ovalbumin and Neuroblastoma

Amyloids are highly organized fibril aggregates arise from inappropriately folded form of the protein or polypeptide precursors under both physiological as well as simulated ambience. Amyloid synthesis is a multistep process that involves formation of several metastable intermediates. Among various intermediate species, the as-formed soluble oligomers are extremely toxic to the neuronal cells. In the present study, we evaluated cyclosporine A (CsA), an undecapeptide, for its potential to prevent aggregation of model protein ovalbumin (OVA). In an attempt to elucidate involved operative mechanism, the preliminary studies delineate that CsA affects both primary nucleation as well as other secondary pathways involved in OVA fibrillation process. By its specific interaction with amyloid intermediates, the cyclic peptide CsA seems to regulate the lag phase of the fibrillation process in concentration dependent manner. The present study further suggests that exposure to CsA during lag phase ensues in reversal of OVA fibrillation process. On the contrary, mature OVA fibril remained impervious to the CsA treatment. The cyclic undecapeptide CsA was also found to successfully alleviate amyloid induced toxicity in neuroblastoma cells.

Topics: Amyloid; Cell Proliferation; Cyclosporine; Humans; Immunosuppressive Agents; Neuroblastoma; Ovalbumin; Peptides, Cyclic; Protein Aggregation, Pathological; Tumor Cells, Cultured

Interleukin (IL-) 27 is a member of IL-12 cytokine family with Th1-promoting and anti-inflammatory effects. IL-27 has been shown to facilitate tumor-specific cytotoxic T lymphocyte (CTL) induction against various tumors. However, IL-27 suppresses cytokine production of lymphocytes and antigen-presenting function of dendritic cells (DCs). To examine the in vivo role of IL-27 in generation of anti-tumor immunity, we examined IL-27-mediated antitumor-effects using WSX-1 (IL-27 receptor alpha chain)-deficient (WSX-1(-/-)) mice. In WSX-1(-/-) mice inoculated with B16 melanoma cells, tumor growth was higher than in wild-type (WT) mice. Accordingly, tumor-specific CTL generation was lower in WSX-1(-/-) mice than in WT mice. CTL induction in WSX-1(-/-) mice was not restored by transfer of WT DCs pulsed with TRP2 peptide, indicating that IL-27 is directly required for generation of tumor-specific CTLs. However, when transferred into tumor-bearing mice, WSX-1(-/-) DCs pulsed with TRP2 peptide was more potent than WT DCs in tumor growth inhibition and generation of CTLs, indicating suppressive effects of IL-27 on DC function. Finally, the combination of WT CD8(+) T cells and KO DCs is more potent in generation of antigen-specific CTLs than any other combinations. Expression of perforin gene and percentages of tumor-specific CD8(+) T cells were also the highest in the combination of WT CD8+ T cells and WSX-1(-/-) DCs. It was thus revealed that IL-27 promotes CTL generation while suppressing DC function during generation of tumor immunity. The combination of WT T cells and IL-27 signal-defective DCs may have therapeutic potential against tumors.

Topics: Angiogenesis Inhibitors; Animals; Dendritic Cells; Interleukin-17; Interleukin-2; Melanoma; Melanoma, Experimental; Mice; Mice, Knockout; Neoplasms; Neuroblastoma; Ovalbumin; Receptors, Cytokine; Receptors, Interleukin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; T-Lymphocytes, Cytotoxic; Th1 Cells

The temperature dependency of anandamide uptake into cells implies an active mechanism but this is still a matter of considerable debate. We have therefore re-examined the temperature-sensitive uptake of anandamide in ND7/23 mouse neuroblastoma x rat dorsal root ganglion neurone hybrid cells and RBL2H3 rat basophilic leukaemia cells.. Cellular uptake of [(3)H] anandamide was measured in the presence of bovine serum albumin at different incubation temperatures and times. Rates of uptake were also measured in wells alone. Free anandamide concentrations were calculated by published methods.. Anandamide showed a time-dependent saturable uptake into ND7/23 cells. The uptake was greater at 37 degrees C than at 4 degrees C for a given added anandamide concentration following a 5 min incubation. However, this temperature-dependency reflected temperature-dependent effects on the concentration of anandamide available for uptake, rather than the uptake process itself. A similar conclusion could be drawn for the rapid ( approximately 1 min) uptake of anandamide into RBL2H3 cells. In contrast, re-analysis of published data for P19 cells indicated a clear temperature-dependency of the uptake at long (15 min) incubation times. The level of anandamide retained by wells alone provided a better measure of free anandamide concentrations than calculated values.. ND7/23 cells may be a useful model system for the study of anandamide uptake. The temperature-dependent uptake of anandamide may reflect effects on free anandamide concentrations rather than on the uptake process itself.

Topics: Animals; Arachidonic Acids; Basophils; Cell Line; Cell Line, Tumor; Endocannabinoids; Ganglia, Spinal; Hybrid Cells; Kinetics; Mice; Neuroblastoma; Ovalbumin; Polyunsaturated Alkamides; Rats; Regression Analysis; Serum Albumin, Bovine; Temperature

Internalization, recycling, and resensitization of the human delta-opioid receptor (hDOR) were studied in the neuroblastoma cell line SK-N-BE, endogenously expressing this receptor. Conventional and confocal fluorescence microscopy observations, corroborated by Scatchard analysis, indicated that after a 100 nM Eto treatment, 60 to 70% of hDOR were rapidly internalized (t(1/2) < 15 min). This agonist-triggered internalization was reversible for a treatment not exceeding 1 h and became irreversible for prolonged treatment (4 h), leading probably to the degradation and/or down-regulation of the receptor. The rapid internalization of hDOR was totally blocked in the presence of heparin, known as an inhibitor of G protein-coupled receptor kinases (Benovic et al., 1989), a result indicating that phosphorylation by these kinases is a critical step in desensitization (Hasbi et al, 1998) and internalization of hDOR (present study) in SK-N-BE cell line. Blockade of internalization by agents not interferring with phosphorylation, as hypertonic sucrose or concanavalin A, also blocked the resensitization (receptor functional recovering) process. Furthermore, blockade of dephosphorylation of the internalized hDOR by okadaic acid totally suppressed its recycling to the plasma membrane and its subsequent resensitization. These results indicate that regulatory events leading to desensitization, internalization, and recycling in a functional state of hDOR involve phosphorylation by a G protein-coupled receptor kinase, internalization via clathrin-coated vesicles, and dephosphorylation by acid phosphatases.

Topics: Brain Neoplasms; Concanavalin A; Cyclic AMP; Diprenorphine; Heparin; Humans; Hypertonic Solutions; Immunohistochemistry; Microscopy, Confocal; Narcotic Antagonists; Neuroblastoma; Ovalbumin; Phosphorylation; Radioligand Assay; Receptors, Opioid, delta; Sucrose; Tumor Cells, Cultured

A monoclonal antibody to homovanillic acid (HVA) was prepared by synthesis of a HVA-protein conjugate (HVA-ovalbumin) as an immunogen, immunization of mice, and the subsequent hybridization technique. Monoclonal antibodies were screened on the basis of sensitivity, specificity, and accuracy. An indirect ELISA was developed for quantification of HVA in human urine. The assay was characterized and shown to have high specificity, with cross-reactivities to vanillylmandelic acid and normetanephrine at 0.18% and <0.1%, respectively. The assay coefficients of variation were <10% within the working range of 0.5-40 mg/L. Initial results from testing urine samples of patients with neuroblastoma and other diseases were validated by HPLC, suggesting that this ELISA method is a reliable and convenient system for quantification of HVA in urine and can be used in the mass screening of neuroblastoma in infants.

Topics: Adolescent; Animals; Antibodies, Monoclonal; Child; Child, Preschool; Chromatography, High Pressure Liquid; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Epitopes; Hemocyanins; Homovanillic Acid; Humans; Immunization; Infant; Infant, Newborn; Male; Mice; Neuroblastoma; Ovalbumin; Reproducibility of Results; Sensitivity and Specificity

Recent immunocytochemical and biochemical studies have shown the intracellular uptake of alpha-foetoprotein (AFP) by most neural crest and neural tube derivatives of developing mammals and birds. The neural crest origin of neuroblastomas has been known for a long time. While many mouse neuroblastoma cell lines can express several neuronal properties, other lines lack specialized neural functions and may re-express embryonal or foetal antigens, suggesting some reversion towards an earlier stage of differentiation. We have therefore tested the C-1300 Jackson mouse neuroblastoma cell line for its ability to incorporate AFP. The results obtained confirm the significant internalization of protein by these cells, both in vitro and in vivo. External photoscans of mice bearing tumours after injection with [131I]-AFP have proven the usefulness of the protein as a radiotracer for neuroblastoma localization.

Topics: alpha-Fetoproteins; Animals; Cell Line; Liver; Male; Mice; Mice, Inbred Strains; Neuroblastoma; Ovalbumin; Radionuclide Imaging; Serum Albumin