Immunoreactive IL-13RA2 in human and canine brain tumor specimens/cells by purified MAb’s of Peptide 1

<p><b><i>A</i></b>, Human glioblastoma (G), <b><i>B</i></b>, oligodendroglioma (O), astrocytoma (A), normal brain (NB), and G14 human GBM tumor lysate; meningioma (M), <b><i>C</i></b>, tissue lysates immunoreactivity using Western blots. Canine astrocytoma, glioblastoma and normal brain, <b><i>D</i></b>; oligodendroglioma, gliosarcoma (GSO) and mixed astro-oligo (AO), <b><i>E</i></b>; and choroid plexus papilloma (CPP) and meningioma, <b><i>F</i></b> tissue lysates immunoreactivity using western blots. Western blot of cell lines and parent tumor tissue obtained from dogs with spontaneous GBM, <b><i>G</i></b>. Immunoprecipitation of IL-13RA2 from U-251 MG cell lysate using either MAb 2G12C3, MAb 2G12E2 or a polyclonal antibody (R&D Systems #AF146); the polyclonal antibody was used for the receptor detection after immunoprecipitation<b><i>, H</i></b>.</p

Immunoreactivity of monoclonal antibodies induced by Peptide 1 and recognition of synthetic and recombinant immunogens.

<p><b><i>A</i></b>, Western blot of U-251 MG and T98G human GBM cell lysates using media of 3G12C3 hybridoma cells. ELISA was conducted using either recombinant IL-13RA2-Fc, <b><i>B</i></b> or the synthetic Peptide 1, <b><i>C</i></b>. </p

MAb 1E10B9 binds to live GBM cells.

<p>Flow cytometry on human U-251 MG cells, canine GBM G06-A cells, and human T98G cells using MAb 1E10B9, <b><i>A.</i></b> Internalization of MAb 1E10B9 by G48a human GBM cells, <b><i>B.</i></b> Internalization of MAb 1E10B9 by U-251, G06-A, and T98G cells after 4-hr incubation, <b><i>C</i></b>.</p

Imunoreactivity of MAbs obtained with immunization using Peptide 3.

<p>Reactivity of MAbs raised against Peptide 3 in ELISA using recombinant IL-13RA2-Fc, <b><i>A</i></b>; and the synthetic Peptide 3, <b><i>B</i></b>. Detection of IL-13RA2, but not of IL-13RA1-Fc with MAbs 1E10B9 and 1E10F9, <b><i>C</i></b>. Immunoreactive IL-13RA2 in Western blot of U-251 MG and T98G cell lysates using MAb 1E10B9, <b><i>D</i></b>. Expression of IL-13RA2 detected by immunohistochemistry using MAb 1E10B9 in human GBM specimens and normal brain, <b><i>E</i></b>, and canine GBMs and normal brain, <b><i>F</i></b>.</p

Quantitative TaqMan RT-PCR comparing expression of IL-13RA2 in canine primary brain tumors.

<p>Elevated expression, relative to normal canine brain cortex, is seen predominantly in high grade glial tumors, essentially mirroring protein expression determined by western blotting. Off scale values are marked with an asterisk and value. MEN – meningioma; AST – astrocytoma; GBM – glioblastoma multiforme; OLIGO – oligodendroglioma.</p

Production and testing of canIL-13.E13K and canIL-13.E13K based cytotoxin.

<p>Superimposition of canIL-13 and huIL-13 molecules, (3D reconstruction using JMol), <b><i>A.</i></b> Purified canIL-13.E13K and canIL-13.E13K cytotoxin, (10% SDS-PAGE), <b><i>B</i><i> and </i><i>C</i></b>. Activation of TF-1 cells proliferation by cytokines, <b><i>D</i></b>. Cytotoxicity of canIL-13 cytotoxin and its neutralization on BTCOE 4795 human GBM cells, <b><i>E</i></b>. P<0.015 and <0.007 for differences between the cytokines (in <b><i>D</i></b>) and the cytotoxin killing vs. neutralization with canIL-13.E13K alone (in <b><i>E</i></b>) using an unpaired t-test. Cytotoxicity of canIL-13.E13K cytotoxin on canine GBM G06-A cells, <b><i>F</i></b>. Cytotoxicity of canIL-13.E13K cytotoxin on human GBM established (U-251 MG), <b><i>G</i></b>, and low passage human GBM cells (BTCOE 4795), <b><i>H</i></b>. CTL – control. Vertical bars represent SEM and if not seen, they are smaller than the points.</p

Alignment of human and canine sequences of IL-13RA2.

<p><b><i>A</i></b>, The sequences were obtained from the NCBI database. The regions of complete sequence identity between the two species that were utilized as immunogens are boxed. <b><i>B</i></b>, Ribbon structure of IL-13RA2 in contact with its natural ligand, IL-13. The three immunogenic peptides used for raising monoclonal antibodies are shown in green. Peptide 1 is located in the extracellular domain of the receptor, Peptide 2 in the vicinity of the ligand binding to the receptor and Peptide 3 is within the extracellular, near transmembrane domain.</p

Imunoreactivity of MAbs obtained with immunization using Peptide 2.

<p>Reactivity of MAbs raised against Peptide 2 in ELISA using recombinant IL-13RA2-Fc, <b><i>A</i></b>; and Peptide 2, <b><i>B</i></b>. Detection of recombinant IL-13RA2, but not of IL-13RA1-Fc with MAbs 6D3E9, 4G9G3 and 3D4G10, The proteins were loaded at 0.5 µg/lane. <b><i>C</i></b>. Immunofluorescence in G48a cells using MAb 6D3E9, <b><i>D</i></b>.</p

Supplemental Material, DS1_VET_10.1177_0300985819868731 - Pathologic Features of the Intervertebral Disc in Young Nova Scotia Duck Tolling Retrievers Confirms Chondrodystrophy Degenerative Phenotype Associated With Genotype

Supplemental Material, DS1_VET_10.1177_0300985819868731 for Pathologic Features of the Intervertebral Disc in Young Nova Scotia Duck Tolling Retrievers Confirms Chondrodystrophy Degenerative Phenotype Associated With Genotype by Brian G. Murphy, Peter Dickinson, Denis J. Marcellin-Little, Kevin Batcher, Stephen Raverty and Danika Bannasch in Veterinary Pathology</p

Dose summation and image registration strategies for radiobiologically and anatomically corrected dose accumulation in pelvic re-irradiation

Re-irradiation (reRT) is a promising technique for patients with localized recurrence in a previously irradiated area but presents major challenges. These include how to deal with anatomical change between two courses of radiotherapy and integration of radiobiology when summating original and re-irradiation doses. The Support Tool for Re-Irradiation Decisions guided by Radiobiology (STRIDeR) project aims to develop a software tool for use in a commercial treatment planning system to facilitate more informed reRT by accounting for anatomical changes and incorporating radiobiology. We evaluated three approaches to dose summation, incorporating anatomical change and radiobiology to differing extents. In a cohort of 21 patients who previously received pelvic re-irradiation the following dose summation strategies were compared: (1) Rigid registration (RIR) and physical dose summation, to reflect the current clinical approach, (2) RIR and radiobiological dose summation in equivalent dose in 2 Gy fractions (EQD2), and (3) Patient-specific deformable image registration (DIR) with EQD2 dose summation. RIR and physical dose summation (Strategy 1) resulted in high cumulative organ at risk (OAR) doses being ‘missed’ in 14% of cases, which were highlighted by EQD2 dose summation (Strategy 2). DIR (with EQD2 dose summation; Strategy 3) resulted in improved OAR overlap and distance to agreement metrics compared to RIR (with EQD2 dose summation; Strategy 2) and was consistently preferred in terms of clinical utility. DIR was considered to have a clinically important impact on dose summation in 38% of cases. Re-irradiation cases require individualized assessment when considering dose summation with the previous treatment plan. Fractionation correction is necessary to meaningfully assess cumulative doses and reduce the risk of unintentional OAR overdose. DIR can add clinically relevant information in selected cases, especially for significant anatomical change. Robust solutions for cumulative dose assessment offer the potential for future improved understanding of cumulative OAR tolerances.</p