Radioimmunoassay For Detection Research Articles

Comparison of Gas Chromatography and Liquid Chromatogram Detecting Pesticide Residue

The existing methods of detecting pesticide residue include gas chromatography, high performance liquid chromatography, gas chromatograph-mass, liquid chromatograph-mass, capillary electrophoresis, radioimmunoassay, biosensor and rapid detection on the spot. The paper analyzes the comparison of gas chromatography and liquid chromatogram detecting pesticide residue, for achieving the development tendency and the future goal of analyzing pesticide residue.

Optimized treatment with RF thermotherapy and immunotherapy combined with CyberKnife for advanced high-risk tumors: A clinical trial report

This study was conducted to evaluate the application value of optimized treatment with radiofrequency (RF) thermotherapy and immunotherapy combined with CyberKnife for advanced high-risk tumors. The database of 1,013 patients with 2,136 tumor lesions and 1,237 target areas who underwent treatment with CyberKnife between November, 2010 and November, 2012, was retrospectively reviewed. We randomly assigned 505 eligible patients (observation group) to RF thermotherapy and adoptive immunotherapy with cytokine-induced killer cells and the remaining 508 patients (control group) to no adjuvant treatment. The patients in the two groups were recorded on efficacy assessment according to imageological examination, World Health Organization criteria, Karnofsky performance status, or radioimmunoassay (RIA) detection. The effective rate of the observation group was 75.05%, whereas that of the control group was 58.06% (P<0.05). The results revealed that CyberKnife combined with hyperthermia and biological therapy are highly effective in improving the local tumor control rate. Further analysis of the Karnofsky score and RIA detection confirmed that this type of combination therapy significantly improved the quality of life. The optimized treatment of RF thermotherapy and immunotherapy combined with CyberKnife may act synergistically in eliminating tumor cells, confirming the efficacy of this type of treatment for patients with advanced malignant tumors.

Distinct systemic distribution of salusin-α and salusin-β in the rat

Salusin-α and salusin-β are multifunctional bioactive peptides that were initially predicted using in silico analyses. These peptides should be concomitantly biosynthesized from prosalusin in humans. However, little information is available yet on the biosynthesis and mode of presence of salusin-α and salusin-β in non-human species. In the present study, we examined whether salusin-α and salusin-β are conserved in the rat and whether salusin-α and salusin-β show distinct systemic distributions. Immunohistochemical analysis of rat tissues using a specific anti-rat salusin-α antibody detected immunoreactivity extensively in neuronal cells and fibers, and abundantly in the epithelial tissues throughout the organs. This distribution contrasts sharply with that of salusin-β, which is mainly localized to the neuroendocrine and hematopoietic systems. Western blot analysis of rat spleen extracts showed the presence of cleaved fragments corresponding to putative rat salusin-α. Reverse-phase and gel filtration high performance liquid chromatography analyses coupled with radioimmunoassay detection of rat urine extracts revealed a major immunoreactive component that co-eluted with synthetic putative rat salusin-β. These data support the processing of rat prosalusin into salusin-α and salusin-β despite absent dibasic amino acids between the two.

Lipopolysaccharide Increases Plasma Levels of Corticotropin-Releasing Hormone in Rats

Background: Corticotropin-releasing hormone (CRH) is expressed in the brain, immune cells and the gut, where gene expression is upregulated by lipopolysaccharide (LPS) 6 h after injection. Whether these changes are reflected by increased circulating levels of CRH and adrenocorticotropic hormone (ACTH) is unknown. Methods: LPS (100 µg/kg) was injected intraperitoneally in conscious rats, and blood processed for CRH using the new RAPID (reduced temperatures, acidification, protease inhibition, isotopic exogenous controls and dilution) method compared with EDTA blood with or without plasma methanol extraction. Hormone levels were measured by commercial radioimmunoassay. Results: The RAPID method improved blood recovery of ¹²⁵I-CRH in vitro compared to EDTA only added to the blood without or with methanol extraction (90.8 ± 2.0 vs. 66.9 ± 2.6 and 47.5 ± 2.0%, respectively; p < 0.001 vs. RAPID). Basal CRH levels from blood processed by the RAPID method were 28.9 ± 2.8 pg/ml, and by other methods below the radioimmunoassay detection limit (<10 pg/ml). At 6 h after LPS, CRH plasma levels increased significantly by 2.9 times, and in the proximal colon tended to decrease (–27.6 ± 5.7%; p > 0.05), while circulating levels were unchanged at 3 or 4 h. ACTH levels rose compared to control rats (135.3 ± 13.8 vs. 101.4 ± 6.0 pg/ml; p < 0.05) 30 min after the increase in CRH, while at 3 or 6 h after LPS, the levels were not changed. Conclusion: Intraperitoneal LPS induces a delayed rise in plasma CRH levels associated with an elevation in ACTH plasma levels 30 min later, suggesting that under conditions of immune challenge, CRH of peripheral origin may also contribute to pituitary activation, as detected using the RAPID method of blood processing, which improves CRH recovery.

Release of salusin-β from human monocytes/macrophages

Salusin-α and salusin-β are related bioactive peptides biosynthesized from the same precursor, prosalusin. Despite the potent hemodynamic and proatherosclerotic activities of salusin-β, its exact distribution and biological functions remain largely undetermined because of technical difficulties associated with its unique physicochemical characteristics, such as marked adhesiveness to polypropylene and polystyrene. By circumventing these problems, we recently established a specific radioimmunoassay for detecting immunoreactive human salusin-β. In the current study, we demonstrated the release of salusin-β from the human monoblastic leukemia cell lines, THP-1 and U937. Dilution curves of extracted conditioned media from both cells were parallel with those of standard human salusin-β by radioimmunoassay. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection of the culture supernatants revealed a major immunoreactive component that co-eluted with authentic salusin-β. Both cell lines secreted salusin-β-like immunoreactivity (LI) into serum-free media as a function of time (1234.3 ± 122.7 and 186.7 ± 9.1 fmol/10 5 cells per 24 h). When THP-1 and U937 cells differentiated into macrophages after incubation with 2- O-tetradecanoylphorbol-13-acetate (TPA), they secreted far greater amounts of salusin-β-LI into the culture supernatant (3351.9 ± 899.3 and 1545.8 ± 183.3 fmol/10 5 cells per 24 h). TPA treatment accelerated the processing of prosalusin into its cleaved fragments, suggesting that the increased secretion of salusin-β-LI in THP-1-derived macrophages was caused by the enhanced intracellular processing of prosalusin. Stimulation with the inflammatory cytokines, tumor necrosis factor alpha (TNF-α) and lipopolysaccharide (LPS), resulted in increased secretion of salusin-β without inducing expression of the gene for preprosalusin, suggesting that TNF-α and LPS stimulated the release of salusin-β. These data demonstrate that salusin-β, which induces macrophage foam cell formation, is secreted in its authentic form from human monocytes/macrophages.

Presence of immunoreactive salusin-β in human plasma and urine

Salusin-α and salusin-β are multifunctional bioactive peptides originally identified using bioinformatics analyses. Salusin-β has been shown to exert potent hypotensive, bradycardic, and pro-atherosclerotic effects. The form in which it exists in biological fluids remains undetermined due to technical difficulties originating from its unexpected physicochemical properties. Here we show that salusin-β peptide adheres to polypropylene and polystyrene, so that the aliquoted peptide dissolved in distilled water may rapidly disappear from the solution. By circumventing these features and using an antibody against C-terminal portion of salusin-β, we have successfully established a specific radioimmunoassay suitable for detection of immunoreactive human salusin-β. We have characterized the molecular form of salusin-β in human plasma and urine. The assay detected immunoreactive salusin-β concentrations as low as 5 fmol/tube and the concentration required for 50% inhibition of binding was 122 fmol/tube. Cross-reactivities with salusin-α and other bioactive peptides were negligible. Reverse-phase high performance liquid chromatography coupled with the radioimmunoassay detection after extraction from plasma and urine and using an octyl-silica column, revealed a major immunoreactive component that co-eluted with authentic salusin-β. Salusin-β-like immunoreactivity in normal human urine ranged from 0.23 to 2.22 nmol/l (mean ± SD, 1.16 ± 0.84 nmol/l, n = 10). These data present the first evidence that salusin-β circulates and is excreted in its authentic form, thereby verifying the initially predicted processing sites for salusin-β in humans.

Choc anaphylactique dû à la succinylcholine compliqué de rhabdomyolyse

A young woman, with a contact dermatitis to nickel, experienced a life threatening anaphylaxis after induction of general anaesthesia for an emergency curettage. Hypersensitivity reaction involving IgE antibodies against succinylcholine was proven by positive skin test and radioimmunoassay detection of antibodies against succinylcholine and quaternary ammonium. After restoration of haemodynamics in a few hours, the patient had rhabdomyolysis predominant at lower left leg, which required a fasciotomy. An haemodialysis session was necessary, renal function being normalized thereafter. Several months later, the patient suffers from dysesthesia on the leg and foot. Muscular ischaemia was the consequence of the shock, the external compression due to the compression stockings probably being an additional factor.

Vasoactive intestinal peptide inhibits TNF-α-induced apoptotic events in acinar cells from nonobese diabetic mice submandibular glands

IntroductionThe role of apoptotic secretory epithelium as a pro-inflammatory triggering factor of exocrine dysfunction is currently explored in Sjogren's syndrome patients and in the nonobese diabetic (NOD) mouse model. Vasoactive intestinal peptide (VIP) has anti-inflammatory effects in various models of chronic inflammation. Our goal was to analyse the effect of TNF-α on apoptotic mediators in isolated acinar cells from NOD submandibular gland and their modulation by VIP.MethodsAcinar cells were isolated from submandibular glands of 16-week-old NOD females with salivary flow decline. Age-matched BALB/c females or eight-week-old NOD females were used as controls. Apoptotic mediators and TNF-α receptor expression were assessed by immunoblotting and RT-PCR, caspase 3 activity was assessed by optical density at 405 nm with Ac-DEVD-pNA as a substrate and chromatin condensation by Hoechst stain. They were evaluated in resting conditions and after a 3.5 or 6 hours of culture with TNF-α. VIP effects in acinar cells were assessed at 100 nM in TNF-α-treated cultures and VIP receptor functional assays by radio immunoassay (cAMP) or enzymatic detection (amylase).ResultsNOD acinar cells at 16 weeks present an increased expression of TNF-α receptor1 together with increased Bax, tumour protein 53-induced nuclear protein1α (TP53INP1α), caspase 3 activity and chromatin condensation. Acini from NOD mice were more sensitive to TNF-α-induced increases of apoptotic mediators than control cells. VIP inhibited TNF-α-induced apoptotic events through functional VPAC1 receptors coupled to the protein kinase A (PKA) signalling pathway.ConclusionsOur results indicate that acinar cells isolated from submandibular glands of NOD mice with salivary dysfunction are more sensitive to apoptosis induced by TNF-α which could be prevented by VIP through a PKA-mediated pathway.

Biosynthesis and secretion of salusin-α from human cells

Salusins originally identified using bioinformatics analyses have been shown to act on the cardiovascular and endocrine systems. Although the hypotensive activity of salusin-α is limited, it exerts a significant anti-atherosclerotic effect via suppression of foam cell formation in human monocyte-derived macrophages by down-regulating acyl-CoA:cholesterol acyltransferase-1. Furthermore, serum salusin-α levels show a close negative correlation with the severity of atherosclerotic diseases. However, biosynthesis and secretion of salusin-α peptide from cultured mammalian cells have not been demonstrated to date. We examined the expression, synthesis and release of salusin-α in human-derived cell lines. Preprosalusin mRNA and protein were detected ubiquitously in all cells tested, whereas the processing of preprosalusin into salusin-α peptide is dependent upon each cell type. Immunohistochemical study revealed the most abundant salusin-α-like immunoreactivity to be present in HeLa cells which released salusin-α-like immunoreactivity into the culture supernatant. Analysis of extracted conditioned media from HeLa cells by reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection revealed a single immunoreactive component that co-eluted with authentic salusin-α. These results present the first evidence that salusin-α is biosynthesized and released from human-derived cells.

Plasma and urine aldosterone to plasma renin activity ratio in the diagnosis of primary aldosteronism

To establish the best cut-off value of the aldosterone (ALD)/plasma renin activity (PRA) ratio when screening patients for primary aldosteronism. One hundred and six patients with primary aldosteronism and 100 essential hypertensive patients were investigated in rigorous standardized conditions. The ALD/PRA cut-off values were calculated from both the plasma and urine ALD/PRA ratio and analyzed by receiver operating characteristic (ROC) curve. In patients with PRA below 0.2 ng/ml/h [our radioimmunoassay detection limit], values were calculated both with PRA levels set at 0.2 ng/ml/h ('adjusted') and with PRA levels detected ('unadjusted') in the assay. ROC analysis on the ALD/PRA ratio indicated that the best performance was obtained when the plasma ALD (ng/dl)/PRA ratio was used in comparison with that observed in the urine ALD (microg/day)/PRA ratio. In patients with primary aldosteronism, as a whole group, the cut-off value of 69 corresponded to the best compromise value between sensitivity (96%) and specificity (85%), with and without PRA adjustment. In patients with aldosterone-producing adenoma, the cut-off to obtain 100% sensitivity with high specificity (85%) proved to be 69, with and without PRA adjustment. In patients with bilateral adrenal hyperplasia, both with and without PRA adjustment, the best compromise between sensitivity (94%) and specificity (86%) was a cut-off value of 71. The best cut-off to identify patients with primary aldosteronism, corresponding to 69, was obtained by using the plasma ALD/PRA ratio. Adjustment of PRA to 0.2 ng/ml/h does not interfere with calculation of the plasma ALD/PRA ratio cut-off.

Presence of immunoreactive salusin-α in human serum and urine

Salusins, identified from a full-length enriched human cDNA library by bioinformatics analyses, show mitogenic, neuromodulatory and hemodynamic activities in rats. They are expressed in a wide variety of human tissues, but their precise structures and levels in human body fluids remain unknown. We developed a radioimmunoassay suitable for the detection of immunoreactive human salusin-α and characterized the molecular forms and concentrations of salusin-α in human serum and urine. The assay allowed for measurement of immunoreactive salusin-α concentrations as low as 1 fmol/tube after extraction of serum with an octyl-silica column, and the concentration required for 50% inhibition of binding was 40 fmol/tube. Cross-reactivities with salusin-β and other bioactive peptides were negligible. Salusin-α-like immunoreactivity in normal human serum and urine ranged from 11.0 to 40.4 pmol/l (mean ± S.D., 23.3 ± 8.1 pmol/l, n = 31) and from 18.6 to 367.3 pmol/l (mean ± S.D., 156.8 ± 95.8 pmol/l), respectively. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection revealed a major immunoreactive component that coeluted with authentic salusin-α. These data indicate the presence of salusin-α in human serum and urine, thereby verifying the initially predicted processing sites for salusin-α in humans.

Effects and Mechanism of Changes of Local Neurotransmitters in Rats' Pylorus and Bile Reflux to the Stomach with Stress Ulcer

Stress ulcer occurs primarily in severe conditions, with a high incidence and mortality in intensive care units. However, studies on the association between stress ulcer and bile reflux to the stomach with stress ulcer are still inconclusive. Therefore, our research aimed to determine whether or not bile reflux exists during stress ulcer and then to investigate the effects and mechanism of changes of pyloric local neurotransmitters on bile reflux in such circumstances so as to provide a new pathway for clinical intervention. Cold water immersion was used to copy the stress ulcer model of rats. Sixty-five adult Sprague-Dawley rats of either sex were randomly divided into three groups: the normal control group (n = 10), the stress group (n = 30), and the antagonist group (n = 25). The gastric ulcer index, pH, and bile acid of gastric juice were measured before and after stress. Radio Immunoassay Detection Kit and Biochemic Detection Kit were used to measure local contents of CGRP (calcitonin gene-related peptide) and nitric oxide, respectively, in rats' pylorus. The local contents of nitric oxide in rats' pylorus reached a maximum at 1 hr after stress. The bile acid and pH of gastric juice peaked at 2 hr after stress and the ulcer index peaked at 4 hr after stress. But the local contents of CGRP in rats' pylorus decreased to the minimum at 4 hr after stress. The bile acid and ulcer index in the L-NAME group were significantly lower than in the antagonist control group. However, the bile acid in the hCGRP8-37 group was less than in the antagonist control group. Compared with hCGRP8-37 group, there was a significant reduction in bile acid in the L-NAME group. There was a significant reduction in the ulcer index of the hCGRP8-37 group compared with the L-NAME group and the antagonist control group. There was a certain kind of positive correlation between nitric oxide in rats' pylorus and bile acid to the stomach, for nitric oxide could loosen the pyloric sphincter and increase the bile acid to the stomach. L-NAME might reduce the local nitric oxide contents in rats' pylorus so that bile acid to the stomach might be decreased, obviously with a looser tight pyloric sphincter. Meanwhile, the CGRP in rats' pylorus was negatively associated with the ulcer index, hence CGRP might protect gastric mucosa under stress conditions.

Frog Corticotropin‐Releasing Hormone (CRH): Isolation, Molecular Cloning, and Biological Activity

Corticotropin-releasing hormone (CRH) was isolated from the brain of the European green frog, Rana esculenta, by combining HPLC purification with radioimmunoassay (RIA) detection. The amino acid sequence SEEPPISLDLTFHLLREVLEMARAEQIAQQAHSNRKLMDII was identical with the sequence of bullfrog (R. catesbeiana) CRH that was deduced from a cDNA encoding the CRH precursor. Synthetic frog CRH enhanced the release of thyroid-stimulating hormone (TSH) from dispersed bullfrog pituitary cells in a concentration-dependent manner. The TSH-releasing activity of a bullfrog hypothalamic extract was decreased by approximately 45% in the presence of the CRH receptor antagonist, alpha-helical CRH(9-41), suggesting that CRH is one of the main TSH-releasing factors present in the bullfrog hypothalamus.

Ontogeny of a novel decapeptide derived from POMC-A in the brain and pituitary of the rainbow trout

Trout POMC-A exhibits a unique C-terminal extension of 25-amino acids which is processed in the pituitary and hypothalamus to generate two novel decapeptides, EQWGREEGEE and ALGERKYHFQ-NH 2. The fibers containing these two decapeptides are widely distributed in the brain, suggesting that they may exert neurotransmitter or neuromodulator activities. In the present study, we have investigated the ontogeny of the decapeptide EQWGREEGEE in the trout pituitary and brain. In the pituitary of 29-day embryos and 33-day alevins, EQWGREEGEE-immunoreactive material was observed in a cluster of cells located in the central and rostral region of the gland, respectively. In 47-day alevins, a second group of cells exhibiting EQWGREEGEE-like immunoreactivity was detected in the caudal region of the pituitary and the intensity of labeling in these cells increased in 61-day fry. In the brain, EQWGREEGEE immunoreactivity was detected in 47-day alevins. In 47- and 61-day larvae, immunoreactive elements were mainly detected in the diencephalon. Characterization of the immunoreactive material by reversed-phase high-performance liquid chromatographic analysis combined with radioimmunoassay detection revealed the existence of two major forms which exhibited different retention times than synthetic EQWGREEGEE. The present study indicates that EQWGREEGEE-related peptides are present in the trout pituitary early during ontogeny and appear in the brain only later, and that processing of the C-terminal extension of POMC-A generates distinct molecular species at different developmental stages. These data suggest that alternative processing of the C-terminal domain of POMC-A gives rise to various peptide products that may exert specific activities during trout development.

Relationship between capsaicin-evoked substance p release and neurokinin 1 receptor internalization in the rat spinal cord

The relationship between substance P release and the activation of its receptor in the spinal cord remains unclear. Substance P release is usually measured by radioimmunoassay, whereas the internalization of the neurokinin 1 (NK1) receptor has been used to assess its activation by noxious stimuli. Our objective was to compare substance P release and NK1 receptor internalization produced by capsaicin in rat spinal cord slices. Superfusion of the slices with capsaicin for 3 min produced a gradual increase in substance P release that peaked 3–7 min afterward, and then decreased to baseline levels. The concentration-response curve for capsaicin was biphasic, with concentrations above 10 μM producing significantly less release. The effective concentration for 50% of response (EC 50) for capsaicin, calculated from its stimulatory phase, was 2.3 μM. However, the potency of capsaicin to elicit NK1 receptor internalization in the same slices was one order of magnitude higher (EC 50=0.37 μM) in lamina I, probably because NK1 receptors become saturated at relatively low concentrations of substance P. The potency of capsaicin to produce internalization was progressively lower in lamina III (EC 50=1.9 μM) and lamina IV (EC 50=14.5 μM), suggesting that neurokinins released in laminae I–II become diluted as they diffuse to the inner dorsal horn. To study the correlation between these two measures, we plotted substance P release against NK1 receptor internalization and fitted a saturation binding function to the points. The correlation was good for laminae I ( R 2=0.82) and III ( R 2=0.78), but it was poor ( R 2=0.35) for lamina IV because NK1 receptor internalization kept on increasing at high concentrations of capsaicin, whereas substance P release decreased. In conclusion, amounts of substance P able to activate NK1 receptors may fall under the threshold of detection of radioimmunoassay. Conversely, radioimmunoassay often detects levels of substance P release well over those required to saturate NK1 receptors in the superficial dorsal horn, but that may be able to activate these receptors in nearby regions of the spinal cord.

Extraction and radioimmunoassay quantitation of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) from human dental pulp tissue

The measurement of neuropeptides in complex biological tissue samples requires efficient and appropriate extraction methods so that immunoreactivity is retained for subsequent radioimmunoassay detection. Since neuropeptides differ in their molecular mass, charge and hydrophobicity, no single method will suffice for the optimal extraction of various neuropeptides. In this study, dental pulp tissue was obtained from 30 human non-carious teeth. Of the three different neuropeptide extraction methods employed, boiling in acetic acid in the presence of protease inhibitors yielded the highest levels of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP). High pressure liquid chromatography (HPLC) analysis of dental pulp tissue verified the authenticity of the neuropeptides extracted.

Hormone concentrations in tobacco roots change during arbuscular mycorrhizal colonization with Glomus intraradices.

• Phytohormones are known to play a pivotal role in various developmental processes in plants and in arbuscular-mycorrhizal (AM) fungal-host symbiosis. This study focuses on characterizing the changes in the concentrations of auxins, cytokinins and gibberellins in tobacco (Nicotiana tabacum) during the early stages of colonization by Glomus intraradices, using advanced analytical detection techniques. • High-pressure liquid chromatography analysis followed by radioimmunoassay detection revealed that AM colonization induced the accumulation of specific zeatin riboside-like and isopentenyl adenosine-like compounds in both roots and shoots. • Use of the gas chromatography-mass spectrometry technique on the same developmental stage revealed that gibberellins (GA) of the earl-13-hydroxylation biosynthetic pathway (GA1 , GA8 , GA19 and GA20 ) were significantly more abundant in roots, but not shoots, of AM inoculated plants than in those of nonmycorrhizal plants. Indoleacetic acid concentrations (total and free) remained unaltered by AM colonization. • This study demonstrates that hormonal changes do occur during AM symbiosis with tobacco, before the fungal benefits manifest.

Agonist-specific Ca 2+ signaling systems, composed of multiple intracellular Ca 2+ stores, regulate gonadotropin secretion

Ca 2+ signals regulate many cellular functions, including hormone secretion. Agonist-specific Ca 2+ signaling may arise from the differential mobilization of multiple Ca 2+ stores. Although they act through the same receptor subtype, two gonadotropin-releasing hormones (sGnRH and cGnRH-II) generate quantifiably different Ca 2+ signals in goldfish gonadotropes, suggesting that their Ca 2+-dependent signaling cascades may differ. We combined electrophysiology, Ca 2+ imaging, and radioimmunoassay detection of gonadotropin (GTH-II) secretion to determine the role of intracellular Ca 2+ stores in GnRH-stimulated exocytosis. Our findings suggest that voltage-gated Ca 2+ channels do not mediate acute GnRH-signaling. Instead, both sGnRH- and cGnRH-II-stimulated GTH-II releases are dependent on Ca 2+ mobilized from TMB–8/CPA-sensitive compartments. However, sGnRH, but not cGnRH-II, utilizes intracellular stores sensitive to caffeine and xestospongin C. We also identified a homeostatic mechanism where reduced extracellular Ca 2+ availability increase GTH-II release by mobilizing Ca 2+ stores. Our results are the first to suggest that several classes of intracellular Ca 2+ stores differentially participate in agonist signaling and homeostasis in gonadotropes.

AUTOCRINE IL-6 PRODUCTION BY HUMAN TRANSITIONAL CARCINOMA CELLS UPREGULATES EXPRESSION OF THE α5β1 FIBRONECTIN RECEPTOR

Purpose: Studies have demonstrated elevated expression and secretion of IL-6 by transitional cell carcinomas (TCC) following bacillus Calmette-Guerin (BCG) therapy. At present, the role of IL-6 on the biology of TCC is poorly understood. This study evaluated the influence of IL-6 expression on a critical variable regulating BCG-tumor interaction, the tumor expression of α5β1 integrin. Materials and Methods: A human TCC cell line (253J) was transfected with an expression vector containing the full-length IL-6 cDNA sequence. Overexpression of IL-6 mRNA and protein was confirmed by Northern analysis and ELISA, respectively. Clones found to overexpress IL-6 were then assayed for α5β1 expression using Northern analysis and flow cytometry. The effect of alterations in α5β1 expression on tumor adherence to fibronectin (FN), and BCG adherence to tumor cells was determined using specific adherence assays. Results: mRNAs for both the α5 and β1 subunits of the FN receptor were increased an average of 9.4 fold and 125.7 fold respectively in the IL-6 overexpressing transfectants relative to the parental 253J cells. Increased mRNA of α5 and β1 was associated with increased cell surface expression of both proteins. Increased protein expression resulted in greater FN substrate binding affinity and increased adherence of BCG to tumor cells. Conclusions: Autocrine expression of IL-6 upregulates the expression of FN receptor subunits in TCC. Increased α5β1 expression increases cellular adherence to FN, and BCG adherence to tumor cells. These results suggest a role for IL-6 in mediating the antitumor activity of BCG by influencing BCG's adherence to TCC.

URINARY INTERLEUKIN-2 MONITORING DURING PROLONGED BACILLUS CALMETTE-GUERIN TREATMENT: CAN IT PREDICT THE OPTIMAL NUMBER OF INSTILLATIONS?

URINARY INTERLEUKIN-2 MONITORING DURING PROLONGED BACILLUS CALMETTE-GUERIN TREATMENT: CAN IT PREDICT THE OPTIMAL NUMBER OF INSTILLATIONS?