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Structural Basis of Affinity Maturation of Antibodies in the 2- Phenyl-5-Oxazolone System
No full textBackground: Affinity maturation of antibodies is the process whereby more efficient antibodies are produced through somatic hypermutation and antigen-guided selection. No extensive study is available at the moment concerning the relationship between somatic mutations and their structural counterpart. The antibody response to 2-phenyl-5-oxazolone has been thoroughly investigated from the genetic point of view. It consists of three antibody classes, with each member of each class derived from a unique pair of VH and VL germline genes by somatic hypermutation. In this project, we are investigating the structure of the VH and VL domains of 10 representative antibodies. Methods: The VH and VL domains of each antibody are being expressed as recombinant scFvs, crystallised, and their structure determined by X-ray crystallography. Results: The structures and models available allow an initial definition of the strategies adopted. In class I, maturation is bound to improvement of surface complementarity, especially at the top of the binding pocket, and in surface charge changes. In class II the maturation strategy seems to be based on the increase of the interacting surface, and on the introduction of a specific bond with the oxazolone ring. In class III, where the low and high affinity antibodies differ by 8 mutations, the increase in affinity is mainly determined by the improvement in the surface complementarity by removal of a bulky phenylalanine, which allows a better tightening of the two sides of the binding site.
Conclusions: These results are relevant to determine the principles underlying affinity maturation of antibodies
Lipoprotein (a) Internalization by RAW 264.7 Cells is Associated with Morphological Changes and Accumulation of Lipids Detectable by Two-photon Scanning Microscopy
No full textLipoprotein (a) [Lp(a)] is an independent cardiovascular risk factor and its pathogenic mechanism
is not completely clear. Lp(a) has been detected in atherosclerotic plaques and macrophages are
one of the major cell types involved in atherogenesis. In order to characterize internalization of
Lp(a) by RAW 264.7 cells, an established model of mouse macrophages, cells were treated with Lp(a) samples purified from plasma by affinity chromatography, and evaluated by western blotting.
By 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, a tetrazolium salt, assay, Lp(a)
was found to be non-cytotoxic for the cells at all the concentrations tested (0.0165-1.65 mg/ml). An
ELISA performed on the lysate of Lp(a)-treated cells allowed to identify the highest intracellular
accumulation of Lp(a) at 72 h treatment. Already at 24 h, however, important morphological
alterations were detected upon Oil red and Nile red staining. A three-dimensional reconstruction
obtained by two-photon scanning microscopy of the intracellular distribution of Nile red stained
structures in treated cells shows preferential uptake of lipids in extra-nuclear regions. These data
are useful to clarify the temporal and spacial aspects of intracellular accumulation of Lp(a) in RAW
264.7 cells and pose new bases for future studies on intracellular Lp(a) accumulation
Anti-Lp(a) Antibody For Diagnosis and Therapy
No full textBackground: The presence of the lipoprotein(a) in plasma was first described by Kare Berg in 1963 as an LDL-like particle. Lp(a) was later identified as a risk factor for atherosclerotic diseases because of its pro-atherogenic, prothrombotic and antifibrinolytic properties. Different epidemiological studies have also suggested that Lp(a) could increase the risk of cardiovascular disease and ischemic stroke if associated with other predisposing factors such as hypercholesterolemia, hypertension, diabetes mellitus and low level of HDL. There is also experimental evidence that lipoproteins have a role in degenerative diseases and not only in atherosclerosis. Today there are few therapeutic approaches for the treatment of hyperlipidemia(a). High-affinity monoclonal antibodies are an attractive therapeutic alternative. Due to their specificity, they have the ability to selectively bind the molecule of interest, and their structure, which includes an Fc region, allows complex binding to the Fc receptor localized on the surface of monocytes and macrophages.
Methods: In this study, we sought to characterize the effect of anti-Lp(A) monoclonal antibody 2E8, directed toward KIV2, in an in vitro system. The ability of the antibody to induce internalization of Lp(a) in murine macrophages (RAW cells) has been tested by an ELISA test and by microscopical evaluation of intracellular lipid accumulation.
Results: The number of foam cells had increased five times compared to the non-MAb control. Conclusions: This system will allow selection of new MAbs generated against human-Lp(a), and a fine characterization of the cellular response triggered by Lp(a)-MAb complexes
Helicobacter pylori L-asparaginase: A promising chemotherapeutic agent
No full textBacterial L -asparaginases are amidohydrolases that catalyse the conversion of L -asparagine to L -aspartate and ammonia and are used as anti-cancer drugs. The current members of this class of drugs have several toxic side effects mainly due to their associated glutaminase activity. In the present study, we report the molecular cloning, biochemical characterisation and in vitro cytotoxicity of a novel L -asparaginase from
the pathogenic strain Helicobacter pylori CCUG 17874. The recombinant enzyme showed a strong preference for L -asparagine over L -glutamine and, in contrast to most L -asparaginases, it exhibited a sigmoidal behaviour towards L -glutamine. The enzyme preserved full activity after 2 h incubation at 45 C. In vitro cytotoxicity assays revealed that different cell lines displayed a variable sensitivity towards the enzyme,
AGS and MKN28 gastric epithelial cells being the most affected. These findings may be relevant both for the interpretation of the mechanisms underlying H. pylori associated diseases and for biomedical applications
Molecular Cloning and Characterisation of Helicobacter Pylori L-Asparaginase: A Cytotoxic Factor with Potential Biomedical Applications
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