Studies of soluble human catalytic antibody with glutathione peroxidase activity

By screening the phage-displayed human single chain antibody library, we have got the specific single chain antibody bound to GSH-S-DNP butyl ester as the hapten. The tertiary structure of the protein was analyzed with the aid of computer, and the results showed the CDR3 region located on the surface of the antibody. The soluble antibody was expressed in E. coli. and the active site serine was converted into selenocysteine with the chemical modifying method, which resulted in the catalytic antibody with GPx activity of 80 U/mu mol. Furthermore, the same Ping-Pong mechanism as the natural GPx was observed when the kinetic behavior of the antibody was studied.

Amplification of antigen-antibody interactions based on biotin labeled protein-streptavidin network complex using impedance spectroscopy

Antibody was covalently immobilized by amine coupling method to gold surfaces modified with a self-assembled monolayer of thioctic acid. The electrochemical measurements of cyclic voltammetry and impedance spectroscopy showed that the hexacyanoferrate redox reactions on the gold surface were blocked due to the procedures of self-assembly of thioctic acid and antibody immobilization. The binding of a specific antigen to antibody recognition layer could be detected by measurements of the impedance change. A new amplification strategy was introduced for improving the sensitivity of impedance measurements using biotin labeled protein- streptavidin network complex. This amplification strategy is based on the construction of a molecular complex between streptavidin and biotin labeled protein. This complex can be formed in a cross-linking network of molecules so that the amplification of response signal will be realized due to the big molecular size of complex. The results show that this amplification strategy causes dramatic improvement of the detection sensitivity of hIgG and has good correlation for detection of hIgG in the range of 2-10 mug/ml. (C) 2001 Elsevier Science B.V. All rights reserved.

Study of molecular order in organo-soluble polyimide thin films

The changes in refractive indices (n(TE) and n(TM)) in a direction normal to the plane of thin films of an organo-soluble polyimide based on 1,4-bis(3,4-dicarboxyphenoxy) benzene dianhydride and 2,2'-dimethyl-4,4'-methylene dianiline were measured by a prism coupler. The results implied that the molecules near the substrate-polyimide interface were much ordered, while those near the polyimide-air interface were less ordered, judging from the variation in the level of negative birefringence with the depth of the films. The molecules are more condensed near the substrate surface, as seen by the average refractive index increasing from the polyimide-air interface to the substrate-polyimide interface, which implies that the condensed states of polyimide molecules change gradually in the depth direction. (C) 1997 Elsevier Science B.V.

Direct deposition and characterization of Langmuir-Blodgett monolayers of soluble polyimides

Langmuir-Blodgett (LB) monolayers of three kinds of soluble polyimides were prepared with the direct deposition method, The monolayer structures were characterized with W-vis absorption spectroscopy, the wide angle X-ray scattering method and electrochemical techniques. The polyimide molecules in the LB monolayers lie orderly on the substrate surfaces with the orientation in which the dianhydride group is normal to the substrate surface and two carbonyl oxygen groups close to the surface. Therefore, the thickness of the three kinds of polyimide LB monolayers are the same because it depends on the distance between the two carbonyl oxygen groups in the same ring. The area of monomeric units are dependent on the length of the diamine group. The model of the molecular packing proposed from the quantomechanical calculation is in good agreement with the experimental results. (C) 1997 Elsevier Science S.A.

Expression of Scophthalmus maximus CD83 correlates with bacterial infection and antigen stimulation

CD83 is a transmembrane glycoprotein of the immunoglobulin (Ig) superfamily and a surface marker for fully matured dendritic cells (DCs) in humans and mice. In teleosts, DC-like cells and their molecular markers are largely unknown. In this report, we described the identification and expressional analysis of a CD83 homologue, SmCD83, from turbot Scophthalmus maximus. The open reading frame of SmCD83 is 639 bp, which is preceded by a S'-untranslated region (UTR) of 87 bp and followed by a 3'-UTR of 1111 bp. The SmCD83 gene is 4716 bp in length, which contains five exons and four introns. The deduced amino acid sequence of SmCD83 shares 40-50% overall identities with the CD83 of several fish species. Like typical CD83, SmCD83 possesses an Ig-like extracellular domain, a transmembrane domain, and a cytoplasmic domain. The conserved disulfide bond-forming cysteine residues and the N-linked glycosylation sites that are preserved in CD83 are also found in SmCD83. Expressional analysis showed that constitutive expression of SmCD83 was high in gill, blood, spleen, muscle, and kidney and low in heart and liver. Bacterial infection and poly(I:C) treatment enhanced SmCD83 expression in kidney in time-dependent manners. Likewise, bacterial challenge caused significant induction of SmCD83 expression in cultured macrophages. Vaccination of turbot with a bacterin and a purified recombinant subunit vaccine-induced significant SmCD83 expression during the first week following vaccination. These results demonstrate that SmCD83 expression correlates with microbial challenge and antigen stimulation, which suggests the possibility that there may exist in turbot DC-like antigen-presenting cells that express SmCD83 upon activation by antigen uptake. In addition, these results also suggest that SmCD83 may serve as a marker for activated macrophages in turbot. (C) 2010 Elsevier Ltd. All rights reserved.

Application of mesoporous silica for the oral delivery of poorly water-soluble drugs

The objective of this thesis was to improve the dissolution rate of the poorly waters-soluble drug, fenofibrate by processing it with a high surface area carrier, mesoporous silica. The subsequent properties of the drug – silica composite were studied in terms of drug distribution within the silica matrix, solid state and release properties. Prior to commencing any experimental work, the properties of unprocessed mesoporous silica and fenofibrate were characterised (chapter 3), this allowed for comparison with the processed samples studied in later chapters. Fenofibrate was a highly stable, crystalline drug that did not adsorb moisture, even under long term accelerated storage conditions. It maintained its crystallinity even after SC-CO2 processing. Its dissolution rate was limited and dependent on the characteristics of the particular in vitro media studied. Mesoporous silica had a large surface area and mesopore volume and readily picked up moisture when stored under long term accelerated storage conditions (75% RH, 40 oC). It maintained its mesopore character after SC-CO2 processing. A variety of methods were employed to process fenofibrate with mesoporous silica including physical mixing, melt method, solvent impregnation and novel methods such as liquid and supercritical carbon dioxide (SC-CO2) (chapter 4). It was found that it was important to break down the fenofibrate particulate structure to a molecular state to enable drug molecules enter into the silica mesopores. While all processing methods led to some increase in fenofibrate release properties; the impregnation, liquid and SC-CO2 methods produced the most rapid release rates. SC-CO2 processing was further studied with a view to optimising the processing parameters to achieve the highest drug-loading efficiency possible (chapter 5). In this thesis, it was that SC-CO2 processing pressure had a bearing on drug-loading efficiency. Neither pressure, duration or depressurisation rate affected drug solid state or release properties. The amount of drug that could be loaded onto to the mesoporous silica successfully was also investigated at different ratios of drug mass to silica surface area under constant SC-CO2 conditions; as the drug – silica ratio increased, the drug-loading efficiency decreased, while there was no effect on drug solid state or release properties. The influence of the number of drug-loading steps was investigated (chapter 6) with a view to increasing the drug-loading efficiency. This multiple step approach did not yield an increase in drug-loading efficiency compared to the single step approach. It was also an objective in this chapter to understand how much drug could be loaded into silica mesopores; a method based on the known volume of the mesopores and true density of drug was investigated. However, this approach led to serious repercussions in terms of the subsequent solid state nature of the drug and its release performance; there was significant drug crystallinity and reduced release extent. The impact of in vitro release media on fenofibrate release was also studied (chapter 6). Here it was seen that media containing HCl led to reduced drug release over time compared to equivalent media not containing HCl. The key findings of this thesis are discussed in chapter 7 and included: 1. Drug – silica processing method strongly influenced drug distribution within the silica matrix, drug solid state and release. 2. The silica surface area and mesopore volume also influenced how much drug could be loaded. It was shown that SC-CO2 processing variables such as processing pressure (13.79 – 41.37 MPa), duration time (4 – 24 h) and depressurisation rate (rapid or controlled) did not influence the drug distribution within the SBA- 15 matrix, drug solid state form or release. Possible avenues of research to be considered going forward include the development and application of high resolution imaging techniques to visualise drug molecules within the silica mesopores. Also, the issues surrounding SBA-15 usage in a pharmaceutical manufacturing environment should be addressed.

An entirely cell-based system to generate single-chain antibodies against cell surface receptors.

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.

Display of cell surface sites for fibronectin assembly is modulated by cell adherence to (1)F3 and C-terminal modules of fibronectin.

BACKGROUND: Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules (7)F3-(10)F3). Interactions of adherent cells with regions of adsorbed fibronectin other than modules (7)F3-(10)F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly. METHODOLOGY/PRINCIPAL FINDINGS: To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to (7)F3-(10)F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules (2)F3-(14)F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module (1)F3 or the C-terminal modules to modules (2)F3-(14)F3 resulted in some activity, and addition of both (1)F3 and the C-terminal modules resulted in a construct, (1)F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs (1)F3-C V0, (1)F3-C V64, and (1)F3-C Delta(V(15)F3(10)F1) were all able to support fibronectin assembly, suggesting that (1)F3 through (11)F1 and/or (12)F1 were important for activity. Coatings in which the active parts of (1)F3-C were present in different proteins were much less active than intact (1)F3-C. CONCLUSIONS: These results suggest that (1)F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells.

Phenotypic characteristics of human type II alveolar epithelial cells suitable for antigen presentation to T lymphocytes.

Type II alveolar epithelial cells (AECII) are well known for their role in the innate immune system. More recently, it was proposed that they could play a role in the antigen presentation to T lymphocytes but contradictory results have been published both concerning their surface expressed molecules and the T lymphocyte responses in mixed lymphocyte cultures. The use of either AECII cell line or fresh cells could explain the observed discrepancies. Thus, this study aimed at defining the most relevant model of accessory antigen presenting cells by carefully comparing the two models for their expression of surface molecules necessary for efficient antigen presentation.

Ontogeny of mouse B lymphocytes and inactivation by antigen of early B lymphocytes

Taking advantage of recent findings about membrane fluidity, the authors studied and compared the biosynthetic capacities of fetal or neonatal mouse B (bone marrow derived) lymphocytes (until 10 days after birth) and adult B lymphocytes. Although both early and adult lymphocytes can synthesize surface immunoglobulins, they have a different physiological behavior after interaction with a ligand (anti immunoglobulin sera or antigen), either in vivo or in vitro. Fetal and neonatal lymphocytes bearing surface immunoglobulins do not reexpress their membrane receptors after capping and endocytosis promoted by anti immunoglobulin sera. On the other hand, adult lymphocytes resynthesize completely their receptors after the same treatment. Furthermore, intrafetal injections of hemocyanin in pregnant mice lead to a striking decrease in the number of hemocyanin binding cells. It seems plausible that this non reexpression of surface immunoglobulins could be the first step in tolerance establishment.

Expression, purification and X-ray crystallographic analysis of the Helicobacter pylori blood group antigen-binding adhesin BabA

Helicobacter pylori is a human pathogen that colonizes about 50% of the world's population, causing chronic gastritis, duodenal ulcers and even gastric cancer. A steady emergence of multiple antibiotic resistant strains poses an important public health threat and there is an urgent requirement for alternative therapeutics. The blood group antigen-binding adhesin BabA mediates the intimate attachment to the host mucosa and forms a major candidate for novel vaccine and drug development. Here, the recombinant expression and crystallization of a soluble BabA truncation (BabA^25-460) corresponding to the predicted extracellular adhesin domain of the protein are reported. X-ray diffraction data for nanobody-stabilized BabA^25-460 were collected to 2.25Å resolution from a crystal that belonged to space group P21, with unit-cell parameters a = 50.96, b = 131.41, c = 123.40Å, α = 90.0, β = 94.8, γ = 90.0°, and which was predicted to contain two BabA^25-460-nanobody complexes per asymmetric unit.

Heparin-binding hemagglutinin, from an extrapulmonary dissemination factor to a powerful diagnostic and protective antigen against tuberculosis.

Interactions of Mycobacterium tuberculosis with macrophages have long been recognized to be crucial to the pathogenesis of tuberculosis. The role of non-phagocytic cells is less well known. We have discovered a M. tuberculosis surface protein that interacts specifically with non-phagocytic cells, expresses hemagglutination activity and binds to sulfated glycoconjugates. It is therefore called heparin-binding hemagglutinin (HBHA). HBHA-deficient M. tuberculosis mutant strains are significantly impaired in their ability to disseminate from the lungs to other tissues, suggesting that the interaction with non-phagocytic cells, such as pulmonary epithelial cells, may play an important role in the extrapulmonary dissemination of the tubercle bacillus, one of the key steps that may lead to latency. Latently infected human individuals mount a strong T cell response to HBHA, whereas patients with active disease do not, suggesting that HBHA is a good marker for the immunodiagnosis of latent tuberculosis, and that HBHA-specific Th1 responses may contribute to protective immunity against active tuberculosis. Strong HBHA-mediated immuno-protection was shown in mouse challenge models. HBHA is a methylated protein and its antigenicity in latently infected subjects, as well as its protective immunogenicity strongly depends on the methylation pattern of HBHA. In both mice and man, the HBHA-specific IFN-gamma was produced by both the CD4(+) and the CD8(+) T cells. Furthermore, the HBHA-specific CD8(+) T cells expressed bactericidal and cytotoxic activities to mycobacteria-infected macrophages. This latter activity is most likely perforin mediated. Together, these observations strongly support the potential of methylated HBHA as an important component in future, acellular vaccines against tuberculosis.

High number of antigen binding cells in unimmunized mice and possible occurrence of multispecific lymphocytes

A high frequency of Tobacco Mosaic virus (TMV) binding cells was found in spleen cells from unimmunized mice (about 3 to 4%). TMV binding is strongly inhibited by previous incubation with anti immunoglobulin antisera. After stripping of membrane receptors, a full recovery for antigen binding capacity can be observed after 24 hr culture. Experiments are presented to exclude artefactual fluorescent cells: interaction of TMV with some non immunoglobulin membrane components; interaction of fluorescent anti TMV antibody with the Fc receptor of B cells; the binding of TMV to cytophilic immunoglobulins. The occurrence of lymphocytes able to bind several non crossreactive antigens is suggested by three lines of evidence: the high number of antigen binding cells in unimmunized mice, presence of surface immunoglobulins on some TMV binding cells after complete capping of TMV receptors and the direct demonstration of lymphocytes binding TMV and hemocyanin at different membrane sites.

Inclusion of poorly soluble drugs in highly ordered mesoporous silica nanoparticles

Silica nanoparticles (MSNs) with a highly ordered mesoporous structures (103A) with cubic Im3 m have been synthesized using triblock copolymers with high poly(alkylene oxide) (EO) segments in acid media. The produced nanoparticles displayed large specific surface area (approximately 765 cm(2)/g) with an average particles size of 120 nm. The loading efficiency was assessed by incorporating three major antiepileptic active substances via passive loading and it was found to varying from 17 to 25%. The state of the adsorbed active agents was further analyzed using differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD). Dissolution studies revealed rapid release profiles within the first 3 h. The viability of 3T3 endothelial cells was not affected in the presence of MSNs indicating negligible cytotoxicity. 2009 Elsevier B.V. All rights reserved.

Low colloidal associations of aluminium and titanium in surface waters of the tropical Atlantic

The distribution of dissolved, soluble and colloidal fractions of Al and Ti was assessed by ultrafiltration studies in the upper water column of the eastern tropical North Atlantic. The dissolved fractions of both metals were found to be dominated by the soluble phase smaller than 10 kDa. The colloidal associations were very low (0.2–3.4%) for Al and not detectable for Ti. These findings are in some contrast to previous estimations for Ti and to the predominant occurrence of both metals as hydrolyzed species in seawater. However, low tendencies to form inorganic colloids can be expected, as in seawater dissolved Al and dissolved Ti are present within their inorganic solubility levels. In addition, association with functional organic groups in the colloidal phase is unlikely for both metals. Vertical distributions of the dissolved fractions showed surface maxima with up to 43 nM of Al and 157 pM of Ti, reflecting their predominant supply from atmospheric sources to the open ocean. In the surface waters, excess dissolved Al over dissolved Ti was present compared to the crustal source, indicating higher solubility and thus elevated inputs of dissolved Al from atmospheric mineral particles. At most stations, subsurface minima of Al and Ti were observed and can be ascribed to scavenging processes and/or biological uptake. The dissolved Al concentrations decreased by 80–90% from the surface maximum to the subsurface minimum. Estimated residence times in the upper 100 m of the water column ranged between 1.6 and 4 years for dissolved Al and between 14 and 17 years for dissolved Ti. The short residence times are in some contrast to the low colloidal associations of Al and Ti and the assumed role of colloids as intermediates in scavenging processes. This suggests that either the removal of both metals occurs predominantly via direct transfer of the hydrolyzed species into the particulate fraction or that the colloidal phase is rapidly turned over in the upper water column.