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Buffalo and cow milk caseins were submitted to hydrolysis either with á -chymotrypsin or with pepsin. Enzymatic peptide modification (EPM) was carried out by using L-methionine ethyl ester in the reaction mixture. As catalyst, á -chymotrypsin or pepsin was used. The incorporation of methionine in to the peptide chains in the presence of á -chymotrypsin showed an optimum value at 0.14 g Met added to the reaction mixture/1 g hydrolysate in both cases. In the case of pepsin used as catalyst, the optimal Met-enrichment was at 0.14 g Met added to the reaction mixture/1 g buffalo casein hydrolysate and at 0.34 g Met/1 g cow casein hydrolysate. The covalent nature of the amino acid incorporation was confirmed by SDS - polyacryl amide gel electrophoresis in the presence of urea. Electrophoretic patterns of the products indicate that transpeptidation plays an essential role in the EPM reaction. Antigenic character of the EPM- products was investigated in vitro by competitive indirect ELISA. Enzymatic peptide modification with methionine enrichment seems to be an efficient method for the reduction of the antigenic/potential allergenic character and for the improvement of the nutritive value of buffalo and cow milk caseins.
Mice (Balb/c), with peanut allergy induced, were subjected to desensitization therapy with the use of pea protein extract (PE) or isolated globulin fractions: legumin (PL) and vicilin (PV). B- and T-cell responses to peanut proteins were analysed by determination of the IgE, IgG1, and IgG2a antibody levels in plasma and the concentration of IL-4, IFN-gamma and IL-10 cytokines secreted by isolated splenocytes.Conducted studies have demonstrated that immunotherapy with proteins resulted in the decrease of total IgE and peanut-specific IgG1 levels and significantly enhanced synthesis of peanut-specific IgG2a in plasma (ELISA method) and at the cellular level (ELISPOT type B). A successful and effective immunotherapy is related to the shift in profile of lymphocytes from Th2 subpopulation towards Th1 subpopulation. In our studies significant increase in the activity of Th1 lymphocytes was observed in groups desensitized with pea protein extracts (PE) and pea legumin fraction (PL). In these groups, significant statistic decrease in IL-4 secreted and increase in IL-10 level were found.Desensitization method with the use of pea proteins being suggested in the presented studies can be an alternative method for specific immunotherapy for people, especially with strong allergic reaction to peanuts; however, this method needs further studies with mouse model.
In this study, we examined the relationship between levels of lactoferrin (LF) and IL-17 in human serum and breast milk and the development of allergy in children. LF and IL-17 levels were determined by ELISA in healthy (n=19) and allergic mothers (n=21) on the 5th day after delivery. Two years later, information on breastfeeding and allergic outcomes was collected by questionnaires from parents of both groups and district child care nurses. Significantly higher concentrations of LF were found in the breast milk of allergic mothers compared to the healthy controls. At 2 years of age, only those three infants became allergic from the atopic group in whose starting breast milk samples a very high LF level (306 μg mg–1 protein) or simultaneously elevated concentrations of LF and IL-17 were measured. These findings indicate that the very early measurement of LF and IL-17 levels in the breast milk of allergic mothers may help to predict the allergy development in their infants.
Non-alcoholic fatty liver disease (NAFLD) is developed mainly by insulin resistance and oxidative stress, but the exact pathogenesis is unknown. Increased prevalence of non-organ specific autoantibodies (NOSA) in NAFLD may be the result of primary immune-mediated mechanism or secondary hepatocellular injury as a consequence of free radical reaction and cytokine production. The importance of NOSA positivity in NAFLD is uncertain. Our aim was to investigate the NOSA prevalence and the redox status as well as the cytokine level in NAFLD patients. Plasma free SH-group concentration, total antioxidant status were measured by colorimetric methods. Free radical–antioxidant balance was determined by a chemiluminometric assay. IL-6 concentration was measured by ELISA in various group of NAFLD patients. NOSA (antinuclear antibody) prevalence was found to be 55% in NAFLD patients. NOSA-positive patients showed a decrease of plasma free SH-group concentration, total antioxidant status and strengthening of free radical reaction. Elevated IL-6 concentrations were measured in both patient groups, but IL-6 concentration was higher in the ANA-negative group with better antioxidant status.
Objective: To determine the relationship between the immunological status and anti-HCV detection of HIV-1 infected individuals in an HIV/AIDS cohort within an African population. Design: Retrospective study of randomly selected HIV/AIDS patients. Methods: The biodata of 1044 consenting HIV infected patients were analyzed retrospectively. These patients had been enrolled following the confirmation of their HIV status by western blot assay after an initial reactive ELISA. Blood obtained from the patients were subjected to serological tests to determine their anti-HCV antibody status using third generation Enzyme Immunoassay (DIA.PRO Diagnostic, Bioprobes srl, Italy). CD4 count was done by flow cytometry (Partec Cyflow, Germany) and viral load by PCR (Roche Amplicor 1.50). The data were analyzed using the Epi Info 2004 statistical software. Results: Ninety out of 1044 patients (8.6%) were positive for anti-HCV antibodies. The rate of HCV infection was directly proportional to the CD4+ group (6.8% vs. 15.4% for < 201 and > 800, respectively, p = 0.026). Conclusion: There is an associated higher chance of detecting anti-HCV in sera of the HIV-1 infected patients whose immunological status are better compared to severely immunocompromised ones.
Abstract
Selenium is an essential component of selenoproteins, enzymes with extensive regulatory and protective effect in organism. Immunological effects of Se are documented and are distinct even above concentrations necessary for maximal activity of selenoenzymes. Therefore, we investigated effect of supplementation by 100 μg of yeast-bound Se on concentrations of thyroid autoantibodies TPOAb and TgAb in the group of 253 seniors living in the Asylum Houses of South Bohemia. Increase of serum selenium from 59 to 150 μg Se/L serum in supplemented group and from 59 to 72 μg Se/L serum in group with placebo were detected by Instrumental Neutron Activation Analysis (INAA) and proved increased Se intake during the trial. Autoantibodies were analyzed by ELISA at the beginning of the trial and after 1 year. Statistical evaluation of results in whole groups (regardless of increased autoantibodies) by ANOVA manifested significant decrease of TPOAb and TgAb in non-supplemented group while supplementation did not effect serum autoantibodies concentrations. Evaluation of groups of seniors created from those with increased autoantibodies, ANOVA demonstrated decrease of TPOAb in both groups but Se supplementation did not affect the decrease. In opposite, TgAb increased significantly and Se supplementation led to higher increase of TgAb. Recent results of possibility to decrease serum concentration of TPOAb proved this effect only for high TPOAb concentrations and for higher Se supplements. From this point of view, it is necessary to conduct subsequent trials with the patients with autoimmune thyreoiditis with different levels of autoantibodies and detect also serum Se levels.
Hungarian cattle herds were surveyed for bovine herpesvirus 1 (BHV-1) infection by ELISA of milk and serum samples. In 1993, 75% of the large cattle herds (consisting of more than 50 cattle) and all small herds (small-scale producers' stocks), while in 1997 90% of the small herds were included in the survey. In the case of large herds, 79.3% of the herds and 64.1% of the samples tested were found to be positive. Of the small herds, 13.5% and 15.7% tested positive in 1993 and 1997, respectively. The majority of large herds were Holstein-Friesian dairy stocks. Small herds with an infection rate markedly exceeding the average were found in those counties where the small herds had been in close contact with the large-scale farms, or where new herds were established by using animals of uncontrolled infectious bovine rhinotracheitis (IBR) status originating from large farms. Attention is called to the importance of maintaining the IBR-free status of small herds that constitute one-third of the Hungarian cattle population.
Serum/plasma fructosamine (SeFa) concentration is a reliable indicator used in human diabetic control. Tests for monitoring the carbohydrate/energy metabolism of (farm) animals are less commonly performed in veterinary laboratories, since most of the reliable determinations, both automated and manual, are relatively expensive. The aim of this study was to develop a precise, money- (and time-) saving automated micro method for measuring SeFa. ELISA microplates (20 µL samples and 200 µL reagents) and an automatic microplate autoreader were used. The classical nitroblue tetrazolium (NBT) stain reagent solution of Johnson et al. (1982) was modified using a SIGMA reagent to render it stable for up to one year. SeFa concentrations measured by the new method in 30 human blood plasma samples were compared with values obtained by the standard (generally used) LaRoche kit procedure. Fifteen cow, 13 dog and 18 chicken plasma samples were assayed by the new automated ‘micro’ method as well as by the manual test tube ‘macro’ method commonly used earlier. The modified reagent was applied for both methods. The coefficient of correlation (r) between the results obtained by the two methods was consistently between 0.94 and 0.98 (p < 0.001).
The objective of this study was to investigate whether baboon females respond to an ovarian stimulation protocol incorporating pituitary suppression with a GnRH agonist (GnRHa) and either highly purified human FSH (hphFSH) or recombinant human FSH (rhFSH) with follicular development and oocyte maturation. A modified human ovulation induction protocol was applied to 5 adult female baboons with a history of regular menstrual cycles (33–34 days). A long-acting GnRHa implant containing goserelin acetate was placed subcutaneously (s.c.) on Days 22–24 of their menstrual cycle. Concentrations of serum oestradiol (E2), progesterone (P4) and human FSH were obtained by ELISA. Menses occurred ∼ 10 days after GnRHa implantation. Daily hphFSH or rhFSH (75 IU i.m.) treatments were started ∼ 10 days following menses. When the majority of follicles were ≥ 5 mm in diameter and the E2 levels had reached a maximum, hCG (2000 IU i.m.) was administered to induce final maturation of oocytes and ovulation. Thirty to 34 h after hCG administration, transabdominal follicular aspiration was performed using a variable frequency transvaginal transducer with ultrasound. A total of 71 oocytes were collected from 4 animals (average: 17). The meiotic maturity of oocytes was evaluated 3 h after retrieval. Ninety-one percent of oocytes were in metaphase 2 and of grades I and II which are appropriate for in vitro insemination.
In contrast to most of the soluble cytokine receptor antagonists properties, the soluble IL-6 receptor (sIL-6R) occurring in various body fluids of healthy persons and patients with various diseases is an agonist. The enhancing effect is due to its ability to form complex with IL-6 and to bind to gp130 making constitutively IL-6 receptor negative cells responsive for IL-6. The generation as well as the functional role of soluble IL-6 receptor is poorly understood. Earlier, we found that the sIL-6R levels in sera of patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) were higher than those of the control group measured by ELISA sandwich technology. In the present study we detected different levels of sIL-6R in the supernatants of lymphocyte cultures of healthy persons and patients with RA as well as SLE. Moreover, we found, that in vitro dexamethasone treatment stimulated generation of sIL-6R in both healthy persons and in active SLE, while it strongly suppressed production of sIL-6R in both RA groups. At mRNA level, we found that in SLE both the IL-6R mRNA encoding the membrane spanning and alternatively spliced (soluble) variants increased. Surprisingly, the strong decrease of sIL6R protein in RA was not found at mRNA level.
