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Interleukin-13 Receptor Chain: A Novel Tumor-associated Transmembrane Protein in Primary Explants of Human Malignant Gliomas

Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics, Evaluation and Research, United States Food and Drug Administration, Bethesda, Maryland 20892 [B. H. J., R. K. P.], and Center for Surgery Research, Cleveland Clinic Foundation, Cleveland, Ohio 44195 [G. E. P.]

	ABSTRACT

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Human malignant glioma cell lines express high levels of interleukin-13 receptor (IL-13R). However, the subunit structure of this receptor in primary brain tumor cells is not known. Herein, we examined the subunit composition of IL-13R by analyzing the expression of four different putative subunits of IL-13R complex in 25 primary explants of malignant brain tumors. Reverse transcription-PCR (RT-PCR) of RNA from these tumor cells, normal astrocytes, and normal brain tissue showed that transcripts of IL-13R chain were present in greater abundance in malignant glioma cells compared with normal astrocytes or normal brain tissues. The transcripts for two other chains (e.g., IL-13R' and IL-4Rß), on the other hand, yielded similar PCR positivity in brain tumors as well as in normal samples, whereas transcripts for c chain were absent in all brain tumor cells and normal tissues. The specificity of RT-PCR products for these genes was confirmed by oligo liquid hybridization analysis using a radiolabeled sequence-specific internal probe. Indirect immunofluorescence studies for different receptor chains confirmed the RT-PCR results and demonstrated a striking difference in the level of expression of IL-13R protein between normal astrocytes and malignant astrocytoma cells. These studies establish the IL-13R subunit as a novel tumor-specific protein that may be useful as a tumor marker, a target for cytotoxin/immunotoxin, or alternatively, a tumor-associated antigen for active, specific immunotherapy.

	Introduction

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Malignant astrocytoma/GBM² is the third leading cause of cancer-related deaths in the United States in adolescents and children (1) . Even in the early stages, two hallmarks of astrocytic neoplasms are diffuse infiltration and lack of clear demarcation between tumor and neighboring normal brain tissues. Several biological as well as immunological markers, including overexpressed growth factor receptors, have been associated with brain tumors that potentially may act as tumor-associated or -specific antigen. These include chromosomal abnormalities within chromosomes 7, 9, 10, 13, and 19 (2) , surface expression of the high-affinity form of fibroblast growth factor receptor 1-ß (3) , EGFR (4) , and TfR (5) . It is believed that these altered receptors or markers provide a cell-growth advantage and possibly contribute to glial cell malignancy. Our previous studies identified abundant expression of receptors for IL-4 and IL-13, two Th2-lymphocyte-derived and -related immunoregulatory cytokines, on brain tumor cells (6, 7, 8, 9) . Normal tissues obtained from human brain did not express detectable IL-4R or IL-13R (7 , 10) .

The structures of IL-4 and IL-13 receptors have been studied extensively. IL-4 receptor complex exists in two different types. Type I IL-4 receptors are composed of IL-4Rß (also known as IL-4R) and IL-2R subunits (c), whereas type II receptors have IL-4Rß and IL-13R'subunits (11 , 12) . IL-13R are also known to exist at least two different types. Type I IL-13R comprise IL-13R' (also known as 1), IL-13R (also known as 2), and the IL-4Rß chains, whereas type II IL-13R consist of IL-4Rß and IL-13R' chains (12, 13, 14, 15, 16, 17) . The role of common chain in the formation of IL-13R complex is not clear. It has been shown that introduction of c can decrease IL-13 and IL-4 binding and interfere in functioning of both receptors in cells that usually do not express this chain (17, 18, 19) . These and other studies have shown that the IL-4Rß and IL-13' chains are shared between IL-4 and IL-13R complexes (12, 13, 14, 15, 16) . Furthermore, both chains are required for signal transduction through type II IL-4R and both type I/II IL-13R (12) . Although both IL-4 and IL-13R are expressed on tumor cells, the significance of expression of these receptors on tumor cells still is not known. It is also not known which chains of these receptors are present on brain tumor cells. In the present study, we investigated primary brain tumor explants for the expression of mRNA for various receptor subunits of IL-13/IL-4 receptor by a sensitive RT-PCR-based assay. In addition, surface expression of these receptor proteins on primary brain tumor explant cells has been examined by indirect immunofluorescence assays.

	Materials and Methods

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Tissue Collection and Cell Culture.
Fresh brain tumor samples were collected from Cleveland Clinic, Cleveland, OH. Pathological grading of tumors was performed according to the Berger and Vogel modification of the Ringhertz grading system. The samples were minced and suspended in HBSS containing 4 mg of DNase I, 40 mg of collagenase type IV, and 100 units of hyaluronidase type V (all from Sigma Chemicals Co., St. Louis, MO) at room temperature for 3 h. The single-cell suspension was filtered through no. 100 nylon mesh, washed twice in HBSS, and added to fibronectin-coated tissue culture flasks. Cells were cultured in medium that consisted of 84% DMEM, 10% X-VIVO 15 (Bio-Whittaker, Walkersville, MD), 5% human AB serum (Sigma), 1% G5 (Life Cell Technologies, Grand Island, NY), and 10 µg/ml hydrocortisone. The cells were maintained in astrocyte growth medium (AGM bullet kit; Clonetics-BioWhittaker, Walkersville, MD) after the first passage. NHAs were also obtained and maintained in AGM bullet kit (both from Clonetics-BioWhittaker). HuOL normal human oligodendrocytes, two cortex cell lines derived from two different areas of a patient’s brain with Rasmussen’s encephalitis, and NT2 neuronal cell lines were kindly provided by Dr. Kathy Carbone of the Center for Biologics, Evaluation and Research, United States Food and Drug Administration.

RNA Extraction.
Primary glioma explant cells in the second passage were detached with trypsin-EDTA, pelleted, washed with 1x PBS and used for RNA extraction using RNAeasy RNA extraction kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Briefly, 5 x 10⁶ cells were pelleted and lysed in lysis buffer. The cell lysis was added to silica spin columns after the addition of an equal volume of 70% ethanol, and centrifuged for 20 s. The columns were washed with wash buffer according to the manufacturer’s instruction, and RNA was eluted with RNase-free water. The RNA was quantitated after the absorbance was measured at 260 and 280 nm in a spectrophotometer and stored at -70°C.

RT-PCR.
The total RNA derived from 25 primary brain tumors, one normal human astrocyte cell culture, and normal brain tissues (Clonetech, Palo Alto, CA) were subjected to RT-PCR analysis. ß-actin mRNA amplification from these samples served as an internal control. The optimal RT-PCR conditions for each chain and the primers used in amplification technique have been published previously (13) . Five hundred ng of total RNA from various tissues were reverse-transcribed using the RNA PCR kit according to the manufacturer’s instructions (Perkin-Elmer Corp., Norwalk, CT). Ten µl of reverse-transcribed products were amplified according to manufacturer’s instruction and amplified for 30 cycles, using the GeneAmp PCR system 9700 (Applied Biosystem-Perkin-Elmer, Norwalk, CT). The amplification products were resolved in 2% agarose gel, stained with ethidium bromide, visualized in a transilluminator, and photographed.

Oligo Liquid Hybridization for RT-PCR Products.
The sequence identities of the RT-PCR products of the IL-13R and ' chains were confirmed by oligo liquid hybridization with specific internal probes complementary to sequence +445 to +461 of IL-13R and +988 to 1005 of IL-13R' cDNA (13) . The probes were end-labeled with [-³²P]ATP (Amersham Pharmacia Biotech, Piscataway, NJ) using a DNA 5'end-labeling kit (Boehringer Mannheim GmbH, Indianapolis, IN). Ten µl of PCR products were mixed with 5 µl of ³²P-end-labeled probe (150,000 cpm) and a few drops of mineral oil to prevent water loss during hybridization steps. The tubes were hybridized by incubating at 95°C for 5 min, followed by additional incubation at 42°C and 40°C in a water bath for the IL-13R and IL-13R' chains, respectively, for 1 h. After brief centrifugation and the addition 5 µl of 5x DNA sample loading buffer, the hybridized products were separated on 12% PAGE in 0.5x Tris-borate EDTA as running buffer. The gel was then wrapped with plastic wrap and placed in a autoradiographic cassette with intensifying screen and exposed to a X-O-matic X-ray film for 16 h at -70°C for autoradiography.

Immunofluorescence Assay.
Twenty thousand cells were cultured in chambered glass slide (Lab Tek-Nalge Nunc International, Naperville, IL) for 48h. The cells were washed twice with PBS and fixed with cold methanol:acetone (1:1, v/v) and incubated at -20°C for 2 h. The slides were then washed and rehydrated with PBS and used for indirect immunofluorescence analysis. Monoclonal antibodies for IL-13, IL-13', and GFAP were obtained from Diaclone (Besancon, France) and Sigma. Polyclonal rabbit antiserum for IL-4R was obtained from Immunex Corporation (Seattle, WA), and polyclonal rabbit anti-c antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rehydrated cells in the chambered slide were incubated with 1% BSA and 5% either goat or horse serum in PBS to block nonspecific binding of antibody. The slides were washed with PBS and incubated with either the specified primary antibody (1:1500 for the monoclonal antibody to IL-13R or IL-13R' and the rabbit polyclonal antibody to c, and 1:100 for polyclonal rabbit antiserum for IL-4Rß) or mouse IgG1 for IL-13R and IL-13R' or rabbit serum or rabbit IgG for the IL-4Rß and c chains for 2 h at room temperature. Slides were then washed three times for 5 min with PBS at room temperature. A secondary antibody that had either tetramethylrhodamine isothiocyanate or FITC tag were diluted in PBS containing 0.1% BSA, and slides were stained according to the manufacturer’s recommendation. After three washes with PBS, slides were dried and layered with Vectashield antifluorescence fading mounting medium (Vector Laboratories, Burlingame, CA) and a coverslip. The slides were viewed in a Nikon fluorescence microscope using appropriate filters.

Statistical Analysis.
The incidence of IL-13R positivity in GBM and oligodendroglioma patients was analyzed statistically using the unpaired Student’s t test.

	Results and Discussion

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As shown in Table 1 , 18 of 25 (72%) brain tumor samples expressed IL-13R chain, whereas all samples expressed both IL-13R' and IL-4Rß chains. Fig. 1A shows the RT-PCR results of a set of 12 representative samples. Normal astrocytes or brain tissue samples showed low levels of mRNA for the IL-13R chain. However, IL-4Rß and IL-4R' chain mRNA was uniformly present in these cells, and the level of expression was similar to that of tumor cells. Four other normal brain cell lines, derived from normal human oligodendrocytes, NT2 neuronal cell lines, and two cortex tissue-derived cell lines from a patient with encephalitis, showed no or very low levels of mRNA for the IL-13R chain (not shown). Interestingly, whereas a human T-cell line (H9) expressed c chain, its expression was not detected in normal brain or any of the tumor samples. Oligo liquid hybridization confirmed the RT-PCR results and demonstrated gradations in positivity for IL-13R chain in various brain tumor tissues and low level expression in normal astrocytes and brain tissue (Fig. 1B) . Similar to the RT-PCR results, the expression of the IL-13R' chain message was uniform among all samples studied. Thus, a comparison of RT-PCR positivity between tumor samples and correspondingly normal samples (i.e., normal human brain and NHAs) revealed that transcripts of IL-13R appear to be overexpressed in brain tumor cells.

View larger version (82K): [in this window] [in a new window]   Fig. 1. mRNA transcripts for various chains of IL-4 and IL-13 receptors. A, RT-PCR products were resolved in 2% agarose gel and visualized by ethidium bromide staining. Lane 1, brain tumor; Lane 2, normal brain RNA; Lane 3, NHAs; Lane 4, negative control; Lane 5, positive control RNA. PM-RCC RNA served as positive control for IL-13R, IL-13R', and IL-4ß chains (15 , 18) , whereas H9 RNA was used as positive control for c chain (13) . B, oligo liquid hybridization analysis of RT-PCR. Lanes 1–12, brain tumor samples; Lane 13, normal brain; Lane 14, NHAs; Lane 15, negative control; Lane 16, positive control.

View this table: [in this window] [in a new window]   Table 2 Incidence of IL-13R positivity in brain tumor patients

View this table: [in this window] [in a new window]   Table 3 Correlation of IL-13R positivity with age of GBM patients

View larger version (34K): [in this window] [in a new window]   Fig. 2. Immunofluorescence analysis of various receptor chains on brain tumor explant cells. Left panels, brain tumor cells stained with either IgG1 isotype or IgG control; middle panels, NHAs; right panels, brain tumor cells. Cells were stained with mouse monoclonal anti-IL-13R (), mouse monoclonal anti-IL-13R' ('), rabbit polyclonal IL-4Rß (ß) antibody, rabbit polyclonal C (c) antibody, or mouse monoclonal anti-GFAP antibody (GFAP).

IL-13R on brain tumor cells appeared to be of type I. This receptor complex appears to be formed of IL-13R, IL-13R', and IL-4Rß chains. This configuration is similar to the configuration that has been observed on RCC cells and normal fibroblasts (11, 12, 13 , 15, 16, 17) . Whether all three chains simultaneously form an IL-13R complex is not known. It is also not known whether all three chains are required for IL-13 functions on tumor cells. Recently, it has been shown that the EC domain of IL-13R chain is present in the serum and urine of mice (20) . It was hypothesized that this EC domain of IL-13R may serve as a carrier protein for IL-13 and modulate its functions in vivo. Human plasma and urine samples were not shown to generate soluble IL-13R (20) . Whether brain tumor cells produce the EC domain of the IL-13R chain is not known and is the subject of investigation in our laboratory.

The significance of IL-13R chain expression on brain tumor cells has not yet been analyzed in detail. It is possible that IL-13R could serve as a biomarker of malignant astrocytoma for predicting response to therapy or to monitor recurrence. Because IL-13R is overexpressed on 72% of brain tumor cells compared with normal brain or astrocytes, it also is possible that IL-13R is involved in oncogenesis. In that regard, the IL-13R chain may behave like other growth factor receptor, such as EGFRvIII, which is overexpressed in up to 50% of malignant glial tumors (4) . Similarly, enhanced expression of TfR in GBM cells in vitro has been demonstrated. Although TfR is also present in normal endothelial cells, overexpression of this receptor may reflect the increased need of tumor cells for iron (21) . Exploiting this property of EGFR and TfR, many cytotoxins and immunotoxins have been designed that target tumor cells either in vitro or in vivo. Among these, DAB₍₃₈₉₎-EGF fusion protein (made of diphtheria toxin and human EGF) and two immunoconjugates that consisted of an anti-EGFR monoclonal antibody covalently linked to the type 1 ribosomal-inactivating proteins, i.e., ocymoidine and pyramidatine from Saponaria ocymoides and Vaccaria pyramidata, are found to exert specific inhibition of EGFR-expressing target cell proliferation and growth of human tumor cells in nude mice (22 , 23) . Two other immunotoxins, i.e., 454A12-rRA and anti-tfnR-CRM 107, have also been developed that target TfR, among which the latter is found effective in reducing by 50% tumor volume in 60% of patients with malignant brain tumors (5) . We have also produced a chimeric fusion protein comprising IL-13 and a mutated form of Pseudomonas exotoxin (termed IL-13-PE38QQR). This cytotoxin is highly cytotoxic to IL-13R-positive malignancies, including brain tumors in vitro and in vivo (8, 9, 10 , 24, 25, 26) .

Our previous studies have demonstrated that human glioblastoma cell lines express different levels of IL-13R, and IL-13-PE38QQR is extremely cytotoxic to cell lines that expressed higher numbers of IL-13 receptors. However, cell lines that expressed low levels of IL-13R were dramatically less sensitive to this targeted cytotoxin (8) . The molecular reasons for this difference in receptor numbers in different glioblastoma cell lines and the cytotoxicity to IL-13-PE38QQR was not clear. We recently found that these glioblastoma cells lacked expression of IL-13R chain,³ indicating the importance of the IL-13R chain for IL-13 binding and sensitivity to IL-13-PE38QQR. To confirm this hypothesis, we have transfected the IL-13R chain in IL-13R-negative cell lines and found that radiolabeled IL-13 binding and sensitivity to IL-13-PE38QQR were dramatically increased compared with wild-type cells.⁴ In addition, radiolabeled IL-13 did not bind to normal human brain tissues from six samples (10) , and IL-13-PE38QQR was not cytotoxic to NHAs.⁵ These observations suggest that normal brain cells do not bind detectable IL-13 and that the differentially higher expression of IL-13R chain on tumor cells would most likely serve as a primary target for receptor-targeted tumor therapy.

In conclusion, multiple lines of evidence suggest that IL-13R is a novel tumor-associated transmembrane protein in malignant astrocytoma/GBM that may serve as a biomarker of the brain malignancy, a target for receptor-directed cytotoxin therapy, or alternatively, a new tumor-rejection antigen for immunotherapy.

	ACKNOWLEDGMENTS

 
We thank Drs. Raynold Donnelly and Steve Bauer of the Center for Biologics, Evaluation and Research for reviewing the manuscript, Pamela Dover and Dr. Syed Rafat Husain, Laboratory of Molecular Tumor Biology, and Drs. Robert Duncan and Hira Nakhasi, Laboratory of Parasitic Biology and Biochemistry, for their support and help, and Christopher Vargas and his team at Scientific Computing Resource Center, NIH for help in fluorescence imaging.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at NIH Building 29B, Room 2NN10, 29 Lincoln Drive, Bethesda, MD 20892. Phone: (301) 827-0471; Fax: (301) 827-0449; E-mail: Puri{at}cber.fda.gov

² The abbreviations used are: GBM, glioblastoma multiforme; EGFR, epidermal growth factor receptor; TfR, transferrin receptor; IL, interleukin; IL-4R and IL-13R, IL-4 and IL-13 receptors; RT-PCR, reverse transcription-PCR; NHA, normal human astrocyte; GFAP, glial fibrillary acidic protein; EC, extracellular.

³ P. Leland and R. K. Puri, unpublished observations.

⁴ K. Kawakami and R. K. Puri, unpublished observations.

⁵ B. H. Joshi and R. K. Puri, unpublished observations.

Received 10/ 5/99. Accepted 1/17/00.

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				K. Kawakami, M. Kawakami, and R. K. Puri Nitric Oxide Accelerates Interleukin-13 Cytotoxin-Mediated Regression in Head and Neck Cancer Animal Model Clin. Cancer Res., August 1, 2004; 10(15): 5264 - 5270. [Abstract] [Full Text] [PDF]

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				D. A. Todhunter, W. A. Hall, E. Rustamzadeh, Y. Shu, S. O. Doumbia, and D. A. Vallera A bispecific immunotoxin (DTAT13) targeting human IL-13 receptor (IL-13R) and urokinase-type plasminogen activator receptor (uPAR) in a mouse xenograft model Protein Eng. Des. Sel., February 1, 2004; 17(2): 157 - 164. [Abstract] [Full Text] [PDF]

				M. Kawakami, K. Kawakami, J. L. Kasperbauer, L. L. Hinkley, M. Tsukuda, S. E. Strome, and R. K. Puri Interleukin-13 Receptor {alpha}2 Chain in Human Head and Neck Cancer Serves as a Unique Diagnostic Marker Clin. Cancer Res., December 15, 2003; 9(17): 6381 - 6388. [Abstract] [Full Text] [PDF]

				T. F. Liu, S. B. Tatter, M. C. Willingham, M. Yang, J. J. Hu, and A. E. Frankel Growth Factor Receptor Expression Varies among High-Grade Gliomas and Normal Brain: Epidermal Growth Factor Receptor Has Excellent Properties for Interstitial Fusion Protein Therapy Mol. Cancer Ther., August 1, 2003; 2(8): 783 - 787. [Abstract] [Full Text] [PDF]

				A.-h. Wu and W. C. Low Molecular cloning and identification of the human interleukin 13 alpha 2 receptor (IL-13Ra2) promoter Neuro-oncol, July 1, 2003; 5(3): 179 - 187. [Abstract] [PDF]

				K. Kawakami, M. Kawakami, and R. K. Puri IL-13 Receptor-Targeted Cytotoxin Cancer Therapy Leads to Complete Eradication of Tumors with the Aid of Phagocytic Cells in Nude Mice Model of Human Cancer J. Immunol., December 15, 2002; 169(12): 7119 - 7126. [Abstract] [Full Text] [PDF]

				M. Kawakami, K. Kawakami, and R. K. Puri Intratumor Administration of Interleukin 13 Receptor-targeted Cytotoxin Induces Apoptotic Cell Death in Human Malignant Glioma Tumor Xenografts Mol. Cancer Ther., October 1, 2002; 1(12): 999 - 1007. [Abstract] [Full Text] [PDF]

				B. H. Joshi, K. Kawakami, P. Leland, and R. K. Puri Heterogeneity in Interleukin-13 Receptor Expression and Subunit Structure in Squamous Cell Carcinoma of Head and Neck: Differential Sensitivity to Chimeric Fusion Proteins Comprised of Interleukin-13 and a Mutated Form of Pseudomonas Exotoxin Clin. Cancer Res., June 1, 2002; 8(6): 1948 - 1956. [Abstract] [Full Text] [PDF]

				C. Li, W. A. Hall, N. Jin, D. A. Todhunter, A. Panoskaltsis-Mortari, and D. A. Vallera Targeting glioblastoma multiforme with an IL-13/diphtheria toxin fusion protein in vitro and in vivo in nude mice Protein Eng. Des. Sel., May 1, 2002; 15(5): 419 - 427. [Abstract] [Full Text] [PDF]

				S. O. Rahaman, P. Sharma, P. C. Harbor, M. J. Aman, M. A. Vogelbaum, and S. J. Haque IL-13R{alpha}2, a Decoy Receptor for IL-13 Acts As an Inhibitor of IL-4-dependent Signal Transduction in Glioblastoma Cells Cancer Res., February 1, 2002; 62(4): 1103 - 1109. [Abstract] [Full Text] [PDF]

				K. Kawakami, M. Kawakami, P. Leland, and R. K. Puri Internalization Property of Interleukin-4 Receptor {alpha} Chain Increases Cytotoxic Effect of Interleukin-4 Receptor-targeted Cytotoxin in Cancer Cells Clin. Cancer Res., January 1, 2002; 8(1): 258 - 266. [Abstract] [Full Text] [PDF]

				K. Kawakami, M. Kawakami, P. J. Snoy, S. R. Husain, and R. K. Puri In Vivo Overexpression of IL-13 Receptor {alpha}2 Chain Inhibits Tumorigenicity of Human Breast and Pancreatic Tumors in Immunodeficient Mice J. Exp. Med., December 17, 2001; 194(12): 1743 - 1754. [Abstract] [Full Text] [PDF]

				B. H. Joshi, P. Leland, A. Asher, R. A. Prayson, F. Varricchio, and R. K. Puri In Situ Expression of Interleukin-4 (IL-4) Receptors in Human Brain Tumors and Cytotoxicity of a Recombinant IL-4 Cytotoxin in Primary Glioblastoma Cell Cultures Cancer Res., November 1, 2001; 61(22): 8058 - 8061. [Abstract] [Full Text] [PDF]

				K. Kawakami, M. Kawakami, B. H. Joshi, and R. K. Puri Interleukin-13 Receptor-targeted Cancer Therapy in an Immunodeficient Animal Model of Human Head and Neck Cancer Cancer Res., August 1, 2001; 61(16): 6194 - 6200. [Abstract] [Full Text] [PDF]

				G.Y. Gillespie BRAIN TUMOR IMMUNOTHERAPY Neuro-oncol, July 1, 2001; 3(3): 216 - 217. [PDF]

				W. Debinski, B. H. Joshi, G. E. Plautz, and R. K. Puri Correspondence re: B. H. Joshi et al., Interleukin-13 Receptor {alpha} Chain: A Novel Tumor-associated Transmembrane Protein in Primary Explants of Human Malignant Gliomas. Cancer Res., 60: 1168-1172, 2000. Cancer Res., July 1, 2001; 61(14): 5660 - 5660. [Full Text] [PDF]

				Y. Oshima and R. K. Puri Characterization of a Powerful High Affinity Antagonist That Inhibits Biological Activities of Human Interleukin-13 J. Biol. Chem., April 27, 2001; 276(18): 15185 - 15191. [Abstract] [Full Text] [PDF]

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