Simultaneous amperometric immunosensing of the metastasis-related biomarkers IL-13Rα2 and CDH-17 by using grafted screen-printed electrodes and a composite prepared from quantum dots and carbon nanotubes for signal amplification.

This paper describes a dual electrochemical immunoassay for the simultaneous determination of IL-13Rα2 and CDH-17, two biomarkers of emerging relevance in metastatic processes. The sandwich assay uses a screen-printed dual carbon electrode that was electrochemically grafted with p-aminobenzoic acid to allow the covalent immobilization of capture antibodies. A hybrid composed of graphene quantum dots (GQDs) and multiwalled carbon nanotubes (MWCNTs) act as nanocarriers for the detection antibodies and horseradish peroxidase. The use of this hybrid material considerably improves the assay (in comparison to the use of MWCNTs) due to the peroxidase mimicking activity of the GQDs. The method works at a low working potential (0.20 V vs. Ag pseudo-reference electrode) and thus is not readily interfered by unknown electroactive species. The dual immunoassay allows for the selective determination of both biomarkers with LOD values of 1.4 (IL-13sRα2) and 0.03 ng mL-1 (CDH-17). The simultaneous determination of IL-13Rα2 and CDH-17 was accomplished in lysates from breast and colorectal cancer cells with different metastatic potential, and in paraffin-embedded tumor tissues extracts from patients diagnosed with colorectal cancer at different stages. The applicability to discriminate the metastatic potential even in intact cells through the detection of both extracellular receptors has been demonstrated also. The assay can be performed within 3 h, requires small sample amounts (0.5 µg), and has a simple protocol. Graphical abstract Dual amperometric immunosensing of the metastasis-related biomarkers IL-13Rα2 and CDH-17 in human colorectal cancer cells and tissues by using grafted screen-printed electrodes and composites of quantum dots and carbon nanotubes as nanocarriers.

Electrochemical immunosensor for IL-13 Receptor α2 determination and discrimination of metastatic colon cancer cells.

This work describes the first electrochemical immunosensor reported for the determination of IL-13 receptor Rα2 (IL-13Rα2), an emerging relevant biomarker in metastatic colon cancer. The approach involves the formation of sandwich immunocomplexes using specific capture (CAb) and biotinylated detector antibodies (BDAb) further labeled with an streptavidin-horseradish peroxidase (Strep-HRP) polymer, onto carboxylic acid-modified magnetic microbeads (HOOC-MBs). Amperometric detection at disposable carbon screen-printed electrodes (SPCEs) using the (H2O2)/hydroquinone (HQ) system was employed to monitor the affinity reactions. The developed immunosensor exhibits a linear calibration plot over the 3.9-100 ng mL-1 concentration range, a LOD of 1.2 ng mL-1 and excellent selectivity against other non-target proteins. The amperometric immunosensor was applied successfully to quantify for the first time the IL-13Rα2 expression in raw lysates of colon cancer cells and to discriminate the metastatic potential of intact cells through recognition of this target extracellular receptor. In comparison with the commercial Enzyme-Linked ImmunoSorbent Assay (ELISA) kit involving the same immunoreagents, the immunosensor provides a similar LOD in a half-time for the assay.

Detecting Chronic Post-Traumatic Osteomyelitis of Mouse Tibia via an IL-13Rα2 Targeted Metallofullerene Magnetic Resonance Imaging Probe.

Differential diagnosis of chronic post-traumatic osteomyelitis (CPO) from aseptic inflammation remains challenging, since both pathological processes share similar clinical symptoms. Here we utilized a novel targeted metallofullerene nanoparticle based magnetic resonance imaging (MRI) probe IL-13-TAMRA-Gd3N@C80(OH)30(CH2CH2COOH)20 to detect CPO in mouse tibia via overexpressed IL-13Rα2 receptors. The functionalized metallofullerene was characterized by X-ray photoelectron spectroscopy. Upon lipopolysaccharide (LPS) stimulation, macrophage Raw 264.7 cells showed elevated IL-13Rα2 expression via immunofluorescence staining and increased MRI probe binding via built-in TAMRA fluorescence imaging. Trauma was induced in both tibia of mice and bacteria soaked suture was inserted into the right tibia to initiate infection. During the acute phase (1.5 weeks), luminol-bioluminescence imaging revealed much higher myeloperoxidase activity in the infected tibia compared to the sham. In the chronic phase (4 weeks), X-ray radiography illustrated bone deformation in the infected tibia compared to the sham. With T1 weighted sequences, the probe clearly exhibited hyperintensity in the infection foci at both acute and chronic phases, which was not observed in the sham tibia. Histological analysis revealed severe bone structural destruction and massive inflammatory cell infiltration in the infected tibia. Immunohistochemistry confirmed abundant expression of IL-13Rα2 in the infection site. In summary, we developed a noninvasive imaging approach to detect and differentiate CPO from aseptic inflammation using a new IL-13Rα2 targeted metallofullerene MRI probe. In addition, for the first time, IL-13Rα2 was investigated as a unique biomarker in the context of osteomyelitis. Our data established a foundation for the translational application of this MRI probe in the clinical differentiation of CPO.

High expression of IL-13 receptor α2 in colorectal cancer is associated with invasion, liver metastasis, and poor prognosis.

Autocrine secretion of cytokines by metastatic colorectal cancer cells and their role during invasion and liver homing has been poorly characterized. In this study, we used cytokine arrays to analyze the secretomes of poorly and highly metastatic colorectal cancer cells. Compared with poorly metastatic cancer cells, highly metastatic cells expressed increased levels of the immunosuppressive cytokines interleukin (IL)-4 and IL-13 in addition to increased surface expression of the high affinity IL-13 receptor IL-13Rα2, suggesting that IL-13Rα2 mediates IL-13 effects in colorectal cancer cells. Silencing of IL-13Rα2 in highly metastatic cells led to a decrease in adhesion capacity in vitro and a reduction in liver homing and increased survival in vivo, revealing a role for this receptor in cell adhesion, migration, invasion, and metastatic colonization. In support of this, IL-13 signaling activated the oncogenic signaling molecules phosphoinositide 3-kinase, AKT, and SRC in highly metastatic cells. Clinically, high expression of IL-13Rα2 was associated with later stages of disease progression and poor outcome in patients with colorectal cancer. Our findings therefore support a critical role for IL-13Rα2 expression in colon cancer invasion and metastasis.

Determination of single cell surface protein expression using a tagless microfluidic method.

We describe a method to detect the expression of a surface protein in single cells without prior labeling or manipulation using a microfluidic device. When the protein is expressed on a cell surface, it undergoes transient bond formation with an immobilized ligand as the cell is pumped through a microfluidic channel, resulting in a specific decrease in the cell's velocity. We were able to detect the expression of interleukin 13 receptor alpha 2 (IL13Rα2) differentially expressed on LM2 cells, a subline of MDA-MB-231 human breast cancer cells with a unique lung metastatic capability. The detection of cells with high expression of the protein was near 100% sensitive and 100% specific. We also provided proof of principle of multiplexing by tracking the same cell over two, differentially-coated patches. The method is non-destructive and cells can be collected for reanalysis. The system can identify positive cells in a cell mixture. This method will have a potential impact in analyzing cancer cells when only a few are available, such as the case with needle aspirates and when labeling and manipulation result in cell loss.

Overexpression of interleukin-13 receptor-alpha2 in neuroendocrine malignant pheochromocytoma: a novel target for receptor directed anti-cancer therapy.

CONTEXT: Pheochromocytomas and paragangliomas are rare catecholamine-secreting neuroendocrine tumors arising from the adrenal medulla and sympathetic tissues. When complete surgical resection is not an option, the treatment of pheochromocytoma is limited. OBJECTIVE: The objective of the study was to identify and characterize overexpression of IL-13 receptor-alpha2 (IL-13Ralpha2) gene expression in human and murine tumors and verify xenograft mouse pheochromocytoma cell (MPC)-derived tumor's response to a selective cytotoxin. DESIGN/SETTING/PATIENTS: Expression of IL-13Ralpha2 was evaluated in a panel of 25 human pheochromocytoma clinical samples by RT-PCR and eight MPC tumors by indirect immunofluorescence assay and RT-PCR. INTERVENTION: The function of IL-13Ralpha2 in these tumor cells was examined by evaluating tumor sensitivity to a recombinant IL-13-Pseudomonas exotoxin (IL-13PE). Subcutaneous small and large MPC tumors in athymic nude mice (n = 10) were treated intratumorally with IL-13PE (100 m icrog/kg). MAIN OUTCOME MEASURES: IC(50) and tumor size were measured. RESULTS: IL-13PE immunotoxin was highly cytotoxic to IL-13Ralpha2-overexpressing MPC cells (IC(50) <2.5 ng/ml) in vitro. Furthermore, IL-13PE was highly cytotoxic to sc tumors. Our results showed a statistically significant decrease in tumor size as early as 3 d after initial treatment and further suppressed growth of MPC tumors. All tumors displayed a histological evidence of necrosis in response to IL-13 immunotoxin without any adverse effects in host at this dose. CONCLUSIONS: Human and murine neuroendocrine pheochromocytoma overexpress the IL-13Ralpha2 chain, and an IL-13PE-based receptor-directed anticancer approach may prove useful in treatment for metastatic pheochromocytoma patients.

Paeoniflorin: a monomer from traditional Chinese medical herb ameliorates Schistosoma japonicum egg-induced hepatic fibrosis in mice.

Treatment of liver fibrosis associated with Schistosoma japonicum ova-induced granulomas remains a challenging proposition. There is a close relationship between high levels of interleukin-13 (IL-13) and the development of severe schistosome fibrosis. In contrast, IL-13 receptor (R) α2 has an effective role in attenuation of profibrosis. Several Chinese herbs have significant beneficial effects in liver disease. Accordingly, the purpose of the present study was to investigate the therapeutic effect of Paeoniflorin (PAE) on liver fibrosis. A mouse model for liver fibrosis was established, using infection with S. japonicum cercariae via the skin. Liver tissue was used to examine the effect of PAE on hydroxyproline, collagen I and III, and IL-13 and IL-13Rα2. The results showed that PAE has significant suppressive effect on the increase of both hepatic hydroxyproline and collagen I and III, which are the main components of extracellular matrix (ECM). Meanwhile, PAE not only inhibits IL-13 production, it also elevates IL-13Rα2 in PAE-pretreated groups compared with controls. These results suggested that PAE can improve liver fibrosis due to S. japonicum infection. The effect of PAE appears to depend on a decrease of IL-13 and an increase of IL-13Rα2.

Identification of interleukin-13 receptor alpha2 chain overexpression in situ in high-grade diffusely infiltrative pediatric brainstem glioma.

Human malignant glioma cell lines and adult brain tumors overexpress high levels of interleukin-13 receptor alpha2 chain (IL-13Ralpha2). Because the IL-13Ralpha2 chain is an important target for cancer therapy and prognosis for patients with brainstem glioma (BSG) remains dismal, we investigated the expression of this receptor in specimens of diffusely infiltrative pediatric BSG relative to normal brain tissue. Twenty-eight BSG specimens and 15 normal brain specimens were investigated for IL-13Ralpha2 protein expression by immunohistochemical analysis (IHC) using two different antibodies in two different laboratories. Highly sensitive Q-dot-based IHC and in situ hybridization (ISH) assays were also developed to identify IL-13Ralpha2 protein and RNA in these specimens. The results were evaluated independently in two laboratories in a blinded fashion. By Q-dot IHC or a standard IHC assay, 17 of 28 (61%) tumor specimens showed modest to strong staining for IL-13Ralpha2, while 15 normal brain tissue samples showed weak expression for IL-13Ralpha2 protein. Significant interrater agreement between the two laboratories was seen in the assessment of IL-13Ralpha2 intensity. High-level IL-13Ralpha2 RNA expression was detected in tumor samples by Q-dot ISH, but only weak RNA expression was observed in normal brain. Significant agreement between ISH and IHC assays was observed (simple kappa [kappa] estimate=0.358, weighted kappa=0.89, p=0.001). IL-13Ralpha2 protein and mRNA are expressed to significantly higher levels in BSG than in normal brain tissue. Both IHC and ISH represent robust methods to detect expression of the IL-13Ralpha2 receptor in BSG that could represent an important new drug target for treatment of this disease.

IL-13 receptor isoforms: breaking through the complexity.

Interleukin (IL)-13 is an immunoregulatory cytokine secreted predominantly by activated T-helper type 2 (Th2) cells, and it has been identified as crucial in developing allergic inflammatory responses. Its diverse functions are mediated by a complex receptor system including IL-4 receptor alpha (IL-4Ralpha; CD124) and two other cognate cell surface proteins, IL-13Ralpha1 (CD213a1) and IL-13Ralpha2 (CD213a2). IL-13Ralpha1 forms a heterodimer with IL-4Ralpha that is a signaling IL-13 receptor. In contrast, IL-13Ralpha2 has been thought to be a decoy receptor due to its short cytoplasmic tail. IL-13Ralpha2 exists on the cell membrane, intracellularly, and in soluble form. Recent reports revealed that membrane IL-13Ralpha2 may have some signaling capabilities, and soluble IL-13Ralpha2 is a critical endogenous modulator for IL-13 responses. The receptor has more complicated functions than a simple decoy receptor. In this review, we describe the isoforms of IL-13Ralpha2 and discuss newly revealed functions of IL-13Ralpha2.

Allergen-dependent solubilization of IL-13 receptor alpha2 reveals a novel mechanism to regulate allergy.

BACKGROUND: Allergic sensitization affects half of western populations and often precedes the development of allergic disorders including asthma. Despite the critical role of allergens in the pathogenesis of these disorders, little is known about how allergens modulate the immune response. IL-13 receptor alpha2 (IL-13Ralpha2) is a decoy receptor for IL-13. OBJECTIVE: Although the existence of soluble IL-13Ralpha2 has been documented, the mechanisms underlying its generation are unknown. Many allergens possess protease activity; we investigated whether IL-13Ralpha2 is solubilized in response to allergen treatment. METHODS: We evaluated the ability of allergens to solubilize IL-13Ralpha2 in vitro and in vivo and examined the effect on IL-13 signaling and responses. RESULTS: We determined that treatment of cells with house dust mite (HDM) allergen or purified Dermatophagoides pteronyssinus or Dermatophagoides farinae, but not other allergens, resulted in release of soluble IL-13Ralpha2 that was biologically active and inhibited IL-13 signaling. Prolonged exposure to HDM or treatment with mold allergens resulted in IL-13Ralpha2 degradation. This was associated with increased IL-13 signaling. A single treatment of HDM in vivo resulted in release of IL-13Ralpha2 into the bronchoalveolar lavage (BAL) fluid. BAL fluid from humans also contained IL-13Ralpha2; BAL fluid from individuals with asthma contained less IL-13Ralpha2 than that from controls. CONCLUSION: Allergen exposure can directly affect the level of soluble IL-13Ralpha2 in a way that affects IL-13 signaling and responses. CLINICAL IMPLICATIONS: Soluble IL-13Ralpha2 may be an important biomarker of environmental allergen exposure and asthma.