

    NASA Astrophysics Data System (ADS)

    Landahl, Eric C.; Antipova, Olga; Bongaarts, Angela; Barrea, Raul; Berry, Robert; Binder, Lester I.; Irving, Thomas; Orgel, Joseph; Vana, Laurel; Rice, Sarah E.

    2011-09-01

    We describe an instrument to record X-ray diffraction patterns from diseased regions of human brain tissue by combining an in-line visible light fluorescence microscope with an X-ray diffraction microprobe. We use thiazine red fluorescence to specifically label and detect the filamentous tau protein pathology associated with Pick's disease, as several laboratories have done previously. We demonstrate that thiazine red-enhanced regions within the tissue show periodic structure in X-ray diffraction, which is not observed in healthy tissue. One observed periodicity (4.2 Å) is characteristic of cross-beta sheet structure, consistent with previous results from powder diffraction studies performed on purified, dried tau protein.

  11. In Vitro and In Vivo Comparison of Lymphocytes Transduced with a Human CD16 or with a Chimeric Antigen Receptor Reveals Potential Off-Target Interactions due to the IgG2 CH2-CH3 CAR-Spacer

    PubMed Central

    Clémenceau, Béatrice; Valsesia-Wittmann, Sandrine; Jallas, Anne-Catherine; Vivien, Régine; Rousseau, Raphaël; Marabelle, Aurélien; Caux, Christophe; Vié, Henri

    2015-01-01

    The present work was designed to compare two mechanisms of cellular recognition based on Ab specificity: firstly, when the anti-HER2 mAb trastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (ADCC) and, secondly, when HER2 positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (CAR). To compare these two mechanisms, we used the same cellular effector (NK-92) and the same signaling domain (FcεRIγ). The NK-92 cytotoxic cell line was transfected with either a FcγRIIIa-FcεRIγ (NK-92CD16) or a trastuzumab-based scFv-FcεRIγ chimeric receptor (NK-92CAR). In vitro, the cytotoxic activity against HER2 positive target cells after indirect recognition by NK-92CD16 was always inferior to that observed after direct recognition by NK-92CAR. In contrast, and somehow unexpectedly, in vivo, adoptive transfer of NK-92CD16 + trastuzumab but not of NK-92CAR induced tumor regression. Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells. This interaction, leading to blockage of the NK-92CAR in the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain. PMID:26665156

  12. Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis.

    PubMed

    Ohmi, Yuhsuke; Ise, Wataru; Harazono, Akira; Takakura, Daisuke; Fukuyama, Hidehiro; Baba, Yoshihiro; Narazaki, Masashi; Shoda, Hirofumi; Takahashi, Nobunori; Ohkawa, Yuki; Ji, Shuting; Sugiyama, Fumihiro; Fujio, Keishi; Kumanogoh, Atsushi; Yamamoto, Kazuhiko; Kawasaki, Nana; Kurosaki, Tomohiro; Takahashi, Yoshimasa; Furukawa, Koichi

    2016-01-01

    Rheumatoid arthritis (RA)-associated IgG antibodies such as anti-citrullinated protein antibodies (ACPAs) have diverse glycosylation variants; however, key sugar chains modulating the arthritogenic activity of IgG remain to be clarified. Here, we show that reduced sialylation is a common feature of RA-associated IgG in humans and in mouse models of arthritis. Genetically blocking sialylation in activated B cells results in exacerbation of joint inflammation in a collagen-induced arthritis (CIA) model. On the other hand, artificial sialylation of anti-type II collagen antibodies, including ACPAs, not only attenuates arthritogenic activity, but also suppresses the development of CIA in the antibody-infused mice, whereas sialylation of other IgG does not prevent CIA. Thus, our data demonstrate that sialylation levels control the arthritogenicity of RA-associated IgG, presenting a potential target for antigen-specific immunotherapy. PMID:27046227

  13. Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis

    PubMed Central

    Ohmi, Yuhsuke; Ise, Wataru; Harazono, Akira; Takakura, Daisuke; Fukuyama, Hidehiro; Baba, Yoshihiro; Narazaki, Masashi; Shoda, Hirofumi; Takahashi, Nobunori; Ohkawa, Yuki; Ji, Shuting; Sugiyama, Fumihiro; Fujio, Keishi; Kumanogoh, Atsushi; Yamamoto, Kazuhiko; Kawasaki, Nana; Kurosaki, Tomohiro; Takahashi, Yoshimasa; Furukawa, Koichi

    2016-01-01

    Rheumatoid arthritis (RA)-associated IgG antibodies such as anti-citrullinated protein antibodies (ACPAs) have diverse glycosylation variants; however, key sugar chains modulating the arthritogenic activity of IgG remain to be clarified. Here, we show that reduced sialylation is a common feature of RA-associated IgG in humans and in mouse models of arthritis. Genetically blocking sialylation in activated B cells results in exacerbation of joint inflammation in a collagen-induced arthritis (CIA) model. On the other hand, artificial sialylation of anti-type II collagen antibodies, including ACPAs, not only attenuates arthritogenic activity, but also suppresses the development of CIA in the antibody-infused mice, whereas sialylation of other IgG does not prevent CIA. Thus, our data demonstrate that sialylation levels control the arthritogenicity of RA-associated IgG, presenting a potential target for antigen-specific immunotherapy. PMID:27046227

  14. Aggregation of Trp > Glu point mutants of human gamma-D crystallin provides a model for hereditary or UV-induced cataract.

    PubMed

    Serebryany, Eugene; Takata, Takumi; Erickson, Erika; Schafheimer, Nathaniel; Wang, Yongting; King, Jonathan A

    2016-06-01

    Numerous mutations and covalent modifications of the highly abundant, long-lived crystallins of the eye lens cause their aggregation leading to progressive opacification of the lens, cataract. The nature and biochemical mechanisms of the aggregation process are poorly understood, as neither amyloid nor native-state polymers are commonly found in opaque lenses. The βγ-crystallin fold contains four highly conserved buried tryptophans, which can be oxidized to more hydrophilic products, such as kynurenine, upon UV-B irradiation. We mimicked this class of oxidative damage using Trp→Glu point mutants of human γD-crystallin. Such substitutions may represent a model of UV-induced photodamage-introduction of a charged group into the hydrophobic core generating "denaturation from within." The effects of Trp→Glu substitutions were highly position dependent. While each was destabilizing, only the two located in the bottom of the double Greek key fold-W42E and W130E-yielded robust aggregation of partially unfolded intermediates at 37°C and pH 7. The αB-crystallin chaperone suppressed aggregation of W130E, but not W42E, indicating distinct aggregation pathways from damage in the N-terminal vs C-terminal domain. The W130E aggregates had loosely fibrillar morphology, yet were nonamyloid, noncovalent, showed little surface hydrophobicity, and formed at least 20°C below the melting temperature of the native β-sheets. These features are most consistent with domain-swapped polymerization. Aggregation of partially destabilized crystallins under physiological conditions, as occurs in this class of point mutants, could provide a simple in vitro model system for drug discovery and optimization. PMID:26991007

  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. Action of Lambert-Eaton myasthenic syndrome IgG at mouse motor nerve terminals.

    PubMed

    Prior, C; Lang, B; Wray, D; Newsom-Davis, J

    1985-06-01

    We have studied the electrophysiological effects of IgG obtained from four patients with Lambert-Eaton myasthenic syndrome (LEMS) (two with small cell carcinoma), using the mouse passive transfer model. Mice received LEMS or control IgG or plasma, 10 to 60 mg daily. Microelectrode intracellular recordings were made from diaphragm muscle. LEMS IgG and plasma decreased end-plate potential quantal content similarly, confirming IgG as the active factor. LEMS IgG was equally effective in C5-deficient mice, indicating that late complement components are not required. The time course of decline and recovery of quantal content closely followed that of the human IgG in the mouse serum, with time to half-maximal effect of about 1.5 days in each case. Binding/dissociation of IgG or down/up regulation of the antigenic determinants, possibly Ca2+ channels, has a half-life of between 2 and 36 hours. The results confirm our concept that IgG antibody to nerve terminal determinants underlies the disorder of transmitter release in LEMS.

  17. The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation

    PubMed Central

    Lee, Mihwa; Sadowska, Agata; Bekere, Indra; Ho, Diwei; Gully, Benjamin S.; Lu, Yanling; Iyer, K. Swaminathan; Trewhella, Jill; Fox, Archa H.; Bond, Charles S.

    2015-01-01

    SFPQ, (a.k.a. PSF), is a human tumor suppressor protein that regulates many important functions in the cell nucleus including coordination of long non-coding RNA molecules into nuclear bodies. Here we describe the first crystal structures of Splicing Factor Proline and Glutamine Rich (SFPQ), revealing structural similarity to the related PSPC1/NONO heterodimer and a strikingly extended structure (over 265 Å long) formed by an unusual anti-parallel coiled-coil that results in an infinite linear polymer of SFPQ dimers within the crystals. Small-angle X-ray scattering and transmission electron microscopy experiments show that polymerization is reversible in solution and can be templated by DNA. We demonstrate that the ability to polymerize is essential for the cellular functions of SFPQ: disruptive mutation of the coiled-coil interaction motif results in SFPQ mislocalization, reduced formation of nuclear bodies, abrogated molecular interactions and deficient transcriptional regulation. The coiled-coil interaction motif thus provides a molecular explanation for the functional aggregation of SFPQ that directs its role in regulating many aspects of cellular nucleic acid metabolism. PMID:25765647

  18. 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

  19. 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

  20. Fc alpha receptors mediate release of tumour necrosis factor-alpha and interleukin-6 by human monocytes following receptor aggregation.

    PubMed Central

    Patry, C; Herbelin, A; Lehuen, A; Bach, J F; Monteiro, R C

    1995-01-01

    The functional capacity of the human monocyte receptor for the Fc portion of IgA (Fc alpha R) in mediating signal transduction was evaluated by cytokine release. F(ab')2 fragments of anti-Fc alpha R monoclonal antibodies (mAb) were used as specific probes to induce release of tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Multivalent cross-linking by a secondary anti-mouse antibody [F(ab')2 fragments] induced a significant release of TNF-alpha and IL-6 by human blood mononuclear cells, indicating requirements for Fc alpha R aggregation on the cell surface to transmit signals. Both cytokines were released exclusively by adherent cells, identifying monocytes as the responding cells within the mononuclear cell population. This cytokine release could not be due to contaminating endotoxins, because it was not abolished by polymyxin B, a lipopolysaccharide (LPS) inhibitor. Moreover, purified recombinant soluble Fc alpha R inhibited the anti-Fc alpha R mAb-mediated cytokine release from blood monocytes, demonstrating that TNF-alpha and IL-6 were released in a receptor-specific manner. Our data suggest that Fc alpha R, through its capacity to mediate secretion of IL-6, may play an important role in B-cell proliferation and immunoglobulin production. On the other hand, release of TNF-alpha following stimulation of Fc alpha R molecules directly implicates these receptors in amplification and regulation of the inflammatory process occurring during IgA-mediated host defence. PMID:7590867

  1. 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

  2. Effects of garlic extract and of three pure components isolated from it on human platelet aggregation, arachidonate metabolism, release reaction and platelet ultrastructure.

    PubMed

    Apitz-Castro, R; Cabrera, S; Cruz, M R; Ledezma, E; Jain, M K

    1983-10-15

    We studied the effect of the methanol extract of garlic bulbs (EOG) and of three pure components isolated from it (F1, F2, F3), on human platelet aggregation induced by ADP, epinephrine, collagen, thrombin, arachidonate, PAF, and the ionophore A-23187. Incubation of PRP with EOG, either in methanol or in homologous PPP, inhibits platelet aggregation induced by all of the above mentioned agonists. F1, F2, and F3 also inhibit platelet aggregation, however, F3 was about four times more potent. Addition of EOG or F3 to platelets that have already been irreversibly aggregated by 10 microM ADP, induces rapid deaggregation. Inhibition of aggregation was still present after three hours. The inhibitory effect persisted even after the treated platelets were Gel-Filtered (GFP) or separated from plasma through a metrizamide gradient and resuspended in new homologous PPP. Thrombin-induced release of ATP from GFP was inhibited by 75-80% after EOG or F3 treatment. Incorporation of [3-H]-arachidonate by intact platelets was decreased by 50-60% in treated platelets. However, platelets incubated with the inhibitors after incorporation of radiolabeled arachidonate, although did not aggregate, produced, after thrombin activation similar amounts of radiolabeled TXB2 and lipoxygenase products as the controls. Electron microscopy of inhibited platelets, in the presence of thrombin, showed no degranulation but an increase of spherical forms. Our results suggest that the effects described might be mediate by a perturbation of the physicochemical properties of the plasma membrane rather than by affecting arachidonate or calcium metabolism in the cells. Chemical structures of F1, F2 and F3 have been provisionally assigned: F1 is diallytrisulfide, F2 is 2-vinyl-1,3-dithiene, and F3 is most probably allyl 1,5-hexadienyltrisulfide. PMID:6419374

  3. Immunogenicity of Recombinant Human Interferon Beta-1b in Immune-Tolerant Transgenic Mice Corresponds with the Biophysical Characteristics of Aggregates.

    PubMed

    Abdolvahab, Mohadeseh Haji; Fazeli, Ahmad; Halim, Andhyk; Sediq, Ahmad S; Fazeli, Mohammad Reza; Schellekens, Huub

    2016-04-01

    Determining to what extent biophysical characteristics of aggregates affect immunogenicity of therapeutic interferon beta-1b. Three recombinant human interferon beta-1b (rhIFNβ-1b) samples with different levels of aggregates generated by copper oxidation, thermal stress, or left untreated, as well as Avonex(®) drug substance and Betaferon(®) drug product, were injected intraperitoneally in nontransgenic and interferon beta transgenic FVB/N mice 5 times per week for 3 weeks. Antibodies against interferon beta were measured using enzyme-linked immunosorbent assay. UV and fluorescence spectroscopy, dynamic light scattering, size exclusion chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC), fluid imaging microscopy, and resonant mass measurement, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, were used to characterize and quantitate aggregates in the 3 rhIFNβ preparations, to correlate biophysical characteristics with immunogenicity. In immune-tolerant interferon beta transgenic FVB/N mice, Betaferon drug product showed the highest immunogenicity, while Avonex drug substance showed the lowest level of immunogenicity. Of the 3 forms of rhIFNβ-1b, copper-oxidized rhIFNβ-1b showed lower immunogenicity than thermally stressed rhIFNβ-1b, despite containing larger aggregates. Both copper-oxidized rhIFNβ-1b and thermally stressed rhIFNβ-1b exhibited changes in protein structure as shown using fluorescence spectroscopy and RP-HPLC. Nontransgenic, nonimmune-tolerant FVB/N mice generated high antibody titers against all interferon beta samples tested. The level of immunogenicity and the breaking of tolerance in FVB/N transgenic mice are not only related to the level of aggregation but also depend on the size and structure of the aggregates. PMID:26835734

  4. Construction aggregates

    USGS Publications Warehouse

    Langer, W.H.; Tepordei, V.V.; Bolen, W.P.

    2000-01-01

    Construction aggregates consist primarily of crushed stone and construction sand and gravel. Total estimated production of construction aggregates increased in 1999 by about 2% to 2.39 Gt (2.64 billion st) compared with 1998. This record production level continued an expansion that began in 1992. By commodities, crushed stone production increased 3.3%, while sand and gravel production increased by about 0.5%.

  5. Construction aggregates

    USGS Publications Warehouse

    Tepordei, V.V.

    1994-01-01

    Part of a special section on industrial minerals in 1993. The 1993 production of construction aggregates increased 6.3 percent over the 1992 figure, to reach 2.01 Gt. This represents the highest estimated annual production of combined crushed stone and construction sand and gravel ever recorded in the U.S. The outlook for construction aggregates and the issues facing the industry are discussed.

  6. Recombinant Human Semenogelin-1 (Sg1) and Sg1 (1-159) form Detergent Stable Amyloid like Aggregates in vitro.

    PubMed

    Sharma, Neetu; Vishwanath, S; Patel, Basant K

    2016-01-01

    Senile seminal vesicle amyloidosis (SSVA) is associated with deposition of semenogelin-1 (Sg1) protein aggregates in seminal vesicles that may manifest as hematospermia. Sg1 is the predominant protein that entraps spermatozoa which are freed upon fragmentation of Sg1 by the protease prostate specific antigen (PSA), post semen release. Certain small peptide fragments of Sg1 have been reported to form amyloid aggregates in vitro that can enhance HIV infectivity to cell cultures. However, the amyloid deposits in the seminal vesicles are expected to be that of the full length Sg1, as PSA is encountered downstream. So far, amyloid forming ability of full length Sg1 has not been established in vitro. Here, we examined the amyloidogenicity of full length Sg1 and a large fragment Sg1 (1-159), using recombinant proteins and tested if Zinc has any effect on their aggregation. Levels of Zinc, which is essential for health of male reproductive system, gradually decline with age. We succeeded in forming amyloid-like aggregates of Sg1 full length and Sg1 (1-159) fragment showing detergent stability and found that presence of Zn2+ substantially inhibits their amyloid aggregation in vitro. Possibly, high Zn2+ found in seminal plasma of young individuals may have preventive role against aggregation of Sg1 in seminal vesicles.

  7. Conformational Dynamics of the Human Islet Amyloid Polypeptide in a Membrane Environment: Toward the Aggregation Prone Form.

    PubMed

    Skeby, Katrine Kirkeby; Andersen, Ole Juul; Pogorelov, Taras V; Tajkhorshid, Emad; Schiøtt, Birgit

    2016-04-01

    Human islet amyloid polypeptide (hIAPP) is a 37-residue peptide hormone, which upon misfolding changes from the physiologically active monomer into pathological amyloid fibril aggregates in the pancreas of type 2 diabetes mellitus patients. During this process, the insulin-producing pancreatic β-cells are damaged; however, the underlying mechanism of this mode of cytotoxicity remains elusive. It is known that anionic lipids accelerate amyloid fibril formation, implicating the importance of the cellular membrane in the process, and that a pH close to the level in the β-cell secretory granules (pH 5.5) inhibits amyloid fibril formation. Using all-atom molecular dynamics simulations, we have investigated the membrane-associated monomer state of α-helical hIAPP, analyzed specific interactions of hIAPP with a mixed anionic-zwitterionic lipid membrane and examined the influence of pH on the structure and dynamics of hIAPP and its interaction with the membrane. We find that hIAPP primarily interacts with the membrane by forming favorable interactions between anionic lipids and the positively charged residues in the N-terminal part of the peptide. Rationalizing experimental findings, the simulations show that the N-terminal part of the peptide interacts with the membrane in the lipid headgroup region. At neutral pH, the C-terminal part of the peptide, which contains the residues that initiate fibril formation, displays a highly dynamic, unfolded state, which interacts with the membrane significantly less than the N-terminal part. Such an unfolded form can be proposed to contribute to the acceleration of fibril formation. At low pH, protonation of His18 mediates a stronger interaction of the C-terminal part with the membrane, resulting in the immobilization of the C-terminal part on the membrane surface that might constitute a mechanism by which low pH inhibits fibril formation. PMID:26953503

  8. 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

  9. 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. PMID:26960140

  10. Targeting the anionic region of human protease activated receptor 4 (PAR4) inhibits platelet aggregation and thrombosis without interfering with hemostasis

    PubMed Central

    Mumaw, M. M.; de la Fuente, M.; Noble, D. N.; Nieman, M.T.

    2014-01-01

    Summary Background Human platelet activation and aggregation is a complex process. To date, many therapies have been developed targeting proteins that mediate this process to prevent unwanted activation. However, the current standard of care for acute coronary syndromes still has limitations including bleeding risk. Objective The aim of the current study is to evaluate the PAR4 anionic cluster as a viable antiplatelet target using a polyclonal antibody (CAN12). Methods We used western blotting, aggregation, and secretion ex vivo to evaluate the ability of CAN12 to interact with PAR4 and inhibit platelet activation. The effects of CAN12 in vivo were evaluated with the Rose Bengal arterial thrombosis model and two models of hemostasis. Results We show that CAN12 is able to interact with human PAR4 and delay PAR4 cleavage. In addition, CAN12 inhibits thrombin induced human platelet aggregation and secretion in a dose dependent manner. We next determined that the specificity of CAN12 is agonist dependent. In vivo, we determined that CAN12 is able to inhibit arterial thrombosis and using two independent methods, we found that CAN12 does not influence hemostasis. Conclusion Targeting the extracellular anionic cluster on PAR4 is a viable novel strategy as an anti-platelet therapy. PMID:24888424

  11. Effects of Recombinant Human Megakaryocyte Growth and Development Factor (rHuMGDF) on Platelet Production, Platelet Aggregation, and Thrombosis.

    PubMed

    Lott; Nelson; Toombs

    1998-01-01

    Recombinant human megakaryocyte growth and development factor (rHuMGDF) is a c-mpl ligand that promotes the differentiation of CD34+ precursor cells into megakaryocyte, and then platelets. In experimental animals, injection of this and other c-mpl ligands leads to profound increases in the circulating platelet count in a matter of days. However, c-mpl ligands have also been shown to sensitize platelets to aggregating agents in vitro, raising the possibility that c-mpI ligands may have prothrombotic effects in vivo. Therefore, characterizing rHuMGDF in an in vivo model of thrombosis is a necessary and critical step in defining the in vivo pharmacology of this novel and important hernatopoietic factor, a pegylated form of which is currently in clinical trials. To determine the biologically effective doses in the rabbit, daily subcutaneous injections of rHuMGDF at 0.1, 1.0, or 10 µg/kg were administered ever 7 days. Daily injection of 10 µ/kg produced an approximate fourfold increase in platelet count and 1.0 µ/kg doubled platelet count over the injection period, both of which were statistically significant. The serum concentrations of rHuMGDF were determined 10 minutes following a single intravenous injection with 0.1, 1.0, and 10 µ/kg, and were 0.05 +/- 0.02, 0.98 +/- 0.07, and 21.32 +/- 21.35 ng/ml. To determine whether rHuMGDF can sensitize platelets in vivo, platelet aggregometry was performed on platelets isolated from animals immediately before and 10 minutes after they had been injected intravenously with rHuMGDF (0.1, 1.0, and 10 µ/kg). Intravenous injection of 10 µ/kg produced measurable changes in platelet aggregometry ex vivo, as evidenced by an increased sensitivity of platelets to adenosine diphosphate (ADP). To assess. the in vivo prothrombotic potential of rHuMGDF, a rabbit carotid artery model of cyclic flow reduction (CFR) was used to measure the effect of intravenous rHuMGDF administration on the rate of thrombus formation as assessed by CFR

  12. The value of flow cytometry in the measurement of platelet activation and aggregation in human immunodeficiency virus infection.

    PubMed

    Nkambule, Bongani B; Davison, Glenda; Ipp, Hayley

    2015-01-01

    Human immunodeficiency deficiency virus (HIV) infection is associated with chronic inflammation and an increased risk of thrombotic events. Activated platelets (PLTs) play an important role in both thrombosis and inflammation, and HIV has been shown to induce PLT activation by both direct and indirect mechanisms. P-selectin (CD62P) is a well-described marker of PLT activation, and PLT glycoprotein (GP) IV (CD36) has been identified as a marker of PLT aggregation. Data on PLT function in the context of HIV infection remain inconclusive. Laboratory techniques, such as flow cytometry, enable the assessment of PLTs in their physiological state and environment, with minimal artifactual in vitro activation and aggregation. In this study, we describe a novel flow cytometry PLT assay, which enabled the measurement of PLT function in HIV infection. Forty-one antiretroviral-naïve HIV-positive individuals and 41 HIV-negative controls were recruited from a clinic in the Western Cape. Platelet function was evaluated by assessing the response of platelets to adenosine diphosphate (ADP) at two concentrations (0.04 mM, 0.2 mM). The percentage expression and mean fluorescence intensity (MFI) of CD62P and CD36 was used to evaluate platelet function. These were then correlated with platelet (PLT) count; CD4 count; % CD38/8; viral load and D-dimers. The % CD62P levels were higher in HIV-positive patients (HIV % CD62P 11.33[5.96-29.36] vs. control 2.48[1.56-6.04]; p < 0.0001). In addition, the HIV group showed higher CD62P MFI levels (HIV CD62P MFI 3.25 ± 7.23 vs. control 2.35 ± 1.31, p = 0.0292). Baseline levels of %CD36 expression were significantly higher in HIV-positive patients (%CD36 12.41[6.31-21.83] vs. control 6.04[1.34-13.15]; p = 0.0091). However, the baseline CD36MFI showed no significant difference between the two groups (HIV CD36 MFI 3.09 ± 0.64 vs. control 2.44 ± 0.11, p = 0.4591). The HIV group showed higher levels of % CD36

  13. 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

  14. An alternative structural isoform in amyloid-like aggregates formed from thermally denatured human γD-crystallin.

    PubMed

    Moran, Sean D; Zhang, Tianqi O; Zanni, Martin T

    2014-03-01

    The eye lens protein γD-crystallin contributes to cataract formation in the lens. In vitro experiments show that γD-crystallin has a high propensity to form amyloid fibers when denatured, and that denaturation by acid or UV-B photodamage results in its C-terminal domain forming the β-sheet core of amyloid fibers. Here, we show that thermal denaturation results in sheet-like aggregates that contain cross-linked oligomers of the protein, according to transmission electron microscopy and SDS-PAGE. We use two-dimensional infrared spectroscopy to show that these aggregates have an amyloid-like secondary structure with extended β-sheets, and use isotope dilution experiments to show that each protein contributes approximately one β-strand to each β-sheet in the aggregates. Using segmental (13) C labeling, we show that the organization of the protein's two domains in thermally induced aggregates results in a previously unobserved structure in which both the N-terminal and C-terminal domains contribute to β-sheets. We propose a model for the structural organization of the aggregates and attribute the recruitment of the N-terminal domain into the fiber structure to intermolecular cross linking. PMID:24415662

  15. 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

  16. Russell body phenotype is preferentially induced by IgG mAb clones with high intrinsic condensation propensity: relations between the biosynthetic events in the ER and solution behaviors in vitro.

    PubMed

    Hasegawa, Haruki; Woods, Christopher E; Kinderman, Francis; He, Feng; Lim, Ai Ching

    2014-01-01

    The underlying reasons for why some mAb (monoclonal antibody) clones are much more inclined to induce a Russell body (RB) phenotype during immunoglobulin biosynthesis remain elusive. Although RBs are morphologically understood as enlarged globular aggregates of immunoglobulins deposited in the endoplasmic reticulum (ER), little is known about the properties of the RB-inducing mAb clones as secretory cargo and their physical behaviors in the extracellular space. To elucidate how RB-inducing propensities, secretion outputs, and the intrinsic physicochemical properties of individual mAb clones are interrelated, we used HEK293 cells to study the biosynthesis of 5 human IgG mAbs for which prominent solution behavior problems were known a priori. All 5 model mAbs with inherently high condensation propensities induced RB phenotypes both at steady state and under ER-to-Golgi transport block, and resulted in low secretion titer. By contrast, one reference mAb that readily crystallized at neutral pH in vitro produced rod-shaped crystalline bodies in the ER without inducing RBs. Another reference mAb without notable solution behavior issues did not induce RBs and was secreted abundantly. Intrinsic physicochemical properties of individual IgG clones thus directly affected the biosynthetic steps in the ER, and thereby produced distinctive cellular phenotypes and influenced IgG secretion output. The findings implicated that RB formation represents a phase separation event or a loss of colloidal stability in the secretory pathway organelles. The process of RB induction allows the cell to preemptively reduce the extracellular concentration of potentially pathogenic, highly aggregation-prone IgG clones by selectively storing them in the ER. PMID:25484054

  17. Russell body phenotype is preferentially induced by IgG mAb clones with high intrinsic condensation propensity: relations between the biosynthetic events in the ER and solution behaviors in vitro.

    PubMed

    Hasegawa, Haruki; Woods, Christopher E; Kinderman, Francis; He, Feng; Lim, Ai Ching

    2014-01-01

    The underlying reasons for why some mAb (monoclonal antibody) clones are much more inclined to induce a Russell body (RB) phenotype during immunoglobulin biosynthesis remain elusive. Although RBs are morphologically understood as enlarged globular aggregates of immunoglobulins deposited in the endoplasmic reticulum (ER), little is known about the properties of the RB-inducing mAb clones as secretory cargo and their physical behaviors in the extracellular space. To elucidate how RB-inducing propensities, secretion outputs, and the intrinsic physicochemical properties of individual mAb clones are interrelated, we used HEK293 cells to study the biosynthesis of 5 human IgG mAbs for which prominent solution behavior problems were known a priori. All 5 model mAbs with inherently high condensation propensities induced RB phenotypes both at steady state and under ER-to-Golgi transport block, and resulted in low secretion titer. By contrast, one reference mAb that readily crystallized at neutral pH in vitro produced rod-shaped crystalline bodies in the ER without inducing RBs. Another reference mAb without notable solution behavior issues did not induce RBs and was secreted abundantly. Intrinsic physicochemical properties of individual IgG clones thus directly affected the biosynthetic steps in the ER, and thereby produced distinctive cellular phenotypes and influenced IgG secretion output. The findings implicated that RB formation represents a phase separation event or a loss of colloidal stability in the secretory pathway organelles. The process of RB induction allows the cell to preemptively reduce the extracellular concentration of potentially pathogenic, highly aggregation-prone IgG clones by selectively storing them in the ER.

  18. 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.

  19. 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

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

    PubMed

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

    1990-07-01

    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.

  1. Intermolecular disulfide bond formation promotes immunoglobulin aggregation: investigation by fluorescence correlation spectroscopy.

    PubMed

    Nag, Moupriya; Bera, Kallol; Basak, Soumen

    2015-01-01

    Protein aggregation generally results from association between hydrophobic regions of individual monomers. However, additional mechanisms arising from specific interactions, such as intermolecular disulfide bond formation, may also contribute to the process. The latter is proposed to be the initiating pathway for aggregation of immunoglobulin (IgG), which is essential for triggering its immune response. To test the veracity of this hypothesis, we have employed fluorescence correlation spectroscopy to measure the kinetics of aggregation of IgG in separate experiments either allowing or inhibiting disulfide formation. Fluorescence correlation spectroscopy measurements yielded a diffusion time (τ(D)) of ∼200 µsec for Rhodamine-labeled IgG, corresponding to a hydrodynamic radius (R(H)) of 56 Å for the IgG monomer. The aggregation kinetics of the protein was followed by monitoring the time evolution of τ(D) under conditions in which its cysteine residues were either free or blocked. In both cases, the progress curves confirmed that aggregation proceeded via the nucleation-dependent polymerization pathway. However, for aggregation in the presence of free cysteines, the lag times were shorter, and the aggregate sizes bigger, than their respective counterparts for aggregation in the presence of blocked cysteines. This result clearly demonstrates that formation of intermolecular disulfide bonds represents a preferred pathway in the aggregation process of IgG. Fluorescence spectroscopy showed that aggregates formed in experiments where disulfide formation was prevented denatured at lower concentration of guanidine hydrochloride than those obtained in experiments where the disulfides were free to form, indicating that intermolecular disulfide bridging is a valid pathway for IgG aggregation. PMID:25371040

  2. Investigating the feasibility of scale up and automation of human induced pluripotent stem cells cultured in aggregates in feeder free conditions.

    PubMed

    Soares, Filipa A C; Chandra, Amit; Thomas, Robert J; Pedersen, Roger A; Vallier, Ludovic; Williams, David J

    2014-03-10

    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.

  3. Effects of the inflammatory cytokines TNF-α and IL-13 on stromal interaction molecule-1 aggregation in human airway smooth muscle intracellular Ca(2+) regulation.

    PubMed

    Jia, Li; Delmotte, Philippe; Aravamudan, Bharathi; Pabelick, Christina M; Prakash, Y S; Sieck, Gary C

    2013-10-01

    Inflammation elevates intracellular Ca(2+) ([Ca(2+)]i) concentrations in airway smooth muscle (ASM). Store-operated Ca(2+) entry (SOCE) is an important source of [Ca(2+)]i mediated by stromal interaction molecule-1 (STIM1), a sarcoplasmic reticulum (SR) protein. In transducing SR Ca(2+) depletion, STIM1 aggregates to form puncta, thereby activating SOCE via interactions with a Ca(2+) release-activated Ca(2+) channel protein (Orai1) in the plasma membrane. We hypothesized that STIM1 aggregation is enhanced by inflammatory cytokines, thereby augmenting SOCE in human ASM cells. We used real-time fluorescence microscopic imaging to assess the dynamics of STIM1 aggregation and SOCE after exposure to TNF-α or IL-13 in ASM cells overexpressing yellow fluorescent protein-tagged wild-type STIM1 (WT-STIM1) and STIM1 mutants lacking the Ca(2+)-sensing EF-hand (STIM1-D76A), or lacking the cytoplasmic membrane binding site (STIM1ΔK). STIM1 aggregation was analyzed by monitoring puncta size during the SR Ca(2+) depletion induced by cyclopiazonic acid (CPA). We found that puncta size was increased in cells expressing WT-STIM1 after CPA. However, STIM1-D76A constitutively formed puncta, whereas STIM1ΔK failed to form puncta. Furthermore, cytokines increased basal WT-STIM1 puncta size, and the SOCE triggered by SR Ca(2+) depletion was increased in cells expressing WT-STIM1 or STIM1-D76A. Meanwhile, SOCE in cells expressing STIM1ΔK and STIM1 short, interfering RNA (siRNA) was decreased. Similarly, in cells overexpressing STIM1, the siRNA knockdown of Orai1 blunted SOCE. However, exposure to cytokines increased SOCE in all cells, increased basal [Ca(2+)]i, and decreased SR Ca(2+) content. These data suggest that cytokines induce a constitutive increase in STIM1 aggregation that contributes to enhanced SOCE in human ASM after inflammation. Such effects of inflammation on STIM1 aggregations may contribute to airway hyperresponsiveness. PMID:23713409

  4. 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.

  5. Analysis and purification of IgG4 bispecific antibodies by a mixed-mode chromatography.

    PubMed

    Yang, Xiaoyu; Zhang, Ying; Wang, Fengqiang; Wang, Larry Jin; Richardson, Daisy; Shameem, Mohammed; Ambrogelly, Alexandre

    2015-09-01

    Therapeutic non-hinge-modified IgG4 molecules form bispecific hybrid antibodies with endogenous human IgG4 molecules via a process known as Fab-arm exchange (or called half molecule exchange). Analysis of the bispecific hybrids is critical for studies of half molecule exchange. A number of analytical methods are available to detect IgG4 hybrids. These methods mostly necessitate labeling or alteration of the model IgG4 molecules, or rely on time-consuming immunoassays and mass spectrometry. In addition, these methods do not allow isolation of hybrid antibodies. We report here the only analytical method to date that relies on chromatographic separation for detection of hybrids formed from intact antibodies in their native forms using pembrolizumab as an example. This method employs a mixed-mode chromatography using a Sepax Zenix SEC-300 column to separate a bispecific hybrid from the parental antibodies. The simultaneous quantitative monitoring of the newly formed hybrid and parental antibodies was achieved by UV absorption and/or protein fluorescence. The bispecific hybrid antibodies were purified with the same method for further biochemical characterization. The method has allowed monitoring of half molecule exchange between a human serum IgG4 and a tested IgG4 molecule, and has been implemented for the analysis of in vitro as well as in vivo samples.

  6. 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.

  7. Changes of tetanus specific IgG, IgM and IgG subclasses after DPT vaccination.

    PubMed

    Kim, J S; Kim, S J; Shin, K J; Hwang, P H; Cho, S C

    1989-01-01

    We evaluated tetanus specific IgG, IgM, IgG subclasses after DPT vaccination in infants and children. Tetanus toxoid specific IgG, IgM IgG subclasses were measured to characterize the isotope profile of antibody against tetanus toxoid. The values of the tetanus specific IgG in the positive group were significantly increased compared to those of the control group, and were significantly increased after two inoculation. Tetanus specific IgG was very low in adults and neonates. In our tetanus specific IgG subclasses study, forty-five of 56 cases (80%) showed predominantly IgG1 antibody responses to tetanus toxoid, while twenty-five of 56 cases (45%) showed IgG4 responses. Both IgG1 and IgG4 responses were demonstrated in 17 cases (30%). So we suggest that IgG was mainly involved in humoral immune response after DPT vaccination, and IgG1 may play an important role among IgG subclasses. IgG4, alone or together with IgG1, can also play a role in immune response to tetanus toxoid.

  8. Heat sensitivity of porcine IgG.

    PubMed

    Metzger, J J; Bourdieu, C; Rouze, P; Houdayer, M

    1975-09-01

    The sensitivity to heat of porcine IgG was studied. The serum from immunized pigs was heated at 56 degrees C for 30 min as for decomplementation. The elution pattern of the serum proteins on an agarose gel column showed a dramatic change with the appearance of a large peak of the gel-excluded material. This peak contained mainly IgG molecules which still retained its antibody activity. This fact points to misinterpretations which can easily occur in 7S and 19S antibody recognition during the porcine immune response. Correlation is suggested of this property with the large number of interheavy chain disulfide bridges present in porcine IgG.

  9. IgG Subclass Staining in Routine Renal Biopsy Material.

    PubMed

    Hemminger, Jessica; Nadasdy, Gyongyi; Satoskar, Anjali; Brodsky, Sergey V; Nadasdy, Tibor

    2016-05-01

    Immunofluorescence staining plays a vital role in nephropathology, but the panel of antibodies used has not changed for decades. Further classification of immunoglobulin (Ig)G-containing immune-type deposits with IgG subclass staining (IgG1, IgG2, IgG3, and IgG4) has been shown to be of diagnostic utility in glomerular diseases, but their value in the evaluation of renal biopsies has not been addressed systematically in large renal biopsy material. Between January 2007 and June 2014, using direct immunofluorescence, we stained every renal biopsy for the IgG subclasses if there was moderate to prominent glomerular IgG staining and/or IgG-predominant or IgG-codominant glomerular staining. The total number of biopsies stained was 1084, which included 367 cases of membranous glomerulonephritis, 307 cases of lupus nephritis, 74 cases of fibrillary glomerulonephritis, 53 cases of proliferative glomerulonephritis with monoclonal IgG deposits, and 25 cases of antiglomerular basement membrane disease, among others. We found that monoclonality of IgG deposits cannot always be reliably determined on the basis of kappa and lambda light chain staining alone, particularly if concomitant (frequently nonspecific) IgM staining is present. In IgG heavy and heavy and light chain deposition disease (3 cases), subclass staining is very helpful, and in proliferative glomerulonephritis with monoclonal IgG deposits subclass staining is necessary. IgG subclass staining is useful in differentiating primary from secondary membranous glomerulonephritis. In proliferative glomerulonephritis with polyclonal IgG deposition, IgG1 dominance/codominance with concomitant IgG3 and IgG2 but weak or absent IgG4 staining favors an underlying autoimmune disease. IgG subclass staining is a very useful diagnostic method in a selected cohort of renal biopsies, particularly in biopsies with glomerulonephritis with monoclonal IgG deposits.

  10. Stabilization by urea during thermal unfolding-mediated aggregation of recombinant human interleukin-1 receptor (type II): does solvation entropy play a role?

    PubMed

    Remmele, Richard L; Zhang-van Enk, Jian; Phan, Duke; Yu, Lei

    2012-06-21

    The protein denaturing properties of urea are well-known and still the subject of debate. It has been noted that in some cases where urea concentrations are relatively low stabilization is afforded against aggregation. An explanation for this unusual effect has seemingly remained elusive. Evidence is offered to propose urea stabilization is related to its influence on the solvation property of the protein molecules when in contact with an unfolded hydrophobic surface that tends to increase the entropy of the local aqueous solvent. This property of urea is expected to lower the entropic driving force of unfolded-mediated aggregation despite the increase in enthalpy. The data presented from toluene transfer experiments into 2 M urea + 0.1 M sodium phosphate solutions showed that the solvation free energy change was negative up to ∼75 °C. The associated ΔΔH was positive, leading to the conclusion that entropy drives the solvation process within the temperature domain from ∼20° to 75 °C. Using thermodynamic parameters from the toluene solvation experiments, it was possible to accurately determine the T(m) shift of recombinant human interleukin-1 receptor type II (rhuIL-1R(II)). Heating experiments above the apparent T(m) in the same urea/phosphate solution support the thesis that urea inhibits the entropy-driven aggregation process of rhuIL-1R(II), adding yet another molecule to the list of low urea concentration stabilized molecules. PMID:22571594

  11. Seroepidemiology and high negativity 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, Iheanyi Omezuruike; Ofoedu, Valentina; Okerentugba, Phillip O; Frank-Peterside, Nnenna

    2015-01-01

    This study reports the seroepidemiology and high negativity of IgG antibodies against the most common low- and high-risk HPVs among sexually active women of childbearing age in Port Harcourt, Nigeria. A total of 182 consented women (age range 19-45 years) were consecutively recruited to participate in the study. Using a Performa specifically designed for this study, pertinent socio-demographic/behavioral data were collected. Five 5 mL blood samples were also collected (aseptically) from each woman. Plasma of each sample was assayed for HPV-6/11/16/18 virus-like particles using a HPV IgG ELISA kit (Dia.Pro). The study showed a high overall anti-HPV seronegativity of 95.1% among these women. High group-specific seronegativity was also observed which ranged from 90.0 -100.0%. None of the variables evaluated showed statistical association with the HPV seronegativity. This study further confirmed the presence of HPV and susceptibility of a large population of women in their childbearing age to infections with these four HPV genotypes in Nigeria. Our findings therefore advocate for routine and early screening and clinical evaluation of all women of childbearing age for HPV- infection and -related manifestations.

  12. 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.

  13. 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.

  14. Construction aggregates

    USGS Publications Warehouse

    Tepordei, V.V.

    1993-01-01

    Part of a special section on the market performance of industrial minerals in 1992. Production of construction aggregates increased by 4.6 percent in 1992. This increase was due, in part, to the increased funding for transportation and infrastructure projects. The U.S. produced about 1.05 Gt of crushed stone and an estimated 734 Mt of construction sand and gravel in 1992. Demand is expected to increase by about 5 percent in 1993.

  15. 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.

  16. Metal-dependent hydrolysis of myelin basic protein by IgGs from the sera of patients with multiple sclerosis.

    PubMed

    Polosukhina, Dar'ya I; Kanyshkova, Tat'yana G; Doronin, Boris M; Tyshkevich, Olga B; Buneva, Valentina N; Boiko, Alexey N; Gusev, Evgenii I; Nevinsky, Georgy A; Favorova, Olga O

    2006-02-28

    Homogeneous IgG fractions were obtained by chromatography of the sera of patients with multiple sclerosis (MS) on Protein G-Sepharose under conditions that remove non-specifically bound proteins. These IgGs contained several chelated metals, the relative amount of which decreases in the order: Fe>or=Ca>Cu>or=Zn>or=Mg>or=Mn>or=Pb>or=Co>or=Ni. In contrast to homogeneous IgGs of healthy individuals, Abs of MS patients effectively hydrolyzed human myelin basic protein (MBP). A minor metal-dependent fraction was obtained by chromatography of highly purified IgGs from MS patient on Chelex-100. This IgG fraction did not hydrolyze human MBP in the absence of Me(2+) ions but was activated after addition of Me(2+) ions: Mg(2+)>Mn(2+)>Cu(2+)>Ca(2+). Proteolytic activities of IgGs from other MS patients were also activated by other metal ions (Ni(2+), Fe(2+), Co(2+), Zn(2+), Pb(2+), and Co(2+)) and especially Ni(2+). Ni(2+)-activated IgGs were separated into distinct MBP-hydrolyzing fractions by chromatography on HiTraptrade mark Chelating Sepharose charged with Ni(2+). Detection of Mg(2+)-dependent proteolytic activity in the SDS-PAGE area corresponding only to IgG provided direct evidence that IgG from sera of MS patients possesses metal-dependent human MBP-hydrolyzing activity. Observed properties of MS abzymes distinguish them from other known mammalian metalloproteases and demonstrate their pronounced catalytic diversity. Metal-dependent IgGs from MS patients represent the first example of abzymes with metal-dependent proteolytic activity. PMID:16310860

  17. The non-protein amino acid BMAA is misincorporated into human proteins in place of L-serine causing protein misfolding and aggregation.

    PubMed

    Dunlop, Rachael Anne; Cox, Paul Alan; Banack, Sandra Anne; Rodgers, Kenneth John

    2013-01-01

    Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNA(Ala) with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, β-N-methylamino-L-alanine (BMAA), can be misincorporated in place of L-serine into human proteins. We also report that this misincorporation can be inhibited by L-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS. PMID:24086518

  18. Interactions between integrin αIIbβ3 and the serotonin transporter regulate serotonin transport and platelet aggregation in mice and humans

    PubMed Central

    Carneiro, Ana Marin D.; Cook, Edwin H.; Murphy, Dennis L.; Blakely, Randy D.

    2008-01-01

    The essential contribution of the antidepressant-sensitive serotonin (5-HT) transporter SERT (which is encoded by the SLC6A4 gene) to platelet 5-HT stores suggests an important role of this transporter in platelet function. Here, using SERT-deficient mice, we have established a role for constitutive SERT expression in efficient ADP- and thrombin-triggered platelet aggregation. Additionally, using pharmacological blockers of SERT and the vesicular monoamine transporter (VMAT), we have identified a role for ongoing 5-HT release and SERT activity in efficient human platelet aggregation. We have also demonstrated that fibrinogen, an activator of integrin αIIbβ3, enhances SERT activity in human platelets and that integrin αIIbβ3 interacts directly with the C terminus of SERT. Consistent with these findings, knockout mice lacking integrin β3 displayed diminished platelet SERT activity. Conversely, HEK293 cells engineered to express human SERT and an activated form of integrin β3 exhibited enhanced SERT function that coincided with elevated SERT surface expression. Our results support an unsuspected role of αIIbβ3/SERT associations as well as αIIbβ3 activation in control of SERT activity in vivo that may have broad implications for hyperserotonemia, cardiovascular disorders, and autism. PMID:18317590

  19. Production of aggregation prone human interferon gamma and its mutant in highly soluble and biologically active form by SUMO fusion technology.

    PubMed

    Tileva, M; Krachmarova, E; Ivanov, I; Maskos, K; Nacheva, G

    2016-01-01

    The Escherichia coli expression system is a preferable choice for production of recombinant proteins. A disadvantage of this system is the target protein aggregation in "inclusion bodies" (IBs) that further requires solubilisation and refolding, which is crucial for the properties and the yield of the final product. In order to prevent aggregation, SUMO fusion tag technology has been successfully applied for expression of eukaryotic proteins, including human interferon gamma (hIFNγ) that was reported, however, with no satisfactory biological activity. We modified this methodology for expression and purification of both the wild type hIFNγ and an extremely prone to aggregation mutant hIFNγ-K88Q, whose recovery from IBs showed to be ineffective upon numerous conditions. By expression of the N-terminal His-SUMO fusion proteins in the E. coli strain BL21(DE3)pG-KJE8, co-expressing two chaperone systems, at 24 °C a significant increase in solubility of both target proteins (1.5-fold for hIFNγ and 8-fold for K88Q) was achieved. Two-step chromatography (affinity and ion-exchange) with on-dialysis His-SUMO-tag cleavage was applied for protein purification that yielded 6.0-7.0mg/g wet biomass for both proteins with >95% purity and native N-termini. The optimised protocol led to increased yields from 5.5 times for hIFNγ up to 100 times for K88Q in comparison to their isolation from IBs. Purified hIFNγ showed preserved thermal stability and antiproliferative activity corresponding to that of the native reference sample (3 × 10(7)IU/mg). The developed methodology represents an optimised procedure that can be successfully applied for large scale expression and purification of aggregation-prone proteins in soluble native form.

  20. IgG Subclasses and Allotypes: From Structure to Effector Functions

    PubMed Central

    Vidarsson, Gestur; Dekkers, Gillian; Rispens, Theo

    2014-01-01

    Of the five immunoglobulin isotypes, immunoglobulin G (IgG) is most abundant in human serum. The four subclasses, IgG1, IgG2, IgG3, and IgG4, which are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. These regions are involved in binding to both IgG-Fc receptors (FcγR) and C1q. As a result, the different subclasses have different effector functions, both in terms of triggering FcγR-expressing cells, resulting in phagocytosis or antibody-dependent cell-mediated cytotoxicity, and activating complement. The Fc-regions also contain a binding epitope for the neonatal Fc receptor (FcRn), responsible for the extended half-life, placental transport, and bidirectional transport of IgG to mucosal surfaces. However, FcRn is also expressed in myeloid cells, where it participates in both phagocytosis and antigen presentation together with classical FcγR and complement. How these properties, IgG-polymorphisms and post-translational modification of the antibodies in the form of glycosylation, affect IgG-function will be the focus of the current review. PMID:25368619

  1. Phosphorylation regulates fibrillation of an aggregation core peptide in the second repeat of microtubule-binding domain of human tau.

    PubMed

    Inoue, Masafumi; Kaida, Shinji; Nakano, Shun; Annoni, Chiara; Nakata, Eiji; Konno, Takashi; Morii, Takashi

    2014-11-15

    Hyperphosphorylation of the microtubule-associated protein tau is believed to play a crucial role in the neurofibrillary tangles formation in Alzheimer’s disease brain. In this study, fibril formation of peptides containing the critical sequences for tau aggregation VQIINK and a plausible serine phosphorylation site of tau at its C-terminal was investigated. All the peptides formed fibrils with the typical cross-b structural core. However, stability of the fibrils was highly sensitive to the pH conditions for the phosphorylated VQIINK peptide, suggesting a regulatory role of phosphorylation for the amyloid-formation of tau.

  2. Construction aggregates

    USGS Publications Warehouse

    Bolen, W.P.; Tepordei, V.V.

    2001-01-01

    The estimated production during 2000 of construction aggregates, crushed stone, and construction sand and gravel increased by about 2.6% to 2.7 Gt (3 billion st), compared with 1999. The expansion that started in 1992 continued with record production levels for the ninth consecutive year. By commodity, construction sand and gravel production increased by 4.5% to 1.16 Gt (1.28 billion st), while crushed stone production increased by 1.3% to 1.56 Gt (1.72 billion st).

  3. Antitumor Efficacy of Anti-GD2 IgG1 Is Enhanced by Fc Glyco-Engineering.

    PubMed

    Xu, Hong; Guo, Hongfen; Cheung, Irene Y; Cheung, Nai-Kong V

    2016-07-01

    The affinity of therapeutic antibodies for Fcγ receptors (FcγRs) strongly influences their antitumor potency. To generate antibodies with optimal binding and immunologic efficacy, we compared the affinities of different versions of an IgG1 Fc region that had an altered peptide backbone, altered glycans, or both. To produce IgG1 with glycans that lacked α1,6-fucose, we used CHO cells that were deficient in the enzyme UDP-N-acetylglucosamine: α-3-d-mannoside-β-1,2-N-acetylglucosaminyltransferase I (GnT1), encoded by the MGAT1 gene. Mature N-linked glycans require this enzyme, and without it, CHO cells synthesize antibodies carrying only Man5-GlcNAc2, which were more effective in antibody-dependent cell-mediated cytotoxicity (ADCC). Our engineered IgG1, hu3F8-IgG1, is specific for GD2, a neuroendocrine tumor ganglioside. Its peptide mutant is IgG1-DEL (S239D/I332E/A330L), both produced in wild-type CHO cells. When produced in GnT1-deficient CHO cells, we refer to them as IgG1n and IgG1n-DEL, respectively. Affinities for human FcγRs were measured using Biacore T-100 (on CD16 and CD32 polymorphic alleles), their immunologic properties compared for ADCC and complement-mediated cytotoxicity (CMC) in vitro, and pharmacokinetics and antitumor effects were compared in vivo in humanized mice. IgG1n and IgG1n-DEL contained only mannose and acetylglucosamine and had preferential affinity for activating CD16s, over inhibitory CD32B, receptors. In vivo, the antitumor effects of IgG1, IgG1-DEL, and IgG1n-DEL were similar but modest, whereas IgG1n was significantly more effective (P < 0.05). Thus, IgG1n antibodies produced in GnT1-deficient CHO cells may have potential as improved anticancer therapeutics. Cancer Immunol Res; 4(7); 631-8. ©2016 AACR. PMID:27197064

  4. MuSK Myasthenia Gravis IgG4 Disrupts the Interaction of LRP4 with MuSK but Both IgG4 and IgG1-3 Can Disperse Preformed Agrin-Independent AChR Clusters

    PubMed Central

    Koneczny, Inga; Cossins, Judith; Waters, Patrick; Beeson, David; Vincent, Angela

    2013-01-01

    A variable proportion of patients with generalized myasthenia gravis (MG) have autoantibodies to muscle specific tyrosine kinase (MuSK). During development agrin, released from the motor nerve, interacts with low density lipoprotein receptor-related protein-4 (LRP4), which then binds to MuSK; MuSK interaction with the intracellular protein Dok7 results in clustering of the acetylcholine receptors (AChRs) on the postsynaptic membrane. In mature muscle, MuSK helps maintain the high density of AChRs at the neuromuscular junction. MuSK antibodies are mainly IgG4 subclass, which does not activate complement and can be monovalent, thus it is not clear how the antibodies cause disruption of AChR numbers or function to cause MG. We hypothesised that MuSK antibodies either reduce surface MuSK expression and/or inhibit the interaction with LRP4. We prepared MuSK IgG, monovalent Fab fragments, IgG1-3 and IgG4 fractions from MuSK-MG plasmas. We asked whether the antibodies caused endocytosis of MuSK in MuSK-transfected cells or if they inhibited binding of LRP4 to MuSK in co-immunoprecipitation experiments. In parallel, we investigated their ability to reduce AChR clusters in C2C12 myotubes induced by a) agrin, reflecting neuromuscular development, and b) by Dok7- overexpression, producing AChR clusters that more closely resemble the adult neuromuscular synapse. Total IgG, IgG4 or IgG1-3 MuSK antibodies were not endocytosed unless cross-linked by divalent anti-human IgG. MuSK IgG, Fab fragments and IgG4 inhibited the binding of LRP4 to MuSK and reduced agrin-induced AChR clustering in C2C12 cells. By contrast, IgG1-3 antibodies did not inhibit LRP4-MuSK binding but, surprisingly, did inhibit agrin-induced clustering. Moreover, both IgG4 and IgG1-3 preparations dispersed agrin-independent AChR clusters in Dok7-overexpressing C2C12 cells. Thus interference by IgG4 antibodies of the LRP4-MuSK interaction will be one pathogenic mechanism of MuSK antibodies, but IgG1-3 Mu

  5. IgG4-related hepatobiliary disease: an overview.

    PubMed

    Culver, Emma L; Chapman, Roger W

    2016-10-01

    IgG4-related hepatobiliary diseases are part of a multiorgan fibroinflammatory condition termed IgG4-related disease, and include IgG4-related sclerosing cholangitis (IgG4-SC) and IgG4-related hepatopathy. These diseases can present with biliary strictures and/or mass lesions, making them difficult to differentiate from primary sclerosing cholangitis (PSC) or other hepatobiliary malignancies. Diagnosis is based on a combination of clinical, biochemical, radiological and histological findings. However, a gold standard diagnostic test is lacking, warranting the identification of more specific disease markers. Novel assays - such as the serum IgG4:IgG1 ratio and IgG4:IgG RNA ratio (which distinguish IgG4-SC from PSC with high serum IgG4 levels), and plasmablast expansion to recognize IgG4-SC with normal serum IgG4 levels - require further validation. Steroids and other immunosuppressive therapies can lead to clinical and radiological improvement when given in the inflammatory phase of the disease, but evidence for the efficacy of treatment regimens is limited. Progressive fibrosclerotic disease, liver cirrhosis and an increased risk of malignancy are now recognized outcomes. Insights into the genetic and immunological features of the disease have increased over the past decade, with an emphasis on HLAs, T cells, circulating memory B cells and plasmablasts, chemokine-mediated trafficking, as well as the role of the innate immune system.

  6. Anticomplementary Activity of Horse IgG and F(ab')2 Antivenoms

    PubMed Central

    Squaiella-Baptistão, Carla Cristina; Marcelino, José Roberto; Ribeiro da Cunha, Luiz Eduardo; Gutiérrez, José María; Tambourgi, Denise V.

    2014-01-01

    Envenomation by poisonous animals is a neglected condition according to the World Health Organization (WHO). Antivenoms are included in the WHO Essential Medicines List. It has been assumed that immunoglobulin G (IgG) antivenoms could activate the complement system through Fc and induce early adverse reactions (EARs). However, data in the literature indicate that F(ab')2 fragments can also activate the complement system. Herein, we show that several batches of IgG and F(ab')2 antivenoms from the Butantan, Vital Brazil, and Clodomiro Picado Institutes activated the complement classical pathway and induced the production of C3a; however, only those antivenoms from Clodomiro Picado generated C5a. Different protein profiles (IgG heavy chain, protein contaminants, and aggregates) were observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses. Our results show that various antivenoms from different producers are able to activate the classical pathway of the complement system and generate anaphylatoxins, and these findings suggest that factors, such as composition, contaminant proteins, and aggregates, may influence the anticomplementary activity of antivenoms in vitro. Therefore, there is a need to further improve antivenom production methods to reduce their anticomplementary activity and potential to cause EARs. PMID:24445201

  7. Characterization and screening of IgG binding to the neonatal Fc receptor

    PubMed Central

    Neuber, Tobias; Frese, Katrin; Jaehrling, Jan; Jäger, Sebastian; Daubert, Daniela; Felderer, Karin; Linnemann, Mechthild; Höhne, Anne; Kaden, Stefan; Kölln, Johanna; Tiller, Thomas; Brocks, Bodo; Ostendorp, Ralf; Pabst, Stefan

    2014-01-01

    The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from degradation and increases the serum half-life of IgG, thereby contributing to a higher concentration of IgG in the serum. Because altered FcRn binding may result in a reduced or prolonged half-life of IgG molecules, it is advisable to characterize Fc receptor binding of therapeutic antibody lead candidates prior to the start of pre-clinical and clinical studies. In this study, we characterized the interactions between FcRn of different species (human, cynomolgus monkey, mouse and rat) and nine IgG molecules from different species and isotypes with common variable heavy (VH) and variable light chain (VL) domains. Binding was analyzed at acidic and neutral pH using surface plasmon resonance (SPR) and biolayer interferometry (BLI). Furthermore, we transferred the well-accepted, but low throughput SPR-based method for FcRn binding characterization to the BLI-based Octet platform to enable a higher sample throughput allowing the characterization of FcRn binding already during early drug discovery phase. We showed that the BLI-based approach is fit-for-purpose and capable of discriminating between IgG molecules with significant differences in FcRn binding affinities. Using this high-throughput approach we investigated FcRn binding of 36 IgG molecules that represented all VH/VL region combinations available in the fully human, recombinant antibody library Ylanthia®. Our results clearly showed normal FcRn binding profiles for all samples. Hence, the variations among the framework parts, complementarity-determining region (CDR) 1 and CDR2 of the fragment antigen binding (Fab) domain did not significantly change FcRn binding. PMID:24802048

  8. Measurement of Anti-Erythropoiesis-Stimulating Agent IgG4 Antibody as an Indicator of Antibody-Mediated Pure Red Cell Aplasia

    PubMed Central

    Weeraratne, Dohan K.; Kuck, Andrew J.; Chirmule, Narendra

    2013-01-01

    Patients treated with erythropoietin-based erythropoiesis-stimulating agents (ESAs) can develop a rare but life-threatening condition called antibody-mediated pure red cell aplasia (amPRCA). The antibody characteristics in a nephrology patient with amPRCA include high antibody concentrations with neutralizing activity and a mixed IgG subclass including anti-ESA IgG4 antibodies. In contrast, anti-ESA IgG4 antibody is generally not detected in baseline samples and antibody-positive non-PRCA patients. Therefore, we validated a highly sensitive immunoassay on the ImmunoCAP 100 instrument to quantitate anti-ESA IgG4 antibodies using a human recombinant anti-epoetin alfa (EPO) IgG4 antibody as a calibrator. The biotinylated ESA was applied to a streptavidin ImmunoCAP, and bound anti-ESA IgG4 antibodies were detected using a β-galactosidase-conjugated mouse anti-human IgG4 antibody. The validated assay was used to detect anti-ESA IgG4 in amPRCA and non-PRCA patients. The immunoassay detected 15 ng/ml of human anti-EPO IgG4 antibody in the presence of a 200 M excess of human anti-ESA IgG1, IgG2, or IgM antibody and tolerated 2 μg/ml of soluble erythropoietin. All patient samples with confirmed amPRCA had measurable anti-ESA IgG4 antibodies. In addition, 94% (17/18) of non-PRCA patient samples were antibody negative or had below 15 ng/ml of anti-ESA IgG4 antibodies. This novel immunoassay can measure low-nanogram quantities of human anti-ESA IgG4 antibodies in the presence of other anti-ESA antibodies. An increased concentration of anti-ESA IgG4 antibody is associated with the development of amPRCA. We propose that the measurement of anti-ESA specific IgG4 antibodies may facilitate early detection of amPRCA in patients receiving all ESAs structurally related to human erythropoietin. PMID:23114696

  9. Platelet Activation and Thrombus Formation over IgG Immune Complexes Requires Integrin αIIbβ3 and Lyn Kinase.

    PubMed

    Zhi, Huiying; Dai, Jing; Liu, Junling; Zhu, Jieqing; Newman, Debra K; Gao, Cunji; Newman, Peter J

    2015-01-01

    IgG immune complexes contribute to the etiology and pathogenesis of numerous autoimmune disorders, including heparin-induced thrombocytopenia, systemic lupus erythematosus, rheumatoid- and collagen-induced arthritis, and chronic glomerulonephritis. Patients suffering from immune complex-related disorders are known to be susceptible to platelet-mediated thrombotic events. Though the role of the Fc receptor, FcγRIIa, in initiating platelet activation is well understood, the role of the major platelet adhesion receptor, integrin αIIbβ3, in amplifying platelet activation and mediating adhesion and aggregation downstream of encountering IgG immune complexes is poorly understood. The goal of this investigation was to gain a better understanding of the relative roles of these two receptor systems in immune complex-mediated thrombotic complications. Human platelets, and mouse platelets genetically engineered to differentially express FcγRIIa and αIIbβ3, were allowed to interact with IgG-coated surfaces under both static and flow conditions, and their ability to spread and form thrombi evaluated in the presence and absence of clinically-used fibrinogen receptor antagonists. Although binding of IgG immune complexes to FcγRIIa was sufficient for platelet adhesion and initial signal transduction events, platelet spreading and thrombus formation over IgG-coated surfaces showed an absolute requirement for αIIbβ3 and its ligands. Tyrosine kinases Lyn and Syk were found to play key roles in IgG-induced platelet activation events. Taken together, our data suggest a complex functional interplay between FcγRIIa, Lyn, and αIIbβ3 in immune complex-induced platelet activation. Future studies may be warranted to determine whether patients suffering from immune complex disorders might benefit from treatment with anti-αIIbβ3-directed therapeutics.

  10. An atypical IgM class platelet cold agglutinin induces GPVI-dependent aggregation of human platelets.

    PubMed

    Sánchez Guiu, I M; Martínez-Martinez, I; Martínez, C; Navarro-Fernandez, J; García-Candel, F; Ferrer-Marín, F; Vicente, V; Watson, S P; Andrews, R K; Gardiner, E E; Lozano, M L; Rivera, J

    2015-08-01

    Platelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to ex vivo platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin αIIbβ3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A2 production. Patient serum induced temperature- and αIIbβ3-dependent decrease of platelet count in allogeneic donor citrated platelet-rich plasma (PRP), but not in PRP from Glanzmann's thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression, (14)C-serotonin release, and TXA2 production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (FcγRIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding. PMID:25994029

  11. 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.

  12. Generation of Islet-like Cell Aggregates from Human Adipose Tissue-derived Stem Cells by Lentiviral Overexpression of PDX-1

    PubMed Central

    Bahrebar, M.; Soleimani, M.; Karimi, M. H.; Vahdati, A.; Yaghobi, R.

    2015-01-01

    Background: Pancreatic duodenal homeobox1 (PDX-1) is a transcription factor that is important in regulating pancreas development and maintaining β-cell function. β-cell replacement is an effective approach for the treatment of type 1 diabetes. Human adipose-mesenchymal stem cells (hAMSCs) are the ideal population cells for differentiating into insulin-producing cells. Objective: To determine if islet-like cell aggregates production could be generated from hAMSCs by lentiviral overexpression of PDX-1. Methods: After isolation of hAMSCs, characteristics of these cells were identified by flow-cytometic analysis and multilineage differentiation studies. PDX-1 gene delivered into hAMSCs through lentiviral vector for differentiating hAMSCs into insulin-producing cells (IPCs) at the utilized protocol for 14 days. Characteristics of IPCs were evaluated by immunocytofluorescence, dithizone staining, and quantitative reverse transcription PCR. In response to high glucose medium, insulin release was detected by chemiluminescence enzyme immunoassay. Results: The islet-like cell aggregates appeared about 10 days after introduction of PDX-1 into hAMSCs. PDX-1 induced its own expression (auto-induction), a number of islet-related genes such as Ngn3, Nkx2-2, and insulin. The insulin-positive cells were detected in the PDX-1 transduced cells. In response to glucose challenge test, secretion of insulin hormone in the medium with high glucose concentration significantly increased in the PDX-1-transduced cells related to medium with low glucose concentration. Conclusion: Introduction of lentiviral PDX-1 significantly induces hAMSCs to differentiate into islet-like cell aggregates, which may provide a source of adipose stem cells-derived insulin-producing cells for cell replacement therapy in type 1 diabetes. PMID:26082830

  13. Factorial experimental design for the culture of human embryonic stem cells as aggregates in stirred suspension bioreactors reveals the potential for interaction effects between bioprocess parameters.

    PubMed

    Hunt, Megan M; Meng, Guoliang; Rancourt, Derrick E; Gates, Ian D; Kallos, Michael S

    2014-01-01

    Traditional optimization of culture parameters for the large-scale culture of human embryonic stem cells (ESCs) as aggregates is carried out in a stepwise manner whereby the effect of varying each culture parameter is investigated individually. However, as evidenced by the wide range of published protocols and culture performance indicators (growth rates, pluripotency marker expression, etc.), there is a lack of systematic investigation into the true effect of varying culture parameters especially with respect to potential interactions between culture variables. Here we describe the design and execution of a two-parameter, three-level (3(2)) factorial experiment resulting in nine conditions that were run in duplicate 125-mL stirred suspension bioreactors. The two parameters investigated here were inoculation density and agitation rate, which are easily controlled, but currently, poorly characterized. Cell readouts analyzed included fold expansion, maximum density, and exponential growth rate. Our results reveal that the choice of best case culture parameters was dependent on which cell property was chosen as the primary output variable. Subsequent statistical analyses via two-way analysis of variance indicated significant interaction effects between inoculation density and agitation rate specifically in the case of exponential growth rates. Results indicate that stepwise optimization has the potential to miss out on the true optimal case. In addition, choosing an optimum condition for a culture output of interest from the factorial design yielded similar results when repeated with the same cell line indicating reproducibility. We finally validated that human ESCs remain pluripotent in suspension culture as aggregates under our optimal conditions and maintain their differentiation capabilities as well as a stable karyotype and strong expression levels of specific human ESC markers over several passages in suspension bioreactors.

  14. 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.

  15. 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

  16. 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.

  17. Effective Rho-associated protein kinase inhibitor treatment to dissociate human iPS cells for suspension culture to form embryoid body-like cell aggregates.

    PubMed

    Horiguchi, Ayumi; Yazaki, Koyuki; Aoyagi, Mami; Ohnuki, Yoshitsugu; Kurosawa, Hiroshi

    2014-11-01

    Treatment conditions using Y-27632 in the preparation of cell suspension of dissociated human pluripotent stem cells (hiPSCs) were investigated in the context of embryoid body (EB)-like cell aggregates. The effectiveness of a pretreatment with Y-27632 before cell dissociation and that of a Y-27632 treatment during cell dissociation were investigated from the viewpoint of simplicity and robustness. The duration of Y-27632 treatment in the preparation process affected the circularity and agglomeration of dissociated hiPSCs. A single application of pretreatment failed to prevent the onset of blebbing. However, a pretreatment promoted the agglomeration of dissociated hiPSCs when combined with the addition of Y-27632 to cell suspension. Our results indicate that pretreatment enhances the agglomeration potential of dissociated hiPSCs. When cell dissociation was performed in the presence of Y-27632, dissociated hiPSCs possessed the highest circularity and significant agglomerating property. It was shown that treatment with Y-27632 during cell dissociation is a simple and robust method to prepare dissociated hiPSCs for suspension culture to form EB-like cell aggregates.

  18. Bromelain proteases reduce human platelet aggregation in vitro, adhesion to bovine endothelial cells and thrombus formation in rat vessels in vivo.

    PubMed

    Metzig, C; Grabowska, E; Eckert, K; Rehse, K; Maurer, H R

    1999-01-01

    The thiol protease, bromelain, an extract from pineapple stem, was suggested to have antithrombotic and anticoagulant activities in vivo. We studied the effects of bromelain on cell size distribution of isolated human platelets in vitro by Coulter Counter measurements. Preincubation of platelets with bromelain (10 micrograms/mL) completely prevented the thrombin (0.2 U/mL) induced platelet aggregation. Papain was less active in preventing platelet aggregation. In vitro, bromelain (0.1 microgram/mL) reduced the adhesion of bound, thrombin stimulated, fluorescent labeled platelets to bovine aorta endothelial cells. In addition, preincubation of platelets with bromelain, prior to thrombin, activation, reduced the platelet adhesion to the endothelial cells to the low binding value of unstimulated platelets. On the basis of mass concentrations, the proteases papain and trypsin were as effective as bromelain. Using a laser thrombosis model, the in vivo effects of orally and intraveneously applied bromelain on thrombus formation in rat mesenteric vessels were studied. Bromelain, orally applied at 60 mg/kg body weight, inhibited the thrombus formation in a time dependent manner, the maximum being after 2 hours in 11% of arterioles and 6% of venoles. Intravenous application at 30 mg/kg was slightly more active in reducing thrombus formation in arterioles (13%) and venoles (5%), suggesting that orally applied bromelain is biologically active. These results may help to explain some of the clinical effects observed after bromelain treatment in patients with thrombosis and related diseases.

  19. Controlled Dual Growth Factor Delivery From Microparticles Incorporated Within Human Bone Marrow-Derived Mesenchymal Stem Cell Aggregates for Enhanced Bone Tissue Engineering via Endochondral Ossification

    PubMed Central

    Dang, Phuong N.; Dwivedi, Neha; Phillips, Lauren M.; Yu, Xiaohua; Herberg, Samuel; Bowerman, Caitlin; Solorio, Loran D.; Murphy, William L.

    2016-01-01

    Bone tissue engineering via endochondral ossification has been explored by chondrogenically priming cells using soluble mediators for at least 3 weeks to produce a hypertrophic cartilage template. Although recapitulation of endochondral ossification has been achieved, long-term in vitro culture is required for priming cells through repeated supplementation of inductive factors in the media. To address this challenge, a microparticle-based growth factor delivery system was engineered to drive endochondral ossification within human bone marrow-derived mesenchymal stem cell (hMSC) aggregates. Sequential exogenous presentation of soluble transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) at various defined time courses resulted in varying degrees of chondrogenesis and osteogenesis as demonstrated by glycosaminoglycan and calcium content. The time course that best induced endochondral ossification was used to guide the development of the microparticle-based controlled delivery system for TGF-β1 and BMP-2. Gelatin microparticles capable of relatively rapid release of TGF-β1 and mineral-coated hydroxyapatite microparticles permitting more sustained release of BMP-2 were then incorporated within hMSC aggregates and cultured for 5 weeks following the predetermined time course for sequential presentation of bioactive signals. Compared with cell-only aggregates treated with exogenous growth factors, aggregates with incorporated TGF-β1- and BMP-2-loaded microparticles exhibited enhanced chondrogenesis and alkaline phosphatase activity at week 2 and a greater degree of mineralization by week 5. Staining for types I and II collagen, osteopontin, and osteocalcin revealed the presence of cartilage and bone. This microparticle-incorporated system has potential as a readily implantable therapy for healing bone defects without the need for long-term in vitro chondrogenic priming. Significance This study demonstrates the regulation of chondrogenesis

  20. Controlled Dual Growth Factor Delivery From Microparticles Incorporated Within Human Bone Marrow-Derived Mesenchymal Stem Cell Aggregates for Enhanced Bone Tissue Engineering via Endochondral Ossification.

    PubMed

    Dang, Phuong N; Dwivedi, Neha; Phillips, Lauren M; Yu, Xiaohua; Herberg, Samuel; Bowerman, Caitlin; Solorio, Loran D; Murphy, William L; Alsberg, Eben

    2016-02-01

    Bone tissue engineering via endochondral ossification has been explored by chondrogenically priming cells using soluble mediators for at least 3 weeks to produce a hypertrophic cartilage template. Although recapitulation of endochondral ossification has been achieved, long-term in vitro culture is required for priming cells through repeated supplementation of inductive factors in the media. To address this challenge, a microparticle-based growth factor delivery system was engineered to drive endochondral ossification within human bone marrow-derived mesenchymal stem cell (hMSC) aggregates. Sequential exogenous presentation of soluble transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) at various defined time courses resulted in varying degrees of chondrogenesis and osteogenesis as demonstrated by glycosaminoglycan and calcium content. The time course that best induced endochondral ossification was used to guide the development of the microparticle-based controlled delivery system for TGF-β1 and BMP-2. Gelatin microparticles capable of relatively rapid release of TGF-β1 and mineral-coated hydroxyapatite microparticles permitting more sustained release of BMP-2 were then incorporated within hMSC aggregates and cultured for 5 weeks following the predetermined time course for sequential presentation of bioactive signals. Compared with cell-only aggregates treated with exogenous growth factors, aggregates with incorporated TGF-β1- and BMP-2-loaded microparticles exhibited enhanced chondrogenesis and alkaline phosphatase activity at week 2 and a greater degree of mineralization by week 5. Staining for types I and II collagen, osteopontin, and osteocalcin revealed the presence of cartilage and bone. This microparticle-incorporated system has potential as a readily implantable therapy for healing bone defects without the need for long-term in vitro chondrogenic priming. Significance: This study demonstrates the regulation of chondrogenesis

  1. IgG Endopeptidase SeMac does not Inhibit Opsonophagocytosis of Streptococcus equi Subspecies equi by Horse Polymorphonuclear Leukocytes

    PubMed Central

    Liu, Mengyao; Lei, Benfang

    2010-01-01

    The secreted Mac protein made by group A Streptococcus (GAS) inhibits opsonophagocytosis of GAS by human polymorphonuclear leukocytes (PMNs). This protein also has the endopeptidase activity against human immunoglobulin G (IgG), and the Cys94, His262 and Asp284 are critical for the enzymatic activity. The horse pathogen Streptococcus equi subspecies equi produces a homologue of Mac (SeMac). SeMac was characterized to determine whether SeMac has IgG endopeptidase activity and inhibits opsonophagocytosis of S. equi by horse PMNs. The gene was cloned and recombinant SeMac was overexpressed in Escherichia coli and purified to homogeneity. Mice with experimental S. equi infection and horses with strangles caused by S. equi seroconverted to SeMac, indicating that SeMac is produced in vivo during infection. SeMac has endopeptidase activity against human IgG. However, the protein just cleaves a small fraction, which may be IgG1 only, of horse IgG. Replacement of Cys102 with Ser or His272 with Ala abolishes the enzymatic activity of SeMac, and the Asp294Ala mutation greatly decreases the enzymatic activity. SeMac does not inhibit opsonophagocytosis of S. equi by horse PMNs but opsonophagocytosis of GAS by human PMNs. Thus, SeMac is a cysteine endopeptidase with a limited activity against horse IgG and must have other function. PMID:20556207

  2. 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

  3. Diagnostic performance of serum IgG4 level for IgG4-related disease: a meta-analysis.

    PubMed

    Xu, Wen-Long; Ling, Ying-Chun; Wang, Zhi-Kai; Deng, Fang

    2016-01-01

    An elevated serum IgG4 level is one of the most useful factors in the diagnosis of IgG4-related disease (IgG4-RD). In this study, we performed a meta-analysis of the published articles assessing the diagnostic accuracy of serum IgG4 concentrations for IgG4-RD. The databases of MEDLINE/PubMed, EMBASE and Web of Science were systematically searched for relevant studies. Sensitivities and specificities of serum IgG4 in each study were calculated, and the hierarchical summary receiver operating characteristic (HSROC) model with a random effects model were employed to obtain the individual and pooled estimates of sensitivities and specificities. In total, twenty-three studies comprising 6048 patients with IgG4-RD were included in the meta-analysis. The pooled sensitivity was 85% with a 95% confidence interval (CI) of 78-90%; the pooled specificity was 93% with a 95% CI of 90-95%. The HSROC curve for quantitative serum IgG4 lies closer to the upper left corner of the plot, and the area under the curve (AUC) was 0.95 (95% CI 0.93, 0.97), which suggested a high diagnostic accuracy of serum IgG4 for the entity of IgG4-RD. Our study suggests that serum IgG4 has high sensitivity and specificity in the diagnosis of IgG4-RD. PMID:27558881

  4. 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

  5. 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

  6. 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.

  7. 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

  8. Effects of a garlic-derived principle (ajoene) on aggregation and arachidonic acid metabolism in human blood platelets.

    PubMed

    Srivastava, K C; Tyagi, O D

    1993-08-01

    When garlic cloves are chopped or crushed several dialkyl thiosulfinates are rapidly formed by the action of the enzyme alliin lyase or alliinase (EC 4.4.1.4) on S(+)-alkyl-L-cysteine sulfoxides. Allicin (diallyl thiosulfinate or allyl 2-propene thiosulfinate) is the dominant thiosulfinate released. A variety of sulfur containing compounds are formed from allicin and other thiosulfinates depending on the way in which garlic is handled. One such compound identified recently is ajoene which has been reported to possess antithrombotic properties. We present here data on the antiplatelet properties of ajoene together with its effects on the metabolism of arachidonic acid (AA) in intact platelets. Thus, ajoene was found to inhibit platelet aggregation induced by AA, adrenaline, collagen, adenosine diphosphate (ADP) and calcium ionophore A23187; the nature of the inhibition was irreversible. In washed platelets stimulated by labelled arachidonate, ajoene inhibited the formation of thromboxane A2; 12-lipoxygenase product(s) were reduced at higher ajoene concentrations. This garlic-derived substance inhibited the incorporation of labelled AA into platelet phospholipids at higher concentration. In labelled platelets, on stimulation with either calcium ionophore A23187 or collagen, reduced amounts of thromboxane and 12-HETE (12-hydroxyeicosatetraenoic acid) were produced in ajoene-treated platelets compared to control platelets. This substance had no effect on the deacylation of platelet phospholipids. The results suggest that at least one of the mechanisms by which ajoene shows antiplatelet effects could be related to altered metabolism of AA.

  9. In situ characterization of protein aggregates in human tissues affected by light chain amyloidosis: a FTIR microspectroscopy study.

    PubMed

    Ami, Diletta; Lavatelli, Francesca; Rognoni, Paola; Palladini, Giovanni; Raimondi, Sara; Giorgetti, Sofia; Monti, Luca; Doglia, Silvia Maria; Natalello, Antonino; Merlini, Giampaolo

    2016-01-01

    Light chain (AL) amyloidosis, caused by deposition of amyloidogenic immunoglobulin light chains (LCs), is the most common systemic form in industrialized countries. Still open questions, and premises for developing targeted therapies, concern the mechanisms of amyloid formation in vivo and the bases of organ targeting and dysfunction. Investigating amyloid material in its natural environment is crucial to obtain new insights on the molecular features of fibrillar deposits at individual level. To this aim, we used Fourier transform infrared (FTIR) microspectroscopy for studying in situ unfixed tissues (heart and subcutaneous abdominal fat) from patients affected by AL amyloidosis. We compared the infrared response of affected tissues with that of ex vivo and in vitro fibrils obtained from the pathogenic LC derived from one patient, as well as with that of non amyloid-affected tissues. We demonstrated that the IR marker band of intermolecular β-sheets, typical of protein aggregates, can be detected in situ in LC amyloid-affected tissues, and that FTIR microspectroscopy allows exploring the inter- and intra-sample heterogeneity. We extended the infrared analysis to the characterization of other biomolecules embedded within the amyloid deposits, finding an IR pattern that discloses a possible role of lipids, collagen and glycosaminoglycans in amyloid deposition in vivo. PMID:27373200

  10. Interleukin-1beta partially alleviates cyclosporin A-induced suppression of IgG1 isotype response to thyroglobulin in BALB/c mice in vivo.

    PubMed Central

    Dalai, S K; Miriyala, B; Kar, S K

    1998-01-01

    Cyclosporin A (CsA) at 120 mg/kg body weight when injected subcutaneously into BALB/c mice along with thyroglobulin emulsified in incomplete Freund's adjuvant (IFA) was found to suppress antigen-specific IgG titre by 86%. Isotyping revealed that both IgG1 and IgG2a titres were suppressed by 87% and 57%, respectively. But under identical conditions when complete Freund's adjuvant (CFA) was used, the suppression of antigen-specific IgG, IgG1 and IgG2a titres was 50%, 51% and 55%, respectively. Injection of anti-IL-1beta-neutralizing hamster monoclonal antibodies along with thyroglobulin and CsA emulsified in CFA increased the suppression of antigen-specific IgG titre. Under such conditions the IgG1 titre was suppressed more than the IgG2a titre. Recombinant human interleukin-1 receptor antagonist (rhuIL-1ra) also enhanced the suppression caused by CsA in the presence of CFA but control hamster immunoglobulin had no such effect. Recombinant human IL-1beta, when administered along with thyroglobulin and CsA emulsified in IFA, alleviated the suppression of antigen-specific IgG titre and the IgG1 titre was alleviated more than the IgG2a titre. Under identical conditions, rhuIL-1ra did not alleviate CsA-induced suppression. Lymphocytes from the lymph nodes of thyroglobulin-sensitized BALB/c mice when stimulated in vitro by thyroglobulin in the presence of CsA, secreted very little interferon-gamma (IFN-gamma) and IL-4, but on addition of an optimal dose of rhuIL-1beta, IFN-gamma and IL-4 secretion was partially restored. PMID:9767461

  11. Flavonoid Glycosides Inhibit Sortase A and Sortase A-Mediated Aggregation of Streptococcus mutans, an Oral Bacterium Responsible for Human Dental Caries.

    PubMed

    Yang, Woo-Young; Kim, Chang-Kwon; Ahn, Chan-Hong; Kim, Heegyu; Shin, Jongheon; Oh, Ki-Bong

    2016-09-28

    Three flavonoids were isolated from dried flowers of Sophora japonica using repetitive column chromatography and high-performance liquid chromatography. The flavonoids were identified as rutin (1), quercetin-3'-O-methyl-3-O-α-L-rhamnopyranosyl(1 → 6)-β-D-glucopyranoside (2), and quercetin (3) on the basis of spectroscopic analysis and comparison of values reported in the literature. These compounds inhibited the action of sortase A (SrtA) from Streptococcus mutans, a primary etiologic agent of human dental caries. The onset and magnitude of inhibition of saliva-induced aggregation of S. mutans treated with compound 1 was comparable to that of untreated S. mutans with a deletion of the srtA gene. PMID:27291675

  12. Flavonoid Glycosides Inhibit Sortase A and Sortase A-Mediated Aggregation of Streptococcus mutans, an Oral Bacterium Responsible for Human Dental Caries.

    PubMed

    Yang, Woo-Young; Kim, Chang-Kwon; Ahn, Chan-Hong; Kim, Heegyu; Shin, Jongheon; Oh, Ki-Bong

    2016-09-28

    Three flavonoids were isolated from dried flowers of Sophora japonica using repetitive column chromatography and high-performance liquid chromatography. The flavonoids were identified as rutin (1), quercetin-3'-O-methyl-3-O-α-L-rhamnopyranosyl(1 → 6)-β-D-glucopyranoside (2), and quercetin (3) on the basis of spectroscopic analysis and comparison of values reported in the literature. These compounds inhibited the action of sortase A (SrtA) from Streptococcus mutans, a primary etiologic agent of human dental caries. The onset and magnitude of inhibition of saliva-induced aggregation of S. mutans treated with compound 1 was comparable to that of untreated S. mutans with a deletion of the srtA gene.

  13. Activation of complement by an IgG molecule without a genetic hinge.

    PubMed

    Brekke, O H; Michaelsen, T E; Sandin, R; Sandlie, I

    1993-06-17

    The hinge region links the two Fab arms to the Fc portion of the IgG molecule. It mediates flexibility to the molecule and serves as a connecting structure between the two heavy chains. In addition it provides space between the Fab and Fc parts. All three properties have been proposed to be important for the ability of IgG to initiate complement activation leading to complement-mediated cell lysis (CML). Here we report the construction of a hinge-deleted mouse-human chimaeric IgG3 molecule with specificity for the hapten NIP (3-iodo-4-hydroxy-5-nitrophenacetyl), HM-1. HM-1 lacks the genetic hinge, but has an introduced cysteine between Ala 231 (EU numbering) and Pro 232 in the lower hinge encoded by the CH2 exon. The introduced cysteine forms a disulphide bond between the two heavy chains of the molecule. In CML, HM-1 shows a greater activity than IgG3 wild type. This is the first time an IgG molecule without a genetic hinge has been found to be active in CML. We conclude that the hinge functioning as a spacer is not a prerequisite for complement activation. Rather, its major role seems to be to connect the heavy chains to each other in the amino-terminal part of CH2. Because HM-1 is expected to have low Fab-Fc flexibility, this molecular feature is probably of no importance for complement activation.

  14. 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.

  15. 1,4-Benzenediboronic-Acid-Induced Aggregation of Gold Nanoparticles: Application to Hydrogen Peroxide Detection and Biotin-Avidin-Mediated Immunoassay with Naked-Eye Detection.

    PubMed

    Yang, Ya-Chun; Tseng, Wei-Lung

    2016-05-17

    Hydrogen-peroxide (H2O2)-induced growth of small-sized gold nanoparticles (AuNPs) is often implemented for H2O2 sensing and plasmonic immunoassay. In contrast, there is little-to-no information in the literature regarding the application of H2O2-inhibited aggregation of citrate-capped AuNPs. This study discloses that benzene-1,4-diboronic acid (BDBA) was effective in driving the aggregation of citrate-capped AuNPs through an interaction between α-hydroxycarboxylate of citrate and boronic acids of BDBA. The H2O2-mediated oxidation of BDBA resulted in the conversion of boronic acid groups to phenol groups. The oxidized BDBA was incapable of triggering the aggregation of citrate-capped AuNPs. Thus, the presence of H2O2 prohibited BDBA-induced aggregation of citrate-capped AuNPs. The BDBA-induced aggregation of citrate-capped AuNPs can be paired with the glucose oxidase (GOx)-glucose system to design a colorimetric probe for glucose. Moreover, a H2O2·BDBA·AuNP probe was integrated with sandwich immunoassay, biotinylated antibody, and avidin-conjugated GOx for the selective naked-eye detection of rabbit immunoglobulin G (IgG) and human-prostate-specific antigen (PSA). The lowest detectable concentrations of rabbit IgG and human PSA by the naked eye were down to 0.1 and 4 ng/mL, respectively. More importantly, the proposed plasmonic immunoassay allowed the naked-eye quantification of 0-10 ng/mL PSA at an interval of 2 ng/mL in plasma samples. PMID:27091002

  16. 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.

  17. IgG4-related intraocular inflammation masquerading as ciliary body melanoma in a young girl.

    PubMed

    Das, Dipankar; Deka, Panna; Verma, Geeta; Kuri, Ganesh Chandra; Bhattacharjee, Harsha; Bharali, Gayatri; Pandey, Divya; Koul, Akanksha; Das, Bidisha; Deka, Apurba

    2016-08-01

    Immunoglobulin G4 (IgG4-related diseases) affects various tissues and organs of the human body. Orbital, adnexal, and scleral inflammations were already reported in the medical literature. To the best of our knowledge, we report the first case of intraocular IgG4-associated inflammatory mass in the ciliary body mimicking as a melanoma in a 23-year-old female from Northeast India. Characteristic histopathology, immunohistochemistry in the tissue, protein chemistry, and raised serum IgG4 were supportive for the diagnosis. As this newly diagnosed disease has multi-organ affection and little is known about its pathogenesis particularly in eye and adnexa, the present case will open many challenges in clinico-pathological diagnosis and research in the future.

  18. Detection of histidine oxidation in a monoclonal immunoglobulin gamma (IgG) 1 antibody.

    PubMed

    Amano, Masato; Kobayashi, Naoki; Yabuta, Masayuki; Uchiyama, Susumu; Fukui, Kiichi

    2014-08-01

    Although oxidation of methionine and tryptophan are known as popular chemical modifications that occur in monoclonal antibody (mAb) molecules, oxidation of other amino acids in mAbs has not been reported to date. In this study, oxidation of the histidine residue in a human immunoglobulin gamma (IgG) 1 molecule was discovered for the first time by mass spectrometry. The oxidation of a specific histidine located at the CH2 domain of IgG1 occurred under light stress, but it was not observed under heat stress. With the forced degradation study using several reactive oxygen species, the singlet oxygen was attributed to a reactive source of the histidine oxidation. The reaction mechanism of the histidine oxidation was proposed on the basis of the mass spectrometric analysis of IgG1 oxidized in deuterium oxide and hydrogen heavy oxide. PMID:24940720

  19. Voltage-driven translocation behaviors of IgG molecule through nanopore arrays

    PubMed Central

    2013-01-01

    Nanopore-based biosensing has attracted more and more interests in the past years, which is also regarded as an emerging field with major impact on bio-analysis and fundamental understanding of nanoscale interactions down to single-molecule level. In this work, the voltage-driven translocation properties of goat antibody to human immunoglobulin G (IgG) are investigated using nanopore arrays in polycarbonate membranes. Obviously, the background ionic currents are modulated by IgG molecules for their physical place-holding effect. However, the detected ionic currents do ‘not’ continuously decrease as conceived; the currents first decrease, then increase, and finally stabilize with increasing IgG concentration. To understand this phenomenon, a simplified model is suggested, and the calculated results contribute to the understanding of the abnormal phenomenon in the actual ionic current changing tendency. PMID:23676116

  20. IgG4-related intraocular inflammation masquerading as ciliary body melanoma in a young girl

    PubMed Central

    Das, Dipankar; Deka, Panna; Verma, Geeta; Kuri, Ganesh Chandra; Bhattacharjee, Harsha; Bharali, Gayatri; Pandey, Divya; Koul, Akanksha; Das, Bidisha; Deka, Apurba

    2016-01-01

    Immunoglobulin G4 (IgG4-related diseases) affects various tissues and organs of the human body. Orbital, adnexal, and scleral inflammations were already reported in the medical literature. To the best of our knowledge, we report the first case of intraocular IgG4-associated inflammatory mass in the ciliary body mimicking as a melanoma in a 23-year-old female from Northeast India. Characteristic histopathology, immunohistochemistry in the tissue, protein chemistry, and raised serum IgG4 were supportive for the diagnosis. As this newly diagnosed disease has multi-organ affection and little is known about its pathogenesis particularly in eye and adnexa, the present case will open many challenges in clinico-pathological diagnosis and research in the future. PMID:27688285

  1. 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.

  2. Production of α2,6-sialylated IgG1 in CHO cells

    PubMed Central

    Raymond, Céline; Robotham, Anna; Spearman, Maureen; Butler, Michael; Kelly, John; Durocher, Yves

    2015-01-01

    The presence of α2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized α2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgG's effector function. This study presents a method for the efficient Fc glycan α2,6-sialylation of a wild-type and a F243A IgG1 mutant by transient co-expression with the human α2,6-sialyltransferase 1 (ST6) and β1,4-galactosyltransferase 1 (GT) in CHO cells. Overexpression of ST6 alone only had a moderate effect on the glycoprofiles, whereas GT alone greatly enhanced Fc-galactosylation, but not sialylation. Overexpression of both GT and ST6 was necessary to obtain a glycoprofile dominated by α2,6-sialylated glycans in both antibodies. The wild-type was composed of the G2FS(6)1 glycan (38%) with remaining unsialylated glycans, while the mutant glycoprofile was essentially composed of G2FS(6)1 (25%), G2FS(3,6)2 (16%) and G2FS(6,6)2 (37%). The α2,6-linked sialic acids represented over 85% of all sialic acids in both antibodies. We discuss how the limited sialylation level in the wild-type IgG1 expressed alone or with GT results from the glycan interaction with Fc's amino acid residues or from intrinsic galactosyl- and sialyl-transferases substrate specificities. PMID:25875452

  3. Isolation and analysis of complement activating aggregates from synovial fluid of patients with rheumatoid arthritis using monoclonal anti-C3d antibodies.

    PubMed Central

    Bedwell, A E; Elson, C J; Carter, S D; Dieppe, P A; Hutton, C W; Czudek, R

    1987-01-01

    The complement activating aggregates in synovial fluids of patients with rheumatoid arthritis (RA) have been isolated using monoclonal IgM anti-C3d antibodies attached to solid phases, and the content of the material bound has been analysed. High levels of aggregated IgG bearing C3d were found in RA synovial fluids, and IgG was the major immunoglobulin bound from such synovial fluids by anti-C3d Sepharose. A strong correlation was shown between levels of aggregated IgG bearing C3d and complement activation, as judged by C3d levels. Significant (but less strong) relationships were also observed between C3d levels and both complement consuming and C1q binding activity. C3d levels and levels of aggregated IgG bearing C3d were both significantly associated with the numbers of polymorphonuclear leucocytes (PMNs) found in RA synovial fluids. From these results it is concluded that the aggregated immunoglobulins bearing C3d (particularly IgG) isolated from RA synovial fluids are responsible for activating complement and attracting PMNs into the joint space. Radioimmunoassay showed no correlation, however, between levels of aggregated IgG (or IgM) bearing C3d and rheumatoid factor (RF) activity bound by anti-C3d. In addition, the material bound by anti-C3d Sepharose from most synovial fluid polyethylene glycol precipitates did not contain either IgM or IgG RF. Thus both techniques show that the majority of complexes bearing C3d do not contain RF. As the complement fixing aggregates apparently contain only immunoglobulin and complement components the results raise the problem of how the aggregates are formed. It is suggested that RA IgG may remain aggregated after either antigen or antibody (RF) has dissociated from the complex. Images PMID:3492971

  4. Aggregation of Antibody Drug Conjugates at Room Temperature: SAXS and Light Scattering Evidence for Colloidal Instability of a Specific Subpopulation.

    PubMed

    Frka-Petesic, B; Zanchi, D; Martin, N; Carayon, S; Huille, S; Tribet, C

    2016-05-17

    Coupling a hydrophobic drug onto monoclonal antibodies via lysine residues is a common route to prepare antibody-drug conjugates (ADC), a promising class of biotherapeutics. But a few chemical modifications on protein surface often increase aggregation propensity, without a clear understanding of the aggregation mechanisms at stake (loss of colloidal stability, self-assemblies, denaturation, etc.), and the statistical nature of conjugation introduces polydispersity in the ADC population, which raises questions on whether the whole ADC population becomes unstable. To characterize the average interactions between ADC, we monitored small-angle X-ray scattering in solutions of monoclonal IgG1 human antibody drug conjugate, with average degree of conjugation of 0, 2, or 3 drug molecules per protein. To characterize stability, we studied the kinetics of aggregation at room temperature. The intrinsic Fuchs stability ratio of the ADC was estimated from the variation over time of scattered light intensity and hydrodynamic radius, in buffers of varying pH, and at diverse sucrose (0% or 10%) and NaCl (0 or 100 mM) concentrations. We show that stable ADC stock solutions became unstable upon pH shift, well below the pH of maximum average attraction between IgGs. Data indicate that aggregation can be ascribed to a fraction of ADC population usually representing less than 30 mol % of the sample. In contrast to the case of (monodisperse) monoclonal antibodies, our results suggest that a poor correlation between stability and average interaction parameters should be expected as a corollary of dispersity of ADC conjugation. In practice, the most unstable fraction of the ADC population can be removed by filtration, which affects remarkably the apparent stability of the samples. Finally, the lack of correlation between the kinetic stability and variations of the average inter-ADC interactions is tentatively attributed to the uneven nature of charge distributions and the presence of

  5. Aggregation of Antibody Drug Conjugates at Room Temperature: SAXS and Light Scattering Evidence for Colloidal Instability of a Specific Subpopulation.

    PubMed

    Frka-Petesic, B; Zanchi, D; Martin, N; Carayon, S; Huille, S; Tribet, C

    2016-05-17

    Coupling a hydrophobic drug onto monoclonal antibodies via lysine residues is a common route to prepare antibody-drug conjugates (ADC), a promising class of biotherapeutics. But a few chemical modifications on protein surface often increase aggregation propensity, without a clear understanding of the aggregation mechanisms at stake (loss of colloidal stability, self-assemblies, denaturation, etc.), and the statistical nature of conjugation introduces polydispersity in the ADC population, which raises questions on whether the whole ADC population becomes unstable. To characterize the average interactions between ADC, we monitored small-angle X-ray scattering in solutions of monoclonal IgG1 human antibody drug conjugate, with average degree of conjugation of 0, 2, or 3 drug molecules per protein. To characterize stability, we studied the kinetics of aggregation at room temperature. The intrinsic Fuchs stability ratio of the ADC was estimated from the variation over time of scattered light intensity and hydrodynamic radius, in buffers of varying pH, and at diverse sucrose (0% or 10%) and NaCl (0 or 100 mM) concentrations. We show that stable ADC stock solutions became unstable upon pH shift, well below the pH of maximum average attraction between IgGs. Data indicate that aggregation can be ascribed to a fraction of ADC population usually representing less than 30 mol % of the sample. In contrast to the case of (monodisperse) monoclonal antibodies, our results suggest that a poor correlation between stability and average interaction parameters should be expected as a corollary of dispersity of ADC conjugation. In practice, the most unstable fraction of the ADC population can be removed by filtration, which affects remarkably the apparent stability of the samples. Finally, the lack of correlation between the kinetic stability and variations of the average inter-ADC interactions is tentatively attributed to the uneven nature of charge distributions and the presence of

  6. Comprehensive Analysis of the Therapeutic IgG4 Antibody Pembrolizumab: Hinge Modification Blocks Half Molecule Exchange In Vitro and In Vivo.

    PubMed

    Yang, Xiaoyu; Wang, Fengqiang; Zhang, Ying; Wang, Larry; Antonenko, Svetlana; Zhang, Shuli; Zhang, Yi Wei; Tabrizifard, Mohammad; Ermakov, Grigori; Wiswell, Derek; Beaumont, Maribel; Liu, Liming; Richardson, Daisy; Shameem, Mohammed; Ambrogelly, Alexandre

    2015-12-01

    IgG4 antibodies are evolving as an important class of cancer immunotherapies. However, human IgG4 can undergo Fab arm (half molecule) exchange with other IgG4 molecules in vivo. The hinge modification by a point mutation (S228P) prevents half molecule exchange of IgG4. However, the experimental confirmation is still expected by regulatory agencies. Here, we report for the first time the extensive analysis of half molecule exchange for a hinge-modified therapeutic IgG4 molecule, pembrolizumab (Keytruda) targeting programmed death 1 (PD1) receptor that was approved for advanced melanoma. Studies were performed in buffer or human serum using multiple exchange partners including natalizumab (Tysabri) and human IgG4 pool. Formation of bispecific antibodies was monitored by fluorescence resonance energy transfer, exchange with Fc fragments, mixed mode chromatography, immunoassays, and liquid chromatography-mass spectrometry. The half molecule exchange was also examined in vivo in SCID (severe combined immunodeficiency) mice. Both in vitro and in vivo results indicate that the hinge modification in pembrolizumab prevented half molecule exchange, whereas the unmodified counterpart anti-PD1 wt showed active exchange activity with other IgG4 antibodies or self-exchange activity with its own molecules. Our work, as an example expected for meeting regulatory requirements, contributes to establish without ambiguity that hinge-modified IgG4 antibodies are suitable for biotherapeutic applications.

  7. Rapid Assessment of Human Amylin Aggregation and Its Inhibition by Copper(II) Ions by Laser Ablation Electrospray Ionization Mass Spectrometry with Ion Mobility Separation

    PubMed Central

    Donaldson, Robert P.; Jeremic, Aleksandar M.; Vertes, Akos

    2015-01-01

    Native electrospray ionization (ESI) mass spectrometry (MS) is often used to monitor noncovalent complex formation between peptides and ligands. The relatively low throughput of this technique, however, is not compatible with extensive screening. Laser ablation electrospray ionization (LAESI) MS combined with ion mobility separation (IMS) can analyze complex formation and provide conformation information within a matter of seconds. Islet amyloid polypeptide (IAPP) or amylin, a 37-amino acid residue peptide, is produced in pancreatic beta-cells through proteolytic cleavage of its prohormone. Both amylin and its precursor can aggregate and produce toxic oligomers and fibrils leading to cell death in the pancreas that can eventually contribute to the development of type 2 diabetes mellitus. The inhibitory effect of the copper(II) ion on amylin aggregation has been recently discovered, but details of the interaction remain unknown. Finding other more physiologically tolerated approaches requires large scale screening of potential inhibitors. Here, we demonstrate that LAESI-IMS-MS can reveal the binding stoichiometry, copper oxidation state, and the dissociation constant of human amylin–copper(II) complex. The conformations of hIAPP in the presence of copper(II) ions were also analyzed by IMS, and preferential association between the β-hairpin amylin monomer and the metal ion was found. The copper(II) ion exhibited strong association with the –HSSNN– residues of the amylin. In the absence of copper(II), amylin dimers were detected with collision cross sections consistent with monomers of β-hairpin conformation. When copper(II) was present in the solution, no dimers were detected. Thus, the copper(II) ions disrupt the association pathway to the formation of β-sheet rich amylin fibrils. Using LAESI-IMS-MS for the assessment of amylin–copper(II) interactions demonstrates the utility of this technique for the high-throughput screening of potential inhibitors of

  8. Development of an HPLC assay to study the effect of endogenous and exogenous substances on heat-induced aggregation of human serum albumin.

    PubMed

    Saso, L; Casini, M L; Valentini, G; Mattei, E; Panzironi, C; Silvestrini, B

    1998-03-01

    The mechanism of action of non-steroidal anti-inflammatory drugs which are used in high doses in chronic inflammatory conditions is not clearly understood. Their known protein-stabilizing properties could play a significant role. The inhibition of cyclooxygenase may not be essential for their anti-rheumatic activity, since compounds with strong anti-denaturant properties and devoid of anti-inflammatory activity were shown to be effective in an experimental model of rheumatoid arthritis. Hence, to develop new anti-rheumatic drugs it is essential that a simple in vitro method to evaluate the anti-denaturant activity of endogenous and exogenous compounds is available. We developed a new assay, using gel permeation high performance liquid chromatography, to study the effect of endogenous and exogenous compounds on heat-induced aggregation of human serum albumin in conditions in which protein precipitation does not occur. Non-steroidal anti-inflammatory drugs like diclofenac, ibuprofen and naproxen inhibited the aggregation of albumin at low concentrations (EC50 10(-4)-10(-5) mol/l) comparable to those active in a classical turbidimetric method, whereas the effect of weak stabilizers, like sodium cloride and formic, fumaric, maleic, malonic, and succinic acid (EC50 10(-1)-10(-2) mol/l in the Mizushima test) was not detectable. Furthermore, the HPLC assay allowed the examination of a number of coloured substances, including bilirubin, which appeared to be a strong stabilizer of its physiological carrier, albumin. These data could be clinically relevant, since the drugs examined are used at very high doses in rheumatoid arthritis and related conditions, with plasma levels that could cause significant stabilization of serum albumin and perhaps other proteins.

  9. Fabrication and detection of tissue engineered bone aggregates based on encapsulated human ADSCs within hybrid calcium alginate/bone powder gel-beads in a spinner flask.

    PubMed

    Song, Kedong; Yang, Yanfei; Xu, Lili; Tian, Jiaxin; Fan, Jiangli; Jiao, Zeren; Feng, Shihao; Wang, Hong; Wang, Yiwei; Wang, Ling; Liu, Tianqing

    2016-05-01

    Traditional treatment for bone diseases limits their clinical application due to undesirable host immune rejection, limited donator sources and severe pain and suffering for patients. Bone tissue engineering therefore is expected to be a more effective way in treating bone diseases. In the present study, hybrid calcium alginate/bone powder gel-beads with a uniform size distribution, good biocompatibility and osteoinductive capability, were prepared to be used as an in-vitro niche-like matrix. The beads were optimized using 2.5% (w/v) sodium alginate solution, 4.5% (w/v) CaCl2 solution and 5.0mg/mL bone powder using an easy-to-use method. Human ADSCs were cultured and induced into chondrocytes and osteoblasts, respectively. The cells were characterized by histological staining showing the ADSCs were able to maintain their characteristic morphology with multipotent differentiation ability. ADSCs at density of 5 × 10(6)cells/mL were encapsulated into the gel-beads aiming to explore cell expansion under different conditions and the osteogenic induction of ADSCs was verified by specific staining. Results demonstrated that the encapsulated ADSCs expanded 5.6 folds in 10 days under dynamic condition via spinner flask, and were able to differentiate into osteoblasts (OBs) with extensive mineralized nodules forming the bone aggregates over 3 weeks postosteogenic induction. In summary, hybrid gel-beads encapsulating ADSCs are proved to be feasible as a new method to fabricate tissue engineered bone aggregation with potential to treat skeletal injury in the near future. PMID:26952485

  10. 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

  11. 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.

  12. 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

  13. The role of IgG subclass of mouse monoclonal antibodies in antibody-dependent enhancement of feline infectious peritonitis virus infection of feline macrophages.

    PubMed

    Hohdatsu, T; Tokunaga, J; Koyama, H

    1994-01-01

    Antibody-dependent enhancement (ADE) of feline infectious peritonitis virus (FIPV) infection was studied in feline alveolar macrophages and human monocyte cell line U937 using mouse neutralizing monoclonal antibodies (MAbs) directed to the spike protein of FIPV. Even among the MAbs that have been shown to recognize the same antigenic site, IgG 2a MAbs enhanced FIPV infection strongly, whereas IgG 1 MAbs did not. These IgG 2a MAbs enhanced the infection even when macrophages pretreated with the MAb were washed and then inoculated with the virus. Immunofluorescence flow cytometric analysis of the macrophages treated with each of the MAbs showed that the IgG 2a MAbs but not the IgG 1 MAbs bound to feline alveolar macrophages. Treatment of the IgG 2a MAb with protein A decreased the binding to the macrophages and, in parallel, diminished the ADE activity. Although no infection was observed by inoculation of FIPV to human monocyte cell line U937 cells, FIPV complexed with either the IgG 2a MAb or the IgG 1 MAb caused infection in U937 cells which are shown to express Fc gamma receptor (Fc gamma R) I and II that can bind mouse IgG 2a and IgG 1, respectively. These results suggest that the enhancing activity of MAb is closely correlated with IgG subclass and that the correlation is involved in binding of MAb to Fc gamma R on feline macrophage.

  14. Evaluation of intrathecal synthesis of IgG in neurocysticercosis.

    PubMed

    Minelli, César; Takayanagui, Osvaldo M

    2005-11-15

    Neurocysticercosis is a world public health problem. An increase in immunoglobulin G (IgG) concentration in the cerebrospinal fluid of patients with neurocysticercosis has been described but the reasons for this finding are unknown. Our hypothesis is that the increase in IgG concentration in cerebrospinal fluid is due to exclusive intrathecal synthesis of IgG and this process is associated with the inflammatory phases of the disease. We studied IgG concentration in cerebrospinal fluid in 16 patients with neurocysticercosis comparing with a control group of 19 patients to verify which pattern of increase in IgG concentration in cerebrospinal fluid occurs in neurocysticercosis. In the neurocysticercosis group, intrathecal synthesis of IgG was detected in 12 (75%) and 5 (31.2%) patients by quantitative and qualitative methods, respectively. When compared with the control group the neurocysticercosis patients had the same pattern of intrathecal synthesis of IgG as multiple sclerosis patients. Intrathecal synthesis of IgG was not associated with any variable indicative of an inflammatory process. We conclude that the increase in IgG concentration in neurocysticercosis is due to intrathecal synthesis, as is also the case for multiple sclerosis, and that this process is not related to the inflammatory stage of NCC.

  15. Taurine and platelet aggregation

    SciTech Connect

    Nauss-Karol, C.; VanderWende, C.; Gaut, Z.N.

    1986-03-01

    Taurine is a putative neurotransmitter or neuromodulator. The endogenous taurine concentration in human platelets, determined by amino acid analysis, is 15 ..mu..M/g. In spite of this high level, taurine is actively accumulated. Uptake is saturable, Na/sup +/ and temperature dependent, and suppressed by metabolic inhibitors, structural analogues, and several classes of centrally active substances. High, medium and low affinity transport processes have been characterized, and the platelet may represent a model system for taurine transport in the CNS. When platelets were incubated with /sup 14/C-taurine for 30 minutes, then resuspended in fresh medium and reincubated for one hour, essentially all of the taurine was retained within the cells. Taurine, at concentrations ranging from 10-1000 ..mu..M, had no effect on platelet aggregation induced by ADP or epinephrine. However, taurine may have a role in platelet aggregation since 35-39% of the taurine taken up by human platelets appears to be secreted during the release reaction induced by low concentrations of either epinephrine or ADP, respectively. This release phenomenon would imply that part of the taurine taken up is stored directly in the dense bodies of the platelet.

  16. The global landscape of cognition: hierarchical aggregation as an organizational principle of human cortical networks and functions

    PubMed Central

    Taylor, P.; Hobbs, J. N.; Burroni, J.; Siegelmann, H. T.

    2015-01-01

    Though widely hypothesized, limited evidence exists that human brain functions organize in global gradients of abstraction starting from sensory cortical inputs. Hierarchical representation is accepted in computational networks, and tentatively in visual neuroscience, yet no direct holistic demonstrations exist in vivo. Our methods developed network models enriched with tiered directionality, by including input locations, a critical feature for localizing representation in networks generally. Grouped primary sensory cortices defined network inputs, displaying global connectivity to fused inputs. Depth-oriented networks guided analyses of fMRI databases (~17,000 experiments;~1/4 of fMRI literature). Formally, we tested whether network depth predicted localization of abstract versus concrete behaviors over the whole set of studied brain regions. For our results, new cortical graph metrics, termed network-depth, ranked all databased cognitive function activations by network-depth. Thus, we objectively sorted stratified landscapes of cognition, starting from grouped sensory inputs in parallel, progressing deeper into cortex. This exposed escalating amalgamation of function or abstraction with increasing network-depth, globally. Nearly 500 new participants confirmed our results. In conclusion, data-driven analyses defined a hierarchically ordered connectome, revealing a related continuum of cognitive function. Progressive functional abstraction over network depth may be a fundamental feature of brains, and is observed in artificial networks. PMID:26669858

  17. Pressure- and temperature-induced unfolding and aggregation of recombinant human interferon-gamma: a Fourier transform infrared spectroscopy study.

    PubMed Central

    Goossens, Koen; Haelewyn, Joost; Meersman, Filip; De Ley, Marc; Heremans, Karel

    2003-01-01

    The effect of hydrostatic pressure on the secondary structure of recombinant human interferon-gamma (rhIFN-gamma) and its biologically inactive truncated form rhIFN-Delta C15 has been studied using Fourier-transform IR (FTIR) spectroscopy. In situ observation of the pressure-induced changes using the diamond anvil cell shows that the alpha-helical structure is mainly transformed into disordered structure at high pressure. Increasing pressure also induces the formation of a gel. Addition of 0.5 M MgCl(2) significantly reduces the pressure stability. Releasing the pressure below 300 MPa results in the formation of intermolecular antiparallel beta-sheets, which is seldom observed. This suggests that the intermolecular beta-sheet of rhIFN-gamma is stabilized by electrostatic interactions that are disrupted at high pressure. For comparison we also studied the effect of temperature. Temperature-induced changes reflect extensive transformation of alpha-helical structure into intermolecular antiparallel beta-sheet, as is usually observed for most proteins. PMID:12425720

  18. Acetal phosphatidic acids: novel platelet aggregating agents.

    PubMed

    Brammer, J P; Maguire, M H; Walaszek, E J; Wiley, R A

    1983-05-01

    1 Palmitaldehyde, olealdehyde and linolealdehyde acetal phosphatidic acids induced rapid shape change and dose-dependent biphasic aggregation of human platelets in platelet-rich plasma; aggregation was reversible at low doses and irreversible at high doses of the acetal phosphatidic acids. The palmitaldehyde congener elicited monophasic dose-dependent aggregation of sheep platelets in platelet-rich plasma.2 The threshold concentration for palmitaldehyde acetal phosphatidic acid (PGAP)-induced platelet aggregation was 2.5-5 muM for human platelets and 0.25-0.5 muM for sheep platelets. PGAP was 4-5 times as potent versus human platelets as the olealdehyde and linolealdehyde acetal phosphatidic acids, which were equipotent.3 PGAP-induced irreversible aggregation of [(14)C]-5-hydroxytryptamine ([(14)C]-5-HT)-labelled human platelets in platelet-rich plasma was accompanied by release of 44.0+/-2.4% (s.e.) of the platelet [(14)C]-5-HT; reversible aggregation was not associated with release. In contrast, PGAP-induced release of [(14)C]-5-HT-labelled sheep platelets was dose-dependent.4 The adenosine diphosphate (ADP) antagonist, 2-methylthio-AMP, and the cyclo-oxygenase inhibitor, aspirin, abolished PGAP-induced second phase aggregation and release in human platelets but did not affect the first, reversible, phase of aggregation. Both the first and second phases of PGAP-induced aggregation were abolished by chlorpromazine, by the phospholipase A(2) inhibitor, mepacrine, and by nmolar concentrations of prostaglandin E(1) (PGE(1)); these agents abolished the second, but not the first phase of ADP-induced aggregation.5 The related phospholipids, lecithin, lysolecithin and phosphatidic acid, at <100 muM, neither induced aggregation of human platelets in platelet-rich plasma, nor modified PGAP-induced aggregation; 1-palmityl lysophosphatidic acid elicited aggregation of human platelets at a threshold concentration of 100 muM.6 It is concluded that the acetal phosphatidic acids

  19. Removal of terminal alpha-galactosyl residues from xenogeneic porcine endothelial cells. Decrease in complement-mediated cytotoxicity but persistence of IgG1-mediated antibody-dependent cell-mediated cytotoxicity.

    PubMed

    Watier, H; Guillaumin, J M; Piller, F; Lacord, M; Thibault, G; Lebranchu, Y; Monsigny, M; Bardos, P

    1996-07-15

    To determine the role of the terminal alpha-galactosyl residue in the endothelial damage mediated by human xenoreactive natural antibodies (IgM and IgG), we treated porcine endothelial cells in culture with green coffee bean alpha-galactosidase. A practically complete removal of terminal alpha-Gal residues (as evaluated by flow cytometry with Bandeiraea simplicifolia isolectin B4) and concomitant exposure of N-acetyllactosamine were obtained without altering cell viability. A dramatic decrease in IgM and IgG binding (from a pool of human sera) was observed, confirming the key role of the alpha-galactosyl residues. The enzyme treatment did not induce any nonspecific immunoglobulin binding sites, but led to the exposure of new epitopes for a minor fraction of IgM. The main residual IgM and IgG binding could be due to xenoantigens other than the alpha-galactosyl residues. When alpha-galactosidase-treated endothelial cells were used as targets in cytotoxicity experiments, they were less susceptible than untreated cells to complement-mediated cytotoxicity induced by fresh human serum. In contrast, they did not acquire resistance to human IgG-dependent cellular cytotoxicity, despite the decrease in IgG binding. Because it is known that antibody-dependent cytotoxicity mediated by CD16+ NK cells is dependent on IgG1 and IgG3, and not on IgG2 or IgG4, which was confirmed by blocking experiments, we studied the binding of all four subclasses to intact and alpha-galactosidase-treated endothelial cells. Two major subclasses, IgG1 and IgG2, bound to untreated endothelial cells, whereas IgG3 binding was low and IgG4 binding was negligible. A decrease in IgG1, IgG2, and IgG3 binding was observed upon alpha-galactosidase treatment, indicating that antibodies belonging to these three subclasses recognize alpha-galactosyl residues. The decrease in IgG2 binding was more pronounced than the decrease in IgG1 binding. Collectively, these data indicate that IgG1 xenoreactive natural

  20. Microstructural evolution of lipid aggregates in nucleating model and human biles visualized by cryogenic transmission electron microscopy.

    PubMed

    Konikoff, F M; Danino, D; Weihs, D; Rubin, M; Talmon, Y

    2000-02-01

    Obtaining reliable information on the physical state and ultrastructure of bile is difficult because of its mixed aqueous-lipid composition and thermodynamic metastability. We have used time-lapse cryogenic transmission electron microscopy (cryo-TEM) combined with video-enhanced light microscopy (VELM) to study microstructural evolution in nucleating bile. A well-characterized model bile and gallbladder biles from cholesterol and pigment gallstone patients were studied sequentially during cholesterol nucleation and precipitation. In model bile, cholesterol crystallization was preceded by the appearance of the following distinct microstructures: spheroidal micelles (3-5 nm), discoidal membrane patches (50-150 nm) often in multiple layers (2-10), discs (50-100 nm), and unilamellar (50-200 nm) and larger multilamellar vesicles (MLVs). The membrane patches and discs appeared to be short-lived intermediates in a micelle-to-vesicle transition. Vesicular structures formed by growth and closure of patches as well as by budding off from vesicles with fewer bilayers. MLVs became more abundant, uniform, and concentric as a function of time. In native bile, all the above microstructures, except discoidal membrane patches, were observed. However, native MLVs were more uniform and concentric from the beginning. When cholesterol crystals appeared by light microscopy, MLVs were always detected by cryo-TEM. Edges of early cholesterol crystals were lined up with micelles and MLVs in a way suggesting an active role in feeding crystal growth from these microstructures. These findings, for the first time documented by cryo-TEM in human bile, provide a microstructural framework that can serve as a basis for investigation of specific factors that influence biliary cholesterol nucleation and crystal formation.

  1. Acetyl eugenol, a component of oil of cloves (Syzygium aromaticum L.) inhibits aggregation and alters arachidonic acid metabolism in human blood platelets.

    PubMed

    Srivastava, K C; Malhotra, N

    1991-01-01

    In continuation of our studies with the oil of cloves--a common kitchen spice and a crude drug for home medicine--we have isolated yet another active component identified as acetyl eugenol (AE); the earlier reported active component being eugenol. The isolated material (IM) was found to be a potent platelet inhibitor; IM abolished arachidonate (AA)-induced aggregation at ca. 12 microM, a concentration needed to abolish the second phase of adrenaline-induced aggregation. Chemically synthesized acetyl eugenol showed similar effects on AA- and adrenaline-induced aggregation. A dose-dependent inhibition of collagen-induced aggregation was also observed. AE did not inhibit either calcium ionophore A23187- or thrombin-induced aggregation. Studies on aggregation and ATP release were done using whole blood (WB). AA-induced aggregation in WB was abolished at 3 micrograms/ml (14.6 microM) which persisted even after doubling the concentration of AA. ATP release was inhibited. Inhibition of aggregation appeared to be mediated by a combination of two effects: reduced formation of thromboxane and increased generation of 12-lipoxygenase product (12-HPETE). These effects were observed by exposing washed platelets to (14C)AA or by stimulating AA-labelled platelets with ionophore A23187. Acetyl eugenol inhibited (14C)TxB2 formation in AA-labelled platelets on stimulation with thrombin. AE showed no effect on the incorporation of AA into platelet phospholipids. PMID:2011614

  2. The association between naturally acquired IgG subclass specific antibodies to the PfRH5 invasion complex and protection from Plasmodium falciparum malaria.

    PubMed

    Weaver, Rupert; Reiling, Linda; Feng, Gaoqian; Drew, Damien R; Mueller, Ivo; Siba, Peter M; Tsuboi, Takafumi; Richards, Jack S; Fowkes, Freya J I; Beeson, James G

    2016-01-01

    Understanding the targets and mechanisms of human immunity to malaria is important for advancing the development of highly efficacious vaccines and serological tools for malaria surveillance. The PfRH5 and PfRipr proteins form a complex on the surface of P. falciparum merozoites that is essential for invasion of erythrocytes and are vaccine candidates. We determined IgG subclass responses to these proteins among malaria-exposed individuals in Papua New Guinea and their association with protection from malaria in a longitudinal cohort of children. Cytophilic subclasses, IgG1 and IgG3, were predominant with limited IgG2 and IgG4, and IgG subclass-specific responses were higher in older children and those with active infection. High IgG3 to PfRH5 and PfRipr were significantly and strongly associated with reduced risk of malaria after adjusting for potential confounding factors, whereas associations for IgG1 responses were generally weaker and not statistically significant. Results further indicated that malaria exposure leads to the co-acquisition of IgG1 and IgG3 to PfRH5 and PfRipr, as well as to other PfRH invasion ligands, PfRH2 and PfRH4. These findings suggest that IgG3 responses to PfRH5 and PfRipr may play a significant role in mediating naturally-acquired immunity and support their potential as vaccine candidates and their use as antibody biomarkers of immunity. PMID:27604417