Bisphenol A and Bisphenol S Oxidative Effects in Sheep Red Blood Cells: An In Vitro Study

Bisphenols (BPs) are plastic components widely used worldwide and occurring in the environment. Exposure to these compounds is known to be harmful for animals and humans at different levels. The aim of this study was to evaluate and compare the oxidative effects of bisphenol A (BPA) and bisphenol S (BPS) in sheep. Reactive oxygen species (ROS) production and correlated structural alterations in sheep erythrocytes were investigated in vitro. Blood samples from four ewes were collected at fasting from the jugular vein using vacuum collection tubes containing EDTA. For ROS assay in erythrocytes, blood was properly diluted and BPA or BPS was added to obtain final bisphenol concentrations in the range between 1 and 300 μM. 2 ′ ,7 ′ -Dichlorodihydrofluorescein diacetate (H2DCF-DA) 3 μM was added to the samples, and fluorescence was read in four replicates using a microplate reader. To evaluate erythrocyte shape, blood smears of blood treated with the different concentrations of BPS and BPA were prepared. A significant increase in ROS production was observed when concentrations of BPS and BPA increased from 1 to 100 μM ( p < 0.05 ). At the higher concentrations of the two studied BPs (300 μM of BPS and 200-300 μM of BPA), a ROS decrease was observed when compared to the control group ( p < 0.01 ). Erythrocytes’ shape alterations were observed in cells treated with BPS and BPA 200-300 μM 4 hours after the beginning of the treatment. This study confirms that BPA and BPS exhibit oxidative effects on sheep erythrocytes. At higher concentrations, BPA was able to modify erythrocytes’ shape, while BPS altered their membrane as a sign of a protein clustering that could lead to eryptosis. These BPs’ effects are consequent to intracellular ROS increase.

Complement Genetic Variants and FH Desialylation in S. pneumoniae-Haemolytic Uraemic Syndrome

Haemolytic Uraemic Syndrome associated with Streptococcus pneumoniae infections (SP-HUS) is a clinically well-known entity that generally affects infants, and could have a worse prognosis than HUS associated to E. coli infections. It has been assumed that complement genetic variants associated with primary atypical HUS cases (aHUS) do not contribute to SP-HUS, which is solely attributed to the action of the pneumococcal neuraminidase on the host cellular surfaces. We previously identified complement pathogenic variants and risk polymorphisms in a few Hungarian SP-HUS patients, and have now extended these studies to a cohort of 13 Spanish SP-HUS patients. Five patients presented rare complement variants of unknown significance, but the frequency of the risk haplotypes in the CFH-CFHR3-CFHR1 region was similar to the observed in aHUS. Moreover, we observed desialylation of Factor H (FH) and the FH-Related proteins in plasma samples from 2 Spanish and 4 Hungarian SP-HUS patients. To analyze the functional relevance of this finding, we compared the ability of native and “in vitro” desialylated FH in: (a) binding to C3b-coated microtiter plates; (b) proteolysis of fluid-phase and surface-bound C3b by Factor I; (c) dissociation of surface bound-C3bBb convertase; (d) haemolytic assays on sheep erythrocytes. We found that desialylated FH had reduced capacity to control complement activation on sheep erythrocytes, suggesting a role for FH sialic acids on binding to cellular surfaces. We conclude that aHUS-risk variants in the CFH-CFHR3-CFHR1 region could also contribute to disease-predisposition to SP-HUS, and that transient desialylation of complement FH by the pneumococcal neuraminidase may have a role in disease pathogenesis.

Characterization of sheep erythrocyte glycosphingolipids recognized by human anti-Forssman antibodies

The FORS histo-blood group system is the most recently discovered carbohydrate-based human blood group system. FORS is a rare blood group system, and most individuals have naturally occurring anti-FORS1 antibodies in plasma. Screening for anti-FORS1 antibodies is often done by hemagglutination assays using FORS1-expressing sheep erythrocytes, since FORS1-positive human erythrocytes are most often not available. Here, we have characterized the non-acid glycosphingolipids from sheep erythrocytes and isolated subfractions, with mass spectrometry, binding of antibodies and lectins, and by enzymatic hydrolysis. This demonstrated the presence of Forssman and Galili pentaosylceramides, and a Galili heptaosylceramide. Two complex glycosphingolipids recognized by human anti-FORS1 antibodies were characterized as a Forssman neolacto hybrid hexaosylceramide (GalNAcα3GalNAcβ3Galβ4GlcNAcβ3Galβ4Glcβ1Cer) and a Forssman Galili hybrid heptaosylceramide (GalNAcα3GalNAcβ3Galα3Galβ4GlcNAcβ3Galβ4Glcβ1Cer). These are novel glycosphingolipid structures, and to our knowledge, the first case of an elongated Galili antigen. Thus, the anti-Forssman antibodies in human serum bind not only to the classical Forssman pentaosylceramide (GalNAcα3GalNAcβ3Galα4Galβ4Glcβ1Cer), but also when the GalNAcα3GalNAcβ3 sequence is presented on a neolacto core chain and even on a Galili carbohydrate sequence.

Antiimmunosuppressive action of 3d-metal gluconates in experimental immunodeficiency

Aim. Evaluation of the effect of 3d-metal gluconates on complement-fixing function of immunoglobulin G and functional activity of complement. Methods. The study was conducted in vivo on 2.5-3 month-old white laboratory mice weighing 25-28 g with secondary immunodeficiency, which was induced by a single intraperitoneal injection of cyclophosphamide, as well as in vitro in a test system using sensitized sheep erythrocytes. Immunological studies were performed in intact animals, and before and after the administration of Mn, Co, Cu, and Zn gluconates to mice with induced immunodeficiency. The content of immunoglobulin G and its complexes with subcomponent of the complement first component C1q was determined in serum by ELISA using specific monoclonal antibodies. Results. Two-week oral administration of 3d-metal gluconates (Mn, Co, Cu, Zn) in a dose of 1/10 LD50 to immunodeficient mice was shown to cause a significant increase in the level of immunoglobulin G and its complexes with C1q. The greatest increase in concentration was observed with the introduction of zinc gluconate. Also by means of sensitized sheep erythrocytes in vitro, cobalt and, to a lesser extent, manganese gluconates were shown to increase the functional activity of C1q. Conclusion. 3d-metal gluconates (Mn, Co, Cu, Zn) demonstrate immunocorrecting properties: increase the content of immunoglobulin G and its complexes with C1q, significantly decreasing as a result of cyclophosphamide effect; cobalt and manganese gluconates have a stimulating effect on the functional activity of complement by its classical pathway, which indicates different mechanisms of immunocorrection action of studied metal gluconates and requires further studies.

Characterization of E. coli pathotypes of bovine and livestockfarm environment origin

In the environment of the farms, feed, fodder and water could be contaminated with fecal material especially which could constitute a reservoir of Enterobacteriaceae bacteria.Total of 80 fecal samples collected from diarrheic calves, normal calves, diarrheic cattle, healthy cattle and shed of cattle were included in this study. Of which 67 (83.75%) isolates were biochemically identified as E.coli. Among 67 E.coli isolates, 12 (17.91%) isolates were of diarrheic cases (10 isolates from 1 month-6 months calf and 2 isolates from diarrheic cow), 51 (76.11%) isolates were of healthy cows (14 isolates from 1 month-6 months calf and 37 isolates from normal cows), 2(2.98%) from water samples, one isolate (1.49%)each from cow manure and air sample at farm respectively, from cow shed. Eleven (16.41%) out of 67 isolates were found to cause lysis on sheep erythrocytes and 55 (82.089%) out of 67 isolates were found to be biofilm producers on Congo red. Twenty-four (35.82%) isolates out of 67 were positive for bfpA gene, eight (11.94%) for eaeC gene, while five (7.46%) for both the genes of E.coli strain.

THE DEVELOPMENT OF TOTAL COMPLEMENT ACTIVITY ASSAY BASED ON COMPLEMENT-DEPENDENT IMMUNE LIPOSOME LYSIS

Background: The purpose of work was development of a fast and reproduced procedure for measurement of the total complement activity (ТСА) in human or animal blood serum. Materials and methods: Steady at storage liposomes preparations, which surface sensitized 2,4-DNP haptens, and the internal volume contains calceine or sulforhodamine 101 are obtained. Complement-dependent immune lysis of liposomes at presence of the anti-2,4-DNP immunoglobulines and complement preparations from animals are investigated. Results: It is shown that the degree of liposomes immune lysis depends on complement concentration in a wide range that can be used for definition of TCA level. Research of blood sera from patients has revealed correlation (r =0,793) between data received with the help of liposome immunolytic systems, and the data of nephelometric analysis with application of suspension sheep erythrocytes. Conclusion: The method allows to define total complement activity in blood serum in 15 minutes without separation of reaction components. This might be useful for measurement ТСА level at patients with various diseases and realization of scientific researches.