Effect of TGF-β2 and anti-TGF-β2 antibody in a new in vivo rodent model of posterior capsule opacification

PURPOSE. This study evaluated the effect of transforming growth factor (TGF)-ß2 and anti-TGF-ß2 antibody in a rodent model of posterior capsule opacification (PCO). METHODS. An extracapsular lens extraction (ECLE) was performed in 72 Sprague-Dawley rats. At the end of the procedure, 10 µL TGF-ß2 (TGF-ß2-treated group), fetal calf serum (FCS)/phosphate- buffered saline (PBS; FCS/PBS-treated control group), a human monoclonal TGF-ß2 antibody (anti-TGF-ß2-treated group), or a null control IgG4 antibody (null antibody-treated control group) was injected into the capsule. Animals were killed 3 and 14 days postoperatively. Eyes were evaluated clinically prior to euthanatization, then enucleated and processed for light microscopy and immunohistochemistry afterward. PCO was evaluated clinically and histopathologically. Student's t-test and ? were used to assess differences between groups. RESULTS. There were no statistically significant clinical or histopathological differences in degree of PCO between the TGF-ß2- and FCS/PBS-treated groups at 3 and 14 days after ECLE. Nor were there differences between the anti-TGF-ß2- and the null antibody-treated groups, with the exception of the histopathology score for capsule wrinkling 3 days after ECLE (P = 0.02). a-Smooth-muscle actin staining was observed in the lens capsular bag only in areas where there was close contact with the iris. CONCLUSIONS. No sustained effect of TGF-ß2 or anti-TGF-ß2 antibody on PCO was found in rodents at the dose and timing administered in this study. Iris cells may play a role in the process of epithelial mesenchymal transition linked to PCO. Copyright © Association for Research in Vision and Ophthalmology.

Rotavirus enterotoxin NSP4 has mucosal adjuvant properties

Rotavirus nonstructural protein 4 (NSP4) is a protein with pleiotropic properties. It functions in rotavirus morphogenesis, pathogenesis, and is the first described viral enterotoxin. Since many bacterial toxins function as potent mucosal adjuvants, we evaluated whether baculovirus-expressed recombinant simian rotavirus SA11 NSP4 possesses adjuvant activity by co-administering NSP4 with keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or ovalbumin (OVA) as model antigens in mice. Following intranasal immunization, NSP4 significantly enhanced both systemic and mucosal immune responses to model immunogens, as compared to the control group, in an antigen-specific manner. Both full-length and a cleavage product of SA11 NSP4 had adjuvant activity, localizing this activity to the C-terminus of the protein. NSP4 forms from virulent and avirulent porcine rotavirus OSU strain, and SA11 NSP4 localized within a 2/6-virus-like particle (VLP) also exhibited adjuvant effects. These studies suggest that the rotavirus enterotoxin NSP4 can function as an adjuvant to enhance immune responses for a co-administered antigen.

Signal enhancement of surface plasmon resonance immunoassay using enzyme precipitation-functionalized gold nanoparticles: A femto molar level measurement of anti-glutamic acid decarboxylase antibody

Colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance (SPR) biosensor. The AuNPs were synthesized and functionalized with HS-OEG(3)-COOH by self assembling technique. Thereafter, the HS-OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti IgG antibody to form an enzyme-immunogold complex. Characterizations were performed by several methods: UV-vis absorption, DLS, HR-TEM and Fr-IR. The Au-anti IgG-HRP complex has been applied in enhancement of SPR immunoassay using a sensor chip constructed by 1:9 molar ratio of HS-OEG(6)-COOH and HS-OEG(3)-OH for detection of anti-GAD antibody. As a result, AuNPs showed their enhancement as being consistent with other previous studies while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. The limit of detection was found as low as 0.03 ng/ml of anti-GAD antibody (or 200 fM) which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

Resonant Rayleigh light scattering response of individual Au nanoparticles to antigen--antibody interaction

A proof-of-concept study was reported on analysis of antigen–antibody recognition based on resonant Rayleigh scattering response of single Au nanoparticles in an imaging chamber. As benefited by a traditional dark-field microscope and a spectrograph, individual Au nanoparticles (30 nm) were observed with high signal-to-noise ratio and they were effectively utilized to monitor changes in refractive index induced by specific binding of the adsorbates. Using PSA antigen as a model, a LSPR ?max shift of about 2.85 nm was recorded for a molecular binding corresponding to 0.1 pg ml-1 of the protein biomarker. This result successfully demonstrates a non-labeling detection system for proteins as well as thousands of different chemical or biological species, and it possesses a great potential as a sensitive, on-chip and multiplexing detection.

Lymphocytic follicles and aggregates are a determinant of mucosal damage and duration of diarrhea

Nonspecific changes (nonspecific chronic inflammation) in patients with chronic diarrhea represent the commonest diagnosis in colorectal biopsy interpretation, but these changes are of little clinical significance.

Differences in Mucosal Gene Expression in the Colon of Two Inbred Mouse Strains after Colonization with Commensal Gut Bacteria

The host genotype has been proposed to contribute to individually composed bacterial communities in the gut. To provide deeper insight into interactions between gut bacteria and host, we associated germ-free C3H and C57BL/10 mice with intestinal bacteria from a C57BL/10 donor mouse. Analysis of microbiota similarity between the animals with denaturing gradient gel electrophoresis revealed the development of a mouse strain-specific microbiota. Microarray-based gene expression analysis in the colonic mucosa identified 202 genes whose expression differed significantly by a factor of more than 2. Application of bioinformatics tools demonstrated that functional terms including signaling/secretion, lipid degradation/catabolism, guanine nucleotide/guanylate binding and immune response were significantly enriched in differentially expressed genes. We had a closer look at the 56 genes with expression differences of more than 4 and observed a higher expression in C57BL/10 mice of the genes coding for Tlr1 and Ang4 which are involved in the recognition and response to gut bacteria. A higher expression of Pla2g2a was detected in C3H mice. In addition, a number of interferon-inducible genes were higher expressed in C3H than in C57BL/10 mice including Gbp1, Mal, Oasl2, Ifi202b, Rtp4, Ly6g6c, Ifi27l2a, Usp18, Ifit1, Ifi44, and Ly6g indicating that interferons may play an essential role in microbiota regulation. However, genes coding for interferons, their receptors, factors involved in interferon expression regulation or signaling pathways were not differentially expressed between the two mouse strains. Taken together, our study confirms that the host genotype is involved in the establishment of host-specific bacterial communities in the gut. Based on expression differences after colonization with the same bacterial inoculum, we propose that Pla2g2a and interferon-dependent genes may contribute to this phenomenon.

Effects of Gold Nanoparticle and Enzyme Precipitation on Enhancement of Surface Plasmon Resonance Immunoassay: A Super Sensitive Measurement of Anti- Glutamic Acid Decarboxylase Antibody

Introduction: In this study, colloidal gold nanoparticle and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation of 3,3'-diaminobenzidine (DAB) in the presence of H2O2 were used to enhance the signal obtained from the surface plasmon resonance biosensor.

Methods: The colloidal gold nanoparticle was synthesized as described by Turkevitch et al., and their surface was firstly functionalized with HS(CH2)11(OCH2CH2)3COOH (OEG3¬-COOH) by self assembling technique. Thereafter, those OEG3-COOH functionalized nanoparticles were covalently conjugated with horseradish peroxidase (HRP) and anti-IgG antibody (specific to the Fc portion of all human IgG subclasses) to form an enzyme-immunogold complex. Characterization was performed by several methods: UV-Vis absorption, dynamic light scattering (DLS), transmission electron microscopy (TEM) and FTIR. The as-prepared enzyme-immunogold complex has been applied in enhancement of SPR immunoassay. A sensor chip used in the experiment was constructed by using 1:10 molar ratio of HS(CH2)11(OCH2CH2)6COOH and HS(CH2)11(OCH2CH2)3OH. The capture protein, GAD65 (autoantigen) which is recognized by anti-GAD antibody (autoantibody) in the sera of insulin-dependent diabetes mellitus patients, was immobilized onto the 1:10 surface via biotin-streptavidin interaction.

Results and conclusions: In the research, we reported the influences of gold nanoparticle and enzyme precipitation on the enhancement of SPR signal. Gold nanoparticle showed its enhancement as being consistent with other previous studies, while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the SPR detection. As the results, anti-GAD antibody could be detected at pg/ml level which is far higher than that of commercial ELISA detection kit. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

Affinity-Purified Respiratory Syncytial Virus Antibodies from Intravenous Immunoglobulin Exert Potent Antibody-Dependent Cellular Cytotoxicity

Mixed infections are one of the major therapeutic challenges, as the current strategies have had limited success. One of the most common and widespread conditions of mixed infection is respiratory syncytial virus-mediated pathology of the respiratory tract in children. There is a dire need for the development of novel therapeutic approaches during mixed infections. Therapeutic intravenous immunoglobulin preparations, obtained from plasma pools of healthy donors have been used in immune deficiencies. This study was thus designed to characterize the functional efficacy of RSV-specific antibodies in IVIg. To explore the functional ability of these affinity-purified RSV-specific antibodies, the antibody-dependent and complement dependent cytotoxicity was determined using peripheral cells of healthy donors. This study demonstrates the existence of highly potent RSV-specific antibodies in IVIg preparations and provides the basis for the use of IVIg as broad-spectrum protective shield to RSV-infected children during mixed infections

CHARACTERISATION OF THE HUMAN ANTIBODY RESPONSE TO THE UK ANTHRAX VACCINE

Monospecific antibody to the haemagglutinin of measles virus

A hybrid between a murine myeloma cell line and spleen cells from a mouse immunized with measles has been produced. Two stable clones produce antibody with identical immunochemical and biological properties. This antibody reacts with the 76,000 mol. wt. protein present in the lysates and on the surface of cells persistently infected with measles. It exhibits HAI and neutralizing activity.

Fasciola hepatica:studies on vaccination of rats and mice with a surface antigen prepared from fluke homogenate by means of a monoclonal antibody

T1 tegumental antigen was isolated from a homogenate of eight- to 10-week-old Fasciola hepatica using a T1-specific monoclonal antibody bound to sepharose in an antibody-affinity column. Rats and mice were vaccinated with T1 antigen in Freund's complete adjuvant, and control groups received equivalent amounts of non-T1 antigen (eluted from the antibody-affinity column) or ovalbumin. On completion of the immunisation programme, serum samples were collected for ELISA and IFA testing. The animals were challenged by oral infection with F hepatica metacercariae or, for several vaccinated rats, by intraperitoneal transplantation of live adult flukes. At autopsy, worm-burden and liver damage was assessed for each animal and the condition of transplanted flukes was examined. Comparison of test and control groups of animals showed that neither T1 nor non-T1 antigens provided significant protection against challenge, although specific antibody responses against the appropriate sensitising antigen were engendered. Flukes transplanted to the peritoneal cavity of immunised rats survived without damage, although they became encased in hollow fibrous capsules of host origin. The results lend support to the pre-existing concept that glycocalyx turnover by discharge of T1 secretory bodies at the apical surface of migrating flukes provides an efficient means of protection for the parasite against host immunity.

Fasciola hepatica:development of monoclonal antibodies against somatic antigens and their characterization by ultrastructural localization of antibody binding

A series of monoclonal antibodies was prepared against tegumental and internal antigens of Fasciola hepatica by immunizing mice with whole adult-fluke homogenates prior to harvesting the splenic lymphocytes for fusion. Preliminary screening by the Indirect Fluorescent Antibody technique indicated the occurrence of discrete groups of monoclonals differing from one another in tissue-specificity but within which IFA labelling patterns were fairly consistent. Representative hybridomas for 5 of these groups were stabilized and used to produce ascites fluid in mice. By application of an immunogold labelling technique it was possible to map the distribution of antigens for which each monoclonal antibody had affinity throughout the tissues of 4-week and 12-week flukes. Several monoclonals specifically labelled antigenic determinants on the important tegumental antigen T1. However the distribution of gold colloid labelling suggested that epitopes other than that normally exposed to the infected host were recognized; and several monoclonals specifically attached to T1 antigen in the tegument of juvenile worms only. The glycocalyx of the gut and excretory system of flukes shared T1 antigenicity with the tegument. Monoclonal antibodies were produced against an internal immunogen associated with ribosomes and heterochromatin in active protein-producing cells, and against interstitial material of adult flukes. Monoclonals against antigens in parenchymal cell cytoplasm and in mature vitelline cells were recognized but the corresponding hybridomas were not stabilized.

Caveolin-1 single nucleotide polymorphism in antineutrophil cytoplasmic antibody associated vasculitis

Immunosuppression is cornerstone treatment of antineutrophil cytoplasmic antibody associated vasculitis (AAV) but is later complicated by infection, cancer, cardiovascular and chronic kidney disease. Caveolin-1 is an essential structural protein for small cell membrane invaginations known as caveolae. Its functional role has been associated with these complications. For the first time, caveolin-1 (CAV1) gene variation is studied in AAV.