Seroprevalence of Toxoplasma gondii Infection among HIV/AIDS Patients in Eastern China

Toxoplasmosis, a neglected tropical disease caused by the protozoan parasite Toxoplasma gondii, occurs throughout the world. Human T. gondii infection is asymptomatic in 80% of the population; however, the infection is life-threatening and causes substantial neurologic damage in immunocompromised patients such as HIV-infected persons. The major purpose of this study was to investigate the seroprevalence of T. gondii infection in subjects infected with HIV/AIDS in eastern China. Our findings showed 9.7% prevalence of anti-T. gondii IgG antibody in HIV/AIDS patients, which was higher than in intravenous drug users (2.2%) and healthy controls (4.7%), while no significant difference was observed in the seroprevalence of anti-Toxoplasma IgM antibody among all participants (P>0.05). Among all HIV/AIDS patients, 15 men (7.7%) and 10 women (15.9%) were positive for anti-T. gondii IgG antibody; however, no significant difference was detected in the seroprevalence of anti-Toxoplasma IgG antibody between males and females. The frequency of anti-Toxoplasma IgG antibody was 8.0%, 13.2%, 5.5%, and 0% in patients with normal immune function (CD4⁺ T-lymphocyte count ≥500 cells/ml), immunocompromised patients (cell count ≥200 and <500 cells/ml), severely immunocompromised patients (cell count ≥50 and <200 cells/ml), and advanced AIDS patients, respectively (cell count <50 cells/ml), while only 3 immunocompromised patients were positive for anti-T. gondii IgM antibody. The results indicate a high seroprevalence of T. gondii infection in HIV/AIDS patients in eastern China, and a preventive therapy for toxoplasmosis may be given to HIV/AIDS patients based on CD4⁺ T lymphocyte count.     

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.     

Cryoprotection of antibody by organic solutes and organic solute/divalent cation mixtures

Antibodies are globular glycoproteins that protect animals from microbial and toxic insult. These proteins have proven to have substantial commercial and research value but are variably susceptible to freeze-thaw damage, thus limiting their usefulness. Several carbohydrates and divalent cations were examined alone and in combination to determine whether they could protect antibody from freeze-thaw damage. The amino acid proline was also tested. Two antibodies, derived from different sources and specific for different antigens, were tested by a direct enzyme-linked immunosorbent assay (ELISA). Confirmation of antibody freeze-lability was obtained by rocket electrophoresis and radial immunodiffusion tests. Neither carbohydrate nor divalent cation alone fully protected antibody activity from freeze-thaw damage. However, several combinations protected antibody activity completely when compared to their effect on antibody maintained at room temperature. In the case of affinity-purified antibody, full protection of antibody activity relative to an untreated control was obtained. In several instances, cryoprotection of antibody by solute-divalent cation combinations was synergistic and not an additive effect of each component. Alkaline phosphatase, an enzyme typically linked to antibody for an ELISA, was not freeze-thaw labile. These results indicate that antibody function can be fully protected from damage due to freeze-thaw treatment by organic solutes in combination with divalent cations.     

Untoward effects associated with practolol: demonstration of antibody binding to epithelial tissue.

An antibody which sticks to the intercellular region of xenogenic epidermal tissue has been shown by indirect immunofluorescence to be present in the serum of patients with practolol-induced eye damage. These antibodies and those found in patients with pemphigus were compared for their ability to bind to isolated epidermal cells. Binding was achieved only with the pemphigus antibody, which suggests that it may have a different specificity from the antibody associated with practolol-induced eye damage.     

Highly parallel characterization of IgG Fc binding interactions

Because the variable ability of the antibody constant (Fc) domain to recruit innate immune effector cells and complement is a major factor in antibody activity in vivo, convenient means of assessing these binding interactions is of high relevance to the development of enhanced antibody therapeutics, and to understanding the protective or pathogenic antibody response to infection, vaccination, and self. Here, we describe a highly parallel microsphere assay to rapidly assess the ability of antibodies to bind to a suite of antibody receptors. Fc and glycan binding proteins such as FcγR and lectins were conjugated to coded microspheres and the ability of antibodies to interact with these receptors was quantified. We demonstrate qualitative and quantitative assessment of binding preferences and affinities across IgG subclasses, Fc domain point mutants, and antibodies with variant glycosylation. This method can serve as a rapid proxy for biophysical methods that require substantial sample quantities, high-end instrumentation, and serial analysis across multiple binding interactions, thereby offering a useful means to characterize monoclonal antibodies, clinical antibody samples, and antibody mimics, or alternatively, to investigate the binding preferences of candidate Fc receptors.     

Immunochemical detection of glyoxal DNA damage.

The relevance of reactive oxygen species (ROS) in the pathogenesis of inflammatory diseases is widely documented. Immunochemical detection of ROS DNA adducts has been developed, however, recognition of glyoxal-DNA adducts has not previously been described. We have generated a polyclonal antibody that has shown increased antibody binding to ROS-modified DNA in comparison to native DNA. In addition, dose-dependent antibody binding to DNA modified with ascorbate alone was shown, with significant inhibition by desferrioxamine, catalase, and ethanol. Minimal inhibition was observed with uric acid, 1,10-phenanthroline and DMSO. However, antibody binding in the presence of EDTA increased 3500-fold. The involvement of hydrogen peroxide and hydroxyl radical in ascorbate-mediated DNA damage is consistent with ascorbate acting as a reducing agent for DNA-bound metal ions. Glyoxal is known to be formed during oxidation of ascorbate. Glyoxylated DNA, that previously had been proposed as a marker of oxidative damage, was recognised in a dose dependent manner using the antibody. We describe the potential use of our anti-ROS DNA antibody, that detects predominantly Fenton-type mediated damage to DNA and report on its specificity for the recognition of glyoxal-DNA adducts.     

Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis

Investigation of events committing cells to death revealed that a concealed NH2-terminal epitope of the pro-apoptotic protein Bak became exposed in vivo before apoptosis. This occurred after treatment of human Jurkat or CEM-C7A T-lymphoma cells with the mechanistically disparate agents staurosporine, etoposide or dexamethasone. The rapid, up to 10-fold increase in Bak-associated immunofluorescence was measured with epitope-specific monoclonal antibodies using flow cytometry and microscopy. In contrast, using a polyclonal antibody to Bak, immunofluorescence was detected both before and after treatment. There were no differences in Bak protein content nor in subcellular location before or after treatment. Immunofluorescence showed Bcl-xL and Bak were largely associated with mitochondria and in untreated cells they coimmunoprecipitated in the presence of nonioinic detergent. This association was significantly decreased after cell perturbation suggesting that Bcl-xL dissociation from Bak occurred on exposure of Bak's NH2 terminus. Multiple forms of Bak protein were observed by two dimensional electrophoresis but these were unchanged by inducers of apoptosis. This indicated that integration of cellular damage signals did not take place directly on the Bak protein. Release of proteins, including Bcl-xL, from Bak is suggested to be an important event in commitment to death.     

The establishment of human anti-TROP2 antibody IgG and its inhibition function on pancreatic cancer cell proliferation

On the basis of anti-TROP2 Fab antibody, this study seek to construct a eukaryotic expression system of human anti-TROP2 antibody IgG, and to analyse the inhibition function of human anti-TROP2 antibody IgG in the cell proliferation of pancreatic cancer. The heavy and light chain genes of anti-TROP2 antibody were amplified respectively to establish the recombinant expression vector of human anti-TROP2 antibody IgG, named pWS-anti-TROP2. The human anti-TROP2 antibody IgG was obtained through transfecting the plasmids into the CHO dhfr^- cell line, selecting the monoclonal cell strains with high amounts of antibody expression by MTX screening and applying Protein G affinity in purification. The identification and immunologic activity of human anti-TROP2 antibody IgG were researched by Western Blot,SDS-PAGE, ELISA, immunofluorescence assay and flow cytometry method (FCM). MTT assay was conducted to analyse the inhibition effect of human anti-TROP2 antibody IgG on BxPC3 cell proliferation. The human anti-TROP2 antibody IgG eukaryotic expression system was established successfully to express human anti-TROP2 antibody IgG, in which the molecular weight of heavy chain and light chain were consistent with expectation, and it could specifically combine with TROP2 protein, the antibody titer reached 1:6,400. The MTT assay results indicated that human anti-TROP2 antibody IgG had a significant effect on inhibiting the proliferation of BxPC3 cell, and the inhibition function can be gradually increased with improved antibody dose and prolonged time. In the study, the human anti-TROP2 antibody IgG eukaryotic expression system was constructed successfully, the antibody could specifically bind to TROP2 protein on the surface of pancreatic cancer cells, and it is shown to have a significant inhibitory action in pancreatic cancer cell proliferation.     

Toll-like receptor 9 ligands enhance mesenchymal stem cell invasion and expression of matrix metalloprotease-13

Human mesenchymal stem cells (hMSCs) are multipotent cells that are found in the bone marrow. Inflammation and tissue damage mobilize MSCs and induce their migration towards the damaged site through mechanisms that are not well defined. Toll-like receptor-9 (TLR9) is a cellular receptor for microbial and vertebrate DNA. Stimulation of TLR9 induces inflammatory and invasive responses in TLR9-expressing cells. We studied here the expression of TLR9 in human MSCs and the effects of synthetic TLR9-agonists on their invasion. Constitutive expression of TLR9 was detected in human MSCs but the expression was suppressed when MSCs were induced to differentiate into osteoblasts. Using standard invasion assays and a novel organotypic culture model based on human myoma tissue, we discovered that stimulation with the TLR9 agonistic, CpG oligonucleotides increased the invasion capacity of undifferentiated MSCs. Simultaneously, an increase in MMP-13 synthesis and activity was detected in the CpG-activated MSCs. Addition of anti-MMP-13 antibody significantly diminished the CpG-induced hMSC invasion. We conclude that treatment with TLR9-ligands increases MSC invasiveness, and this process is at least partially MMP-13-mediated.     

Production of antisera specific to aldosterone: effect of hapten density and of carrier proteins

In order to investigate the influence of hapten density and of carrier proteins on the immunological characteristics of antisera, 4 groups of rabbits were injected with different aldosterone-carboxymethoxime protein conjugates. Six animals immunized with an aldosterone rabbit serum albumin (RSA) conjugate carrying 15 steroid molecules (RSA-2 conjugate) showed markedly higher antibody titers than rabbits injected with a RSA conjugate carrying 8 aldosterone molecules (RSA-1 conjugate). Low antibody titers were found in 8 animals immunized with an aldosterone bovine gamma globulin (BGG) conjugate showing a molar incorporation of 15. In a group of rabbits which was first injected with the RSA-1 conjugate and re-immunized with the RSA-2 conjugate the magnitude of antibody production was not enhanced. No differences in antibody sensitivity or specificity were observed between the 4 groups. It was concluded from these experiments a) that the density of haptenic groups depending on the molar incorporation of haptens and on the molecular weight of the carrier protein had influenced the magnitude of antibody production, b) that hapten density or carrier proteins had no effect on antibody sensitivity or specificity, c) that the magnitude of antibody production cannot be altered by re-immunizing with a more potent antigen.     

Functional intracellular antibody fragments do not require invariant intra-domain disulfide bonds

Intracellular antibody fragments that interfere with molecular interactions inside cells are valuable in investigation of interactomes and in therapeutics, but their application demands that they function in the reducing cellular milieu. We show here a 2.7-Å crystal structure of intracellular antibody folds based on scaffolds developed from intracellular antibody capture technology, and we reveal that there is no structural or functional difference with or without the intra-domain disulfide bond of the variable domain of heavy chain or the variable domain of light chain. The data indicate that, in the reducing in vivo environment, the absence of the intra-domain disulfide bond is not an impediment to correction of antibody folding or to interaction with antigen. Thus, the structural constraints for in-cell function are intrinsic to variable single-domain framework sequences, providing a generic scaffold for isolation of functional intracellular antibody single domains.     

Natural IgG antibody with anti-β-galactosyl specificity suppressed hepatoma cell invasion in culture

The effect of natural IgG antibody recognizing β-galactosyl epitope on hepatoma cell invasion was investigated. Anti-β-galactosyl antibody dose-dependently suppressed hepatoma invasion underneath primarily cultured mesothelial cells monolayer without affecting the proliferation, to the same extent as natural IgG antibody with anti-α-galactosyl specificity, which had already been reported to have an anti-metastatic activity. The inhibitory effect of anti-β-galactosyl antibody was completely canceled by adding lactose (galactose-β-1, 4-glucose) to the medium, indicating that this antibody recognized some antigens with β-galactosyl epitope. Hepatoma cells pretreated with this antibody for 48 h showed reduced invasive activity, while the pretreatment of mesothelial cells with the antibody did not affect hepatoma cells invasion. Anti-β-galactosyl antibody also suppressed hepatoma cells adhesion to mesothelial cells monolayer. These results suggest that natural antibody with anti-β-galactosyl specificity may recognize the β-galactosyl epitope in some adhesion-related molecules on hepatoma cells, thus suppressing adhesion and invasion to mesothelial cells monolayer. These results suggest possible therapeutic uses of this antibody in the treatment of metastatic tumors.     

Critical contribution of VH-VL interaction to reshaping of an antibody: the case of humanization of anti-lysozyme antibody, HyHEL-10

To clarify the effects of humanizing a murine antibody on its specificity and affinity for its target, we examined the interaction between hen egg white lysozyme (HEL) and its antibody, HyHEL-10 variable domain fragment (Fv). We selected a human antibody framework sequence with high homology, grafted sequences of six complementarity-determining regions of murine HyHEL-10 onto the framework, and investigated the interactions between the mutant Fvs and HEL. Isothermal titration calorimetry indicated that the humanization led to 10-fold reduced affinity of the antibody for its target, due to an unfavorable entropy change. Two mutations together into the interface of the variable domains, however, led to complete recovery of antibody affinity and specificity for the target, due to reduction of the unfavorable entropy change. X-ray crystallography of the complex of humanized antibodies, including two mutants, with HEL demonstrated that the complexes had almost identical structures and also paratope and epitope residues were almost conserved, except for complementary association of variable domains. We conclude that adjustment of the interfacial structures of variable domains can contribute to the reversal of losses of affinity or specificity caused by humanization of murine antibodies, suggesting that appropriate association of variable domains is critical for humanization of murine antibodies without loss of function.     

Antibody informatics for drug discovery

More and more antibody therapeutics are being approved every year, mainly due to their high efficacy and antigen selectivity. However, it is still difficult to identify the antigen, and thereby the function, of an antibody if no other information is available. There are obstacles inherent to the antibody science in every project in antibody drug discovery. Recent experimental technologies allow for the rapid generation of large-scale data on antibody sequences, affinity, potency, structures, and biological functions; this should accelerate drug discovery research. Therefore, a robust bioinformatic infrastructure for these large data sets has become necessary. In this article, we first identify and discuss the typical obstacles faced during the antibody drug discovery process. We then summarize the current status of three sub-fields of antibody informatics as follows: (i) recent progress in technologies for antibody rational design using computational approaches to affinity and stability improvement, as well as ab-initio and homology-based antibody modeling; (ii) resources for antibody sequences, structures, and immune epitopes and open drug discovery resources for development of antibody drugs; and (iii) antibody numbering and IMGT. Here, we review “antibody informatics,” which may integrate the above three fields so that bridging the gaps between industrial needs and academic solutions can be accelerated. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Establishment of a monoclonal antibody recognizing ultraviolet light-induced (6-4) photoproducts

We obtained a monoclonal antibody directed against UV-induced DNA damage. Analysis of the antigenic determinant in UV-irradiated DNA recognized by this antibody, 64M-1, revealed that it bound UV-irradiated oligo- or poly-nucleotides containing thymine-thymine or thymine-cytosine sequences. The antibody failed to bind DNA irradiated with 313 nm UV in the presence of acetophenone, which contained predominantly thymine dimers as DNA damage. The binding activity of this antibody to 254-nm UV-irradiated DNA decreased with 313-nm UV irradiation, and the decrease of this binding activity correlated with the decrease of fluorescence corresponding to (6-4) photoproducts. These results suggest that the antigenic determinant recognized by this monoclonal antibody is a (6-4) photoproduct. Using autoradiography with 3H-antibody, we could detect the formation of the (6-4) photoproduct in individual human cells irradiated with 254-nm UV doses as low as 20 J/m2.     

Measuring the impact of Leptoglossus occidentalis (Heteroptera: Coreidae) on seed production in lodgepole pine using an antibody-based assay

We measured the impact of Leptoglossus occidentalis on seed production in lodgepole pine, Pinus contorta variety latifolia Engelmann, using an antibody marker developed to detect residual saliva in fed-on seeds. Nymphs, adult females, and adult males were caged on cones during early, mid- and late season cone development. Individual analysis of 12,887 seeds extracted from 365 cones revealed that 37.3% seeds tested positive for seed bug saliva. The antibody assay was 38 times more effective than radiography at detecting seed bug damage. Radiography can detect partially emptied seed but cannot discriminate between aborted seeds and those emptied by seed bugs. The antibody marker was least sensitive in detecting early season damage compared with mid- and late season damage. We hypothesize that residual saliva in seeds fed on early in the season was either absorbed by the damaged seed or degraded over time. Early season feeding resulted in the greatest number of seeds fused to cone scales and the extraction efficiency for cones exposed to feeding during this time was reduced by 64% compared with control cones. Adding fused seeds to antibody-positive seeds raised the proportion of damaged seeds to 48.3%. At all stages of cone development, adult females were the most destructive life stage, damaging up to two seeds per day late in the season. When seed losses were adjusted to damage per degree-day, female damage was greatest early in the season, while males caused the same amount of damage regardless of cone development period. The results of the antibody assay provide baseline data for developing damage prediction formulae, and establish L. occidentalis as a potentially serious pest in lodgepole pine seed orchards.     

Antibody buffering in the brain

The efficacy of chemotherapy on brain tumors is often hindered by the presence of the blood brain barrier. This barrier keeps many systemically administered substances from entering the cerebrospinal fluid (CSF), while allowing intrathecally administered drugs free passage out of that compartment. Therefore, achieving a therapeutic concentration of a cell cycle inhibitor in the CSF for a time long enough to have a cytotoxic effect on slow-growing tumor cells has proven difficult. The ability of an antibody to prolong ligand half-life and bioactivity has been previously described occurring in the plasma. This phenomenon has not yet been described or exploited for use in the CSF compartment. Antibodies often have a longer residence time in the CSF than small-molecule drugs, so antibody buffering, administration of a drug with its specific antibody, can prolong the bioactive lifetime of a drug in the CSF. Here we describe antibody buffering of the small molecule hapten 2-phenyl-oxazol-5-one-methylene-gamma-amino butyrate in the CSF of a rats. Not only does the presence of an antibody buffer increase the half-life of both total and free hapten in the CSF, but the antibody can be re-charged in situ with fresh hapten, even days after the initial antibody infusion. Antibody buffering may provide a viable option for delivering a stable, bio-available concentration of a drug that is normally rapidly eliminated from the CSF.     

Viral fitness can influence the repertoire of virus variants selected by antibodies

Minority genomes in the mutant spectra of viral quasispecies may differ in relative fitness. Here, we report experiments designed to evaluate the contribution of relative fitness to selection by a neutralizing monoclonal antibody (mAb). We have reconstructed a foot-and-mouth disease virus (FMDV) quasispecies, with two matched pairs of distinguishable mAb-escape mutants as minority genomes of the mutant spectrum. Each mutant of a pair differs from the other by 11-fold or 33-fold in relative fitness. Analysis of the mutant spectra of virus populations selected with different concentrations of antibody in infections in liquid culture medium has documented a dominance of the high fitness counterpart in the selected population. Plaque development as a function of increasing concentration of the antibody has shown that each mutant of a matched pair yielded the same number of plaques, although the high fitness mutant required less time for plaque formation, and attained a larger plaque size at any given time-point. This result documents equal intrinsic resistance to the antibody of each mutant of a matched pair, confirming previous biochemical, structural, and genetic studies, which indicated that the epitopes of each mutant pair were indistinguishable regarding reactivity with the monoclonal antibody. Thus, relative viral fitness can influence in a significant way the repertoire of viral mutants selected from a viral quasispecies by a neutralizing antibody. We discuss the significance of these results in relation to antibody selection, and to other selective forces likely encountered by viral quasispecies in vivo.     

Characterization of a monoclonal antibody to thymidine glycol monophosphate

A monoclonal antibody specific for thymine glycol (TG) in irradiated or OsO4-treated DNA was obtained by immunizing with thymidine glycol monophosphate (TMP-glycol) conjugated to bovine serum albumin by a carbodiimide procedure. Screening by dot-immunobinding and enzyme-linked immunosorbant assay (ELISA) procedures gave eight clones that bound OsO4- treated DNA. One of them, 2.6F.6B.6C, an IgG2a kappa, was characterized further. Hapten inhibition studies with OsO4-treated DNA showed that the antibody was specific for TMP-glycol. Among the various inhibitors tested, inhibition was in the order TMP-glycol greater than 5,6-dihydrothymidine phosphate greater than TMP greater than thymidine glycol greater than TG. Inhibition by 5,6-dihydrothymidine, thymidine, thymine, AMP, and CMP was negligible. In OsO4-treated DNA, as few as 0.5 TG per 10,000 bp were detectable by direct ELISA. Inhibition assays could detect as few as 1.5 TG per 10,000 bp. The antibody was equally reactive with native or denatured DNA containing TG. Among the X-irradiated homopolymers dC, dA, dG, and dT, only dT reacted with the antibody. Using an ELISA, the antibody could detect damage in irradiated DNA at the level of 20 Gy. Thus the antibody is of potential use in assays for DNA damage caused by X rays or other agents that damage DNA by free radical interactions.     

IgG antibody responses in early experimental sparganosis and IgG subclass responses in human sparganosis

Antigenic components in the crude extracts of Spirometra mansoni plerocercoid were analyzed in early experimental infections and in IgG subclass observed in clinical sparganosis. By IgG immunoblot, sera obtained serially from experimental mice, fed 5 spargana each, were reacted with the crude extracts. Protein bands at 36-26 kDa and 103 kDa showed positive reactions since two weeks after infection. In a differential immunoblot, in which a monospecific antibody against sparganum chymase at 36 kDa was pre-treated, the reactions at 36-26 kDa disappeared, indicating that the sparganum chymase and its degradation products invoked IgG antibody reactions. When 69 patients sera of human sparganosis were examined for their IgG subclass responses, IgG4 levels showed the highest reaction which was followed by IgG1. The IgG4 antibody also reacted mainly with 36-31 kDa protease. These results indicate that 36 kDa chymase of S. mansoni plerocercoid is the main antigenic component inducing IgG antibody response in early stage of experimental sparganosis and for specific IgG subclass reactions in human sparganosis.