DETECTION OF TNF INHIBITHORS (SOLUBLE RECEPTORS) IN THE SERA AND TUMOR CYST FLUID OF PATIENTS WITH MALIGNANT ASTROCYTOMAS OF THE BRAIN

Mario Ammirati ¹, Sanjay Rao ², and Gale Granger ³

1 Division of Neurosurgery, Albert Einstein Medical Center, ² Marshfield Clinic Neurosurgery, ³ Department of Molecular Biology and Biochemistry, University of California, Irvine, CA

TABLE OF CONTENTS

1. Abstract

2. Introduction

3. Materials and methods

3.1. Clinical Characteristics of Patients
3.2. Serum Collection
3.3. Tumor Cyst Fluid Collection
3.4. ELISA for TNF and stnf-r's
3.5. Cytolytic Bioassay for TNF Activity and TNF Blocking Factor Activity
3.6. Immunohistochemical Staining of Astrocytoma Tissue Sample
3.7. Statistical Analysis

4. Results

4.1. Serum Levels of TNF and the 55 kda and 75 kda stnf-R's in Malignant Astrocytoma Patients and Normal Individuals
4.2. TNF Levels in Brain Tumor Cyst Fluid Samples
4.3. Levels of the 55 and 75 kda stnf-r's, and TNF Blocking Factor Activity in the Tumor Cyst Fluid From Patients With Malignant Astrocytomas
4.4. Immunohistochemical Staining of Astrocytoma Tissue

5. Discussion

6. Acknowledgements

7. References

Patients with malignant astrocytomas of the brain exhibit varying degrees of immunosuppression with only a few factors responsible for this immunosuppression having been characterized. The soluble forms of the 55 kDa and 75 kDa membrane receptors for tumor necrosis factor (sTNF-R's) have been shown to bind to and inhibit the activity of TNF. The present studies analyze levels of sTNF-R's in the sera and tumor cyst fluids of patients with malignant astrocytomas. Using sensitive ELISA techniques, serum levels of the 55 and 75 kDa sTNF-R's in 17 patients tested were found to be elevated [] 55 kDa of 2.29 ± 2.85 ng/ml and 75 kDa of 4.98 ± 4.03 ng/ml]as compared to 20 normal controls [] 55 kDa of of 1.21 ± 0.91 ng/ml and 75 kDa of 1.85 ± 0.40 ng/ml]although this was only statistically significant for the 75 kDa sTNF-R (P=0.006). Brain tymor cyst fluid samples obtained from eight patients were shown to have very high levels of both sTNF-R's ranging from 4.16 to 17.17 ng/ml for the 55 kDa receptor and 4.83 to 19.96 ng/ml for the 75 kDa receptor. Six of these cyst fluid samples were also tested for their ability to inhibit TNF cytolytic activity using an in vitro assay. All samples tested had TNF inhibitory activity. Immunohistochemical studies on patient tumor samples showed high levels of expression of these receptors both in the cytoplasm and the cell surface of astrocytoma cells.