Auto-Antibodies to β-F1-ATPase and Vimentin in Malignant Mesothelioma

Patients with Malignant Mesothelioma (MM) develop unidentified auto-antibodies to MM tumour antigens. This study was conducted to identify the targets of MM patient auto-antibodies in order to try to understand more of the anti-tumour response and to determine if these antibodies might be helpful for diagnosis or prognostication. Using MM patient sera in a Western immunoblott screening strategy, no common immunoreactive proteins were identified. The sera from one long-term survivor recognised a protein band of 50–60 kDa present in cell lysates from four of five MM cell lines tested. The immunoreactive proteins in this band were identified by 2D electrophoretic separation of a MM cell line protein lysate, followed by analysis of excised immunoreactive proteins on a MALDI TOF mass spectrometer and peptide mass fingerprinting. The immunoreactive proteins identified were vimentin (accession gi55977767) and the ATP synthase (F1-ATPase) beta chain (accession gi114549 and gi47606749). ELISA assays were developed for antibodies to these proteins. Neither vimentin (median and 95% CI 0.346; 0.32–0.468 for MM patients, 0.327; 0.308–0.428 for controls) nor ß-F1-ATPase (0.257; 0.221–0.453 for MM patients, 0.263; 0.22–0.35 for controls) showed significant differences in autoantibody levels between a group of MM patients and controls. Using a dichotomized antibody level (high, low) for these targets we demonstrated that vimentin antibody levels were not associated with survival. In contrast, high ß-F1-ATPase antibody levels were significantly associated with increased median survival (18 months) compared to low ß F1 ATPase antibody levels (9 months; p = 0.049). Immunohistochemical analysis on a MM tissue microarray showed cytoplasmic staining in 28 of 33 samples for vimentin and strong cytoplasmic staining in14 and weak in 16 samples for ß-F1-ATPase. Therefore antibodies to neither vimentin nor ß-F1-ATPase are useful for differential diagnosis of MM, however high antibody levels to ß-F1-ATPase may be associated with increased survival and this warrants further investigation

GATA-3 Has Dual Regulatory Functions in Human Interleukin-5 Transcription

Interleukin-5 (IL-5) is a T-cell cytokine involved in Type 2 diseases and is commonly described as being coordinately regulated with other Type 2 cytokines, such as IL-4 and IL-13. Considering the unique control of eosinophilia by IL-5, such coordinate regulation would be surprising. In fact, the biological specificity of eosinophilia and its control by IL-5 suggests a unique and independent control of IL-5 regulation. In this report we show the binding of GATA-3 to three sites in the human IL-5 promoter in the human T-cell line PER117. The previously identified -70 site and another site at position - 152 are shown to positively regulate IL-5 transcription. More importantly, the site located at -400 acts as a powerful repressor of IL-5 transcription with mutagenesis of this site allowing a high level expression of IL-5 without the activation of other factors normally required for IL-5 expression. Whereas GATA-3 has been proposed to be involved in the regulation of the IL-4/IL-5/ IL-13 locus, we show here that it has another function in controlling IL-5 transcription that supports the observed unique biological function of this cytokine.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Sequence alignment of mass peaks matching to ATP5B.

<p>Clustal alignment of the ATP5B sequence with trypsin digestion peptides identified by MS with a matching sequence of confidence 85% or greater.</p

ß-F1-ATPase ELISA.

<p>Levels of antibodies to ß-F1-ATPase were determined at least in duplicate and individual patient (open diamonds) and control (closed triangles) values are plotted on the graph. The defined cut-off value of two standard deviations above the mean of the control group is depicted by the hashed horizontal line. The index MM patient sample (Patient 145) result is indicated by the closed black diamond symbol. The MM patients indicated by grey symbols were previously identified as being immunopositive to ß-F1-ATPase by SEREX. There was no significant difference (ns) between the groups.</p

Two Dimensional Polyacrylamide Gel Electrophoresis.

<p>The Tris-soluble fraction of the JU77 cell lysate was separated by 2D electrophoresis and proteins stained with Coomassie Blue. Proteins from a parallel gel were transferred to PDVF and incubated with serum from MM Patient 145 diluted 1∶100. Two densely stained IgG reactive spots were defined by the approximate pI range 5 to 7 and molecular weight range 50 to 60 kDa, indicated by the circle. Molecular weights (MW) depicted to the left of figures; isoelectric point (pI) to the top of figures.</p

Vimentin ELISA.

<p>Levels of antibodies to vimentin were determined at least in duplicate and individual patient (open diamonds) and control (closed triangles) values are plotted on the graph. The defined cut-off value of two standard deviations above the mean of the control group (excluding the outlier) is depicted by the hashed horizontal line. The index MM patient sample (Patient 145) result is indicated by the closed black diamond symbol. There was no significant difference (ns) between the groups.</p

Survival of MM patients dichotomized on antibody reactivity.

<p>Kaplan–Meier survival curves of overall survival (months) from diagnosis for patients with (A) anti-vimentin and (B) anti-β-F1-ATPase antibody levels dichotomized below (thick line) and above the median (thin line).</p

Western Immunoblots of MM patient serum with MM tumour cell line lysates.

<p>(A) Representative serum samples from individual MM patients. Patients segregated into short, medium and long term survivors. Patient identification number indicated above each lane. (B) Western immunoblot of cell lysates from five different MM cell lines and the A549 lung adenocarcinoma, U2OS osteosarcoma and HT29 colon carcinoma cell lines incubated with serum from a MM Patient 145. Serum was diluted 1∶100. Molecular weights (MW) depicted to the left of figures.</p

Immunohistochemical tissue localisation of β-F1-ATPpase.

<p>Immunohistochemical staining of representative MM samples using anti-β-F1-ATPpase antibody diluted 1∶100 (A) Positive stained MM tumour sample, arrows indicate representative groups of tumour cells; over 90% of the tumour cells have been stained. (B) Non stained MM tumour sample with positively stained macrophage (indicated by arrow); less than 10% of tumour cells have been stained. (C) Positively stained MM cells present in a pleural effusion sample. Arrows indicate representative positive stained MM cells; over 90% of the tumour cells have been stained. (D) Non stained MM tumour cells in a MM patient pleural effusion; less than 10% of tumour cells have been stained. Photomicrographs were taken at a ×200 magnification.</p