Mouse monoclonal antibodies against the most acutely toxic substances, botulinum neurotoxins (BoNTs) of types A, B, E, and F, was generated and characterized, that recognize their respective toxins in natural toxin complex. Based on these antibodies, we developed sandwich-ELISA for quantitative detection of these toxins. For each respective toxin the detection limit of the assay was: BoNT/A - 0.4 ng/ml, BoNT/B - 0.5 ng/ml; BoNT/E - 0.1 ng/ml; and for BoNT/F - 2.4 ng/ml. The developed assays permitted quantitative identification of the BoNTs in canned meat and vegetables. The BNTA-4.1 and BNTA-9.1 antibodies possessed neutralizing activity against natural complex of the botulinium toxin type A in vivo, both individually and in mixture, the mixture of the antibodies neutralized the higher dose of the toxin. The BNTA-4.1 antibody binds specifically the light chain (the chain with protease activity) of the toxin, whereas BNTA-9.1 interacts with the heavy chain. We believe that the BNTA-4.1 and BNTA-9.1 monoclonal antibodies are prospective candidates for development of humanized therapeutic antibodies for treatment of BoNT/A-caused botulism.
Blood concentration of soluble Fas antigen is higher in patients with benign and malignant tumors in comparison with healthy subjects, which probably suggests its involvement into tumorigenesis. Key Words: tumors; apoptosis; soluble Fas antigenApoptosis, programmed cell death, is an important mechanism maintaining homeostasis in multicellular organisms. Disturbances in cell elimination underlie various pathological states such as malignant tumors, Alzheimer disease, multiple sclerosis, amyotrophic lateral sclerosis, thyroiditis, hepatitis, and autoimmune diseases.Fas/APO-1/CD95 is a type I transmembrane glycoprotein belonging to the TNF/NDF receptor family [5]. Fas is expressed in various tissues, in particular in the thymus, liver, heart, lungs; it is present on activated lymphocytes, natural killers, virus-infected and tumor cells.Similarly to tumor necrosis factor p55, Fas not only induces cell apoptosis upon interaction with specific ligand FasL or agonistic monoclonal antibodies (MCA) to Fas, but also activate transcription factor NtkB [14]. The mechanism triggering cell death via Fas is studied in detail. The interaction of Fas with FasL or agonistic MCA induces activation of caspase-8 or caspase-2 proteases via adapter proteins FADD/ MORT-1 or RIP and RAIDD, respectively [11,14] Molecular cloning studies and nucleotide-sequence analysis have demonstrated that apart from transmembrane Fas receptor, cells produce also soluble Fas (sFas), a product of alternative spicing of full-length Fas mRNA, which inhibits cytotoxicity induced by anti-Fas MCA in vitro [2,3]. Thus, enhanced production of sFas can underlie cell resistance to apoptosis.Elevated blood concentration of sFas was observed in some autoimmune disease: systemic lupus erythematosus [15] The aim of the present study was to determine serum concentration of sFas in patients with malignant tumors of various morphology and localization and to explore the relationship between sFas and these diseases. MATERIALS AND METHODSHybrid cells producing MCA to recombinant full-length Fas (Coultronics) were obtained according to a standard protocol [9]. Splenocytes from immunized BALB/c mice (3 intraperitoneal injections with a 2-week interval) were fused with mouse myeloma cells NSO/1
The initial levels of soluble Fas antigen (sFas), leptin, and vascular endothelium growth factor (VEGF) were measured in the sera of 100 patients with ovarian cancer and benign tumors and in 60 healthy women aged 28-65 years. Serum levels of sFas and VEGF were elevated in the total group of patients with ovarian tumors, while leptin levels were the same as in healthy women. The studied parameters did not depend on the age of patients and healthy women. The levels of sFas and leptin were virtually the same in benign and malignant ovarian tumors, while VEGF concentration was higher in patients with ovarian cancer. The mean serum levels of sFas, VEGF, and leptin in patients with poorly and moderately differentiated serous ovarian cancer were 2-fold higher than in well-differentiated tumors (p<0.05), while serum concentrations of sFas and leptin increased with the disease stage progress in patients with ovarian cancer (p<0.05). According to the data of unifactorial analysis, the increase in serum levels of sFas and VEGF in ovarian cancer patients correlated with short duration of the relapse-free period. Multifactorial analysis showed that the disease stage (p=0.006), presence of ascites (p=0.03), VEGF concentration (p=0.02), and the sFas/leptin coeffi cient (p=0.045) are highly signifi cant independent factors for predicting the relapse-free survival of patients with serous ovarian cancer.Ovarian cancer ranks fi rst in the structure of mortality from malignant tumors of the female reproductive sys tem [2]. Analysis of the Russian and foreign publications showed unsatisfactory results of treatment of patients with ovarian cancer, because by the moment of diagnosis about 70% patients have advanced stages of the tumor process [3]. In addition, ovarian cancer unites the histogenetic variants of tumors, the majo rity of which are characterized by aggressive clinical course and are liable to early metastasizing. The aggressive nature of the tumor manifests by its capacity to invasion and metastasis development. This can be due to histogenesis, differentiation degree, and genome damage, all this leading to activation of the factors closely related to "biological" behavior of the tumor.Studies of biological factors essential for tumor growth and metastasizing will lead to better understanding of some stages in the pathogenesis of ovarian tumors. Inhibition of Fas-dependent apoptosis is an important component in the system of transformed cell defense from antitumor immunity. The Fas receptor triggers apoptosis in the target cell after interactions with its ligand (FasL). Soluble Fas (sFas) distantly
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