Image cytometric analysis of p53 and mdm-2 expression in primary and recurrent mucoepidermoid carcinoma of parotid gland: immunohistochemical study

<p>Abstract</p> <p>Aims and Objectives</p> <p>This study aims to analyze immunocytochemically p53 aberrant expression and mdm-2 expression in primary and recurrent mucoepidermoid carcinoma (MEC) of parotid gland and to ascertain if expression of these markers correlates with tumor behavior, clinical outcome, histological grade and local recurrence.</p> <p>Methods</p> <p>20 cases histologically diagnosed as primary MEC with different grades were included in the study. Out of 20 cases, 7 were classified as grade I, 8 as grade II and 5 as grade III. Immunohistochemical staining of these 20 primary cases as well as 6 recurrent cases with anti-p53 and anti-mdm-2 antibodies was carried out. Area fraction of immunopositivity was estimated by image analysis software.</p> <p>Results</p> <p>16/20 primary cases were p53 +ve (80%). The p53 positive cases included 3 cases classified as grade (I), 8 cases as grade (II) and 5 cases as grade (III). All 6 recurrent cases were p53 +ve. On the other hand, 14/20 primary and only 2/6 recurrent cases were mdm-2 +ve. The mdm-2 +ve primary cases included 2 classified as grade (I), 7 as grade (II) and 5 as grade (III). 12 primary MEC showed co-expression of both p53 and mdm-2 of which 2 cases showed local recurrence.</p> <p>Conclusions</p> <p>these data suggested that expression of p53 and mdm-2 in primary and recurrent MEC correlates with the high histological grade. P53 aberrant expression is not only considered as an early event in MEC carcinogenesis but also correlates to tumor behavior and local recurrence. Mdm-2 overexpression is correlated to pathogenesis of MEC. However, no strong evidence was found between mdm-2 expression and MEC local recurrence.</p

Accurate identification of paraprotein antigen targets by epitope reconstruction

We describe the first successful clinical application of a new discovery technology, epitope-mediated antigen prediction (E-MAP), to the investigation of multiple myeloma. Until now, there has been no reliable, systematic method to identify the cognate antigens of paraproteins. E-MAP is a variation of previous efforts to reconstruct the epitopes of paraproteins, with the significant difference that it provides enough epitope sequence data so as to enable successful protein database searches. We first reconstruct the paraprotein's epitope by analyzing the peptides that strongly bind. Then, we compile the data and interrogate the nonredundant protein database, searching for a close match. As a clinical proof-of-concept, we apply this technology to uncovering the protein targets of para-proteins in multiple myeloma (MM). E-MAP analysis of 2 MM paraproteins identified human cytomegalovirus (HCMV) as a target in both. E-MAP sequence analysis determined that one para-protein binds to the AD-2S1 epitope of HCMV glycoprotein B. The other binds to the amino terminus of the HCMV UL-48 gene product. We confirmed these predictions using immunoassays and immunoblot analyses. E-MAP represents a new investigative tool for analyzing the role of chronic antigenic stimulation in B-lymphoproliferative disorders

B-cell epitopes in GroEL of Francisella tularensis.

The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60), is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft), the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs) specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA. In a mouse model of respiratory tularemia with the highly pathogenic Ft type A strain SchuS4, the Ab64 and N200 IgG2a mAbs, which block each other's binding to and are sensitive to the same two point mutations in FtGroEL, reduced bacterial burden indicating that they target protective GroEL B-cell epitopes. The Ab64 and N200 epitopes, as well as those of three other mAbs with different crossblocking profiles, Ab53, N3, and N30, were mapped by hydrogen/deuterium exchange-mass spectrometry (DXMS) and visualized on a homology model of FtGroEL. This model was further supported by its experimentally-validated computational docking to the X-ray crystal structures of Ab64 and Ab53 Fabs. The structural analysis and DXMS profiles of the Ab64 and N200 mAbs suggest that their protective effects may be due to induction or stabilization of a conformational change in FtGroEL

X-ray crystallographic structures of the antigen-binding sites of Ab53 and Ab64.

<p>(<b>A</b>) Head-on views of the molecular surfaces, colored gray for the H chain and purple for the L chain, with the CDR loops colored and indicated as H1, H2, H3 for the H chain and L1, L2, L3 for the L chain. (<b>B</b>) Ribbon diagrams of the binding-sites, colored gray for VH and purple for VL, and clipped/depth-cued for clarity. Selected side chains are shown in stick and labeled in black for VH residues and purple for VL residues. Solvent molecules present in the structures (sulfate, chloride, tris) are also shown. Hydrogen bonds indicated in the text are shown as dotted lines. (<b>C</b>) Ribbon diagrams of the binding-sites with the docked FtGroEL model, shown in red for Ab53 with the side-chain of R362 and in green for Ab64 with the side-chain of K344. The positions of some of the residues in the docked antibody structures are somewhat shifted compared with the crystal structures in B due to the energy minimization steps used during the docking protocol. Figure made with Maestro (version 9.3.5, Schrödinger, Inc., New York, NY).</p

DXMS-epitopes of FtGroEL mAbs on the molecular surface of Ft GroEL-GroES homology models.

<p>(<b>A</b>) FtGroEL monomers. (<b>B</b>) FtGroEL tetradecamers complexed with GroES. The epitopes of the five mAbs and the binding-site of GroES on GroEL are color-coded as indicated, and are divided into two images to separate the overlapping epitopes. FtGroEL residue Y476 is indicated in black. Note that the N30 epitope is exposed on both the GroES-bound and non-GroES-bound monomers but is blocked from view by the apical domain in the head-on view of the former. The proximity of the N3 and N30 epitopes in the tetradecamer, especially in the non-GroES-bound heptamer, reflects the juxtaposition of epitopes from neighboring monomers.</p

Homology model of FtGroEL.

<p>(<b>A–C</b>) Ribbon diagrams of GroEL tetradecamer complexed with GroES and of the non-GroES-bound and GroES-bound monomers. The positions of ADP (space-filling model in purple), of FtGroEL residues K344 and Y476 (space-filling models), and of the amino (N) and carboxyl (C) termini of the monomers are indicated. The GroES-bound monomer in C top is also shown after a 180° rotation about the z axis in C bottom right, to facilitate comparison with the non-GroES-bound monomer in C bottom left. (<b>D</b>) Linear amino acid sequence (in one-letter code) and secondary structure representation of FtGroEL. α-helices, β-strands, and loops are represented as boxes, thick arrows, and lines, respectively, blue for the equatorial domain, green for the intermediate region, and red for the apical domain. Helix letters and strand numbers are indicated. The FtGroEL amino acid residues involved in the interaction with GroES are highlighted in magenta.</p

Crosscompetition between 14 GroEL mAbs reveals at least three non-overlapping epitope areas in FtGroEL.

<p>(<b>A</b>) Competition ELISA. HRP-conjugated streptavidin was used to detect the binding of biotinylated reporter antibodies (indicated by *) to native GroEL (except for Ab12, which was detected by HRP-conjugated anti-mouse IgG3), in the presence of the indicated competitor mAbs. (<b>B</b>) Data summary. Competition, defined as a ≥80% increase in percent inhibition is indicated by +; a 40–79% increase in percent inhibition is indicated by ±; and ≤39% increase in percent inhibition is indicated by –. Four groups of mAbs based on similarity in crossblocking profiles are colored red, orange, green or blue. NA, not applicable; ND, not done.</p