Soluble saccharides block the inhibition of agonist-induced human platelet aggregation observed after in vitro incubation of human platelet-rich plasma with porcine aortic endothelial cells.

Platelet aggregation is a prominent feature in the hyperacute process of vascularized allografts and xenografts. In a study of extracorporeal connection of pig kidneys to the blood circulation of human volunteers, we observed in one case considerable destruction of human platelets in the pig kidney without signs of hyperacute rejection or microthrombi formation. In the present study, we have investigated the agonist-induced aggregation of human platelets in mixtures with porcine aortic endothelial cells (PAEC). In vitro incubation of human platelet-rich plasma (PRP) with PAEC inhibited platelet aggregation induced by ADP, collagen and arachidonic acid in a time-dependent manner and partially inhibited adrenalin-induced aggregation. Aggregation of the human platelets could not be induced by high concentrations of ADP (20 microM) to overcome the inhibition capacity of the PAEC. The PAEC inhibiting effect could be transferred by the supernatants of PAEC/PRP and PAEC/PPP incubation mixtures. Preincubation of the PAEC with aspirin, but not with NG-methyl-L-Arg, reduced the aggregation inhibitory effect. Control experiments mixing human umbilical vein endothelial cells (HUVEC) and human PRP or mixing porcine PRP and PAEC did not elicit any inhibition of ADP-induced platelet aggregation. The aggregation inhibition effect could partially be blocked by preincubation of PRP with soluble Gal alpha 1-3Gal, Gal alpha 1-3 beta 1-4GlcNAc, lactose, galactose, and glucose, but not by lactosamine, galactosamine, or glucosamine. The Gal alpha 1-3Gal disaccharide was most effective in blocking aggregation inhibition, and to a similar extent as its ability to block the human anti-pig lymphocytotoxicity reaction. In conclusion, the data indicate that PAEC, upon stimulation by human anti-pig xenoantibodies in a nondynamic system, inhibits agonist-induced human platelet aggregation, and that this effect is probably at least partially caused by prostacyclin released from the PAEC.

Effects of Ajoene on lymphocyte and macrophage membrane-dependent functions.

Ajoene, (E, Z) -4, 5, 9-trithiadeca-1, 6, 11-triene 9 oxide, is a compound originally isolated from ethanolic extracts of garlic that impairs platelet aggregation by inhibiting the functional exposure of platelet integrins GPIIb/IIIa. In vitro, Ajoene is toxic for several tumoral cell lines, and exert an antiproliferative effect on T. cruzi and murine malaria parasites. Here we show that Ajoene strongly inhibited the proliferation induced in human lymphocytes by the mitogens phytohemagglutinin (PHA), phorbol myristate acetate (PMA) and anti-CD3, and the capping formation induced in B lymphocytes by anti-IgM antibodies. On macrophages, Ajoene was also found to partially inhibit the lypopolysaccharide-induced production of Tumor Necrosis Factor (TNF), and to decrease the phagocytic activity of thioglycolate-elicited mouse peritoneal macrophages for IgG-opsonized, human erythrocytes. Ajoene also partially prevented the lytic effect of human and rabbit TNF on Actinomycin D-treated WEHI 164 cells. These results strongly suggest that Ajoene is a potent modulator of membrane-dependent functions of immune cells.

Increased lipopolysaccharide-induced tumour necrosis factor levels and death in hypercholesterolaemic rabbits.

Nutritional-induced hypercholesterolaemia in New Zealand rabbits causes increased susceptibility to experimental infections. Rabbits fed cholesterol (0.5 g%) for 8 weeks were injected intravenously with varying doses of Escherichia coli 0127: B8 lipopolysaccharide (LPS; 3-100 micrograms/kg). The levels of cholesterol, triglycerides, tumour necrosis factor (TNF), and the survival rates of treated rabbits were then measured. Rabbits fed either normal chow or chow impregnated with sesame oil were used as controls. LPS induced higher serum TNF levels in hypercholesterolaemic rabbits than in normal rabbits or rabbits fed with chow containing sesame oil. TNF levels rose faster in hypercholesterolaemic rabbits than in normal rabbits, reaching maximum levels at 60 min and 120 min, respectively, after LPS injection. The survival rate of hypercholesterolaemic rabbits (1/11) was lower than in normal rabbits (6/7) or rabbits fed with the sesame oil chow (4/4) at the higher LPS doses. No death occurred at lower doses. One possible interpretation of these results, also supported by neutralization experiments, is that increased TNF secretion in hypercholesterolaemic rabbits raises the host's susceptibility to experimental endotoxaemia and possibly to Gram-negative infection.

Iron overload augments the development of atherosclerotic lesions in rabbits.

Iron, a major oxidant in vivo, could be involved in atherosclerosis through the induction of the formation of oxidized LDL, a major atherogenic factor. This study was designed to test this hypothesis experimentally. Four groups of New Zealand White rabbits were included: iron-overloaded/hypercholesterolemic (group A, n = 8), iron-overloaded (group B, n = 6), hypercholesterolemic (group C, n = 6), and untreated (group D, n = 6). Iron overload was achieved by the intramuscular administration of 1.5 g of iron dextran divided in 30 doses. Hypercholesterolemia was produced by feeding rabbit chow enriched with 0.5% (wt/wt) cholesterol. Serum iron, ferritin, cholesterol, triglycerides, and lipoperoxides in serum were measured throughout the study. Lipoperoxides were measured at the end of the study in liver, aorta, and spleen homogenates. Aortas of groups A and C had multiple lesions; however, group A had greater lesional involvement than group C (P < .05). Lesions were not observed in rabbits fed normal chow (group D). As expected, serum iron and ferritin were above normal levels in groups A and B. Serum cholesterol increased in groups A and C. Lipoperoxides in liver and spleen homogenates of iron-overloaded rabbits were increased. Interestingly, iron deposits were seen by ultrastructural studies in the arterial walls of rabbits in groups A and B. Our study suggests that iron overload augments the formation of atherosclerotic lesions in hypercholesterolemic rabbits.

ABO-haemolytic disease of the newborn (ABO-HDN): factors influencing its severity and incidence in Venezuela.

The objective of this study was to determine the incidence of the Haemolytic Disease of the Newborn due to ABO blood group incompatibility (ABO-HDN) in the population of Caracas attending the Maternity Hospital 'Concepción Palacios'. The relationship between A and B antigens density of cord blood erythrocytes and the cytotoxic activity of antibodies in mothers' sera with the severity of the haemolytic disease, was also studied. From a sample of 245 blood group 'O' mothers, 68 gave birth to full term 'A' or 'B' blood group infants. The evolution of serum bilirubin and the routine haematological values, were followed in all the babies during 72 h after birth, allowing the diagnosis of ABO-HDN in 21 infants. Taking into account that in Venezuela the frequency of blood group 'O' is 59 per cent, it was concluded that in the general population of newborns, 16 per cent present foeto-maternal ABO incompatibility, and the incidence of ABO-HDN was near to 5 per cent. The density of the 'A' and 'B' antigens in cord red cells was studied using an immunoenzymatic assay. No statistically significant association between antigen maturity and severity of the ABO-HDN could be shown. A positive association was found between cytotoxic capacity of mothers' sera and development of ABO-HDN (P < 0.05). Twenty ABO incompatibles children presented moderate late anaemia at 3 weeks of age.

Treatment of ABO hemolytic disease with synthetic blood group trisaccharides.

Thirteen infants, 10 with A-O and 3 with B-O hemolytic disease of the newborn (ABO-HDN), were treated with synthetic A or B blood group trisaccharides (ATS, BTS) which cause dissociation of maternal antibody bound to infant red cells. The clinical outcome was compared with that of a control group of 21 infants treated with phototherapy during the preceding year. Exchange transfusion was required in 2 out of 13 infants in the experimental group and in 7 in the control group. A randomized prospective controlled study is necessary to confirm these results.

Human monoclonal anti-Rh antibodies produced by human-mouse heterohybridomas express the Gal alpha 1-3 Gal epitope.

The presence of the Gal alpha 1-3 Gal structure (Gal epitope) in the carbohydrate component of fifteen human monoclonal antibodies with specificity for the Rh blood group factor and produced by human-mouse heterohybridomas was evaluated. To do that, an antiglobulin-like agglutination test and an enzyme linked immunosorbent assay were performed using an affinity-purified anti-Gal antibody obtained from the serum of an AB blood group donor. Using the antiglobulin reaction, results were obtained showing that only five of the fifteen human monoclonal antibodies tested contained the structure at levels sufficient to allow agglutination. However, all fifteen monoclonals were positive using the more sensitive enzyme linked immunosorbent assay. By means of an indirect immunofluorescence assay the same anti-Gal antibody was used to test the presence of Gal epitopes on the surface of the producer heterohybridomas. Results were obtained indicating that twelve out of the fifteen hybridomas studied do express the Gal structure on its surface. The relevance of these findings is discussed in the context of therapeutic applications of human monoclonal antibodies.

Iron polymers impair the function and maturation of macrophages.

An inhibitory effect of iron salts on various immune functions in vitro has been reported in several laboratories during the last years. This work extends such observations by showing that iron citrate, but not sodium citrate, inhibits the function and maturation of murine macrophages (MOs). However, such inhibition is only observed in the presence of ferric citrate with a metal-to-ligand molar ratio of 1:1, but not with ferric citrate with a metal-to-ligand molar ratio of 1:10 in which the hydrolyzation and polymerization of iron in physiological solutions is prevented. Accumulation of ferric iron on the cytoplasm of MOs was observed, but only in the group of MOs treated with ferric citrate 1:1. Increasing the concentration of serum in the culture medium diminished the inhibitory effect of ferric citrate 1:1. The inhibitory capacity of iron polymer was probably associated to its ability to both interact with the cell constituents of the cytoplasm and stimulate lipid peroxidation.

Evaluation of complement activity by an enzyme immunoassay.

An ELISA-type assay useful for the evaluation of the complement activity in serum is described. Aggregated pooled human IgG (IgGn) prepared so as to exclude large and small aggregates, to resemble soluble circulating immune complexes, was used to coat polystyrene microwells to serve as initiator of complement activation. Fresh serum, at different dilutions, as the source of the complement to be evaluated was added and the plate incubated 90 min at 37 degrees C. Then, a peroxidase-labeled antihuman C3c antibody was added to react with the bound C fragments. This was followed by 2,2'-azino-di-3-ethyl benzothiazoline sulfonic acid (ABTS), as the color reagent used for detection of the enzyme activity. In this system, theoretically, the levels of activating and regulatory complement components are evaluated up to the level of C3 splitting. The assay was applied in healthy volunteers to set normal values and in 15 patients suffering from systemic lupus erythematosus making possible the differentiation of those with normal and low complement levels.

Enzyme-linked immunosorbent assay for the detection of substances that carry blood group A specificity.

An enzyme-linked immunosorbent assay (ELISA) was developed to estimate the amount of material carrying blood group A activity in biologic samples. A soluble synthetic form of the A antigenic determinant (A trisaccharide, ATS) conjugated to peroxidase competes with the blood group A substance present in a biologic sample for anti-A attached to a solid phase by a second antibody coating the plastic micro-wells. A reference curve is constructed by using known quantities of ATS to compete with a fixed amount of ATS-peroxidase conjugate. The A substance activity in a sample is obtained by extrapolating the degree of inhibition of the binding of the ATS-peroxidase conjugate to an equivalent amount of ATS in the reference curve. The assay is reproducible, specific, and sensitive. It has been used in pharmacologic studies to estimate the concentration of ATS in the blood and urine of rats, rabbits, and baboons and in a study with human samples, testing the potential clinical use of ATS to neutralize anti-A when therapeutically indicated. It is also useful for the detection of ABO natural products in secretions, thus allowing the accurate classification of secretor and nonsecretor individuals.

A modified colorimetric method for the measurement of phagocytosis and antibody-dependent cell cytotoxicity using 2,7-diaminofluorene.

A sensitive and rapid colorimetric method for the in vitro determination of phagocytic activity and antibody-dependent cell-mediated cytotoxicity (ADCC) is described. The assay uses red blood cells (RBC) as target cells and relies on the specific oxidation of 2,7-diaminofluorene (DAF) by the pseudoperoxidase activity of hemoglobin (Hb). Generation of fluorene blue (FB), the chromophore formed upon oxidation of DAF, was a linear function of erythrocyte concentration. The oxidation of DAF by peritoneal macrophages (M phi) containing myeloperoxidase was negligible, confirming that the development of color was exclusively due to the pseudoperoxidase activity of Hb. A positive correlation was observed between FB formation and increased phagocytosis of opsonized erythrocytes. Phagocytosis increased as a function of time, reaching a maximum at 90 min of incubation. The phagocytosis of IgG-opsonized erythrocytes was greater than non-opsonized erythrocytes and was inhibited by high concentrations of non-specific human or mouse IgG, showing that phagocytosis was mediated by the Fc gamma receptor of macrophages. The interaction between opsonized RBC and macrophages also evoked an antibody-dependent extracellular lysis, however this process was slower than ingestion. The DAF phagocytosis assay has shown to be very sensitive, simple, rapid and safe.

Infestación por Ascaris: una posible explicación para los altos niveles de IgG anti-A observados en la población venezolana / Ascaris infestation: a possible explanation for the high levels of IgG anti-A found in Venezuela populations

Hemos investigado los niveles de IgG anti-A y los de IgG anti-Ascaris en grupos de nivel socioeconómico alto (NSEA) con un bajo grado de infestación por Ascaris (5,6%), de nivel socioeconómico bajo (NSEB) con un 47,6% por ciento de infestación y de indígenas del Amazonas con un nivel de infestación del 67,5 por ciento. Los títulos de IgG anti-A y anti-B se determinaron por hemaglutinación simple y por la técnica indirecta de Coombs, y los de IgG anti-Ascaris (en su forma adulta) por una técnica inmunoenzimática. Los títulos de IgG anti-A obtenidos por la prueba de Coombs, fueron significativamente mayores en el grupo indígena (media geométrica 1833) y en el de NSEB (659) en relación al grupo de NSEA (225). Aunque hubo una asociación positiva entre la frecuencia de infestación por Ascaris y los niveles de IgG anti-A, al comparar individualmente los títulos de IgG anti-A con los de IgG anti-Ascaris no se obtuvo correlación. Sin embargo, estos resultados no descartan la posibilidad de que componentes de otros estadios del parásito presenten reactividad cruzada con el antígeno A

The effect of cysteine-containing peptides released during meat digestion on iron absorption in humans.

In order to investigate the constituents responsible for the enhancing effect of meat on intestinal iron absorption in humans, two different types of peptic digestion extracts were prepared from 100 g of beef, in which the thiol groups of the resulting peptides were either oxidized (CYS-), or left untreated (CYS+). The absorption of radioiron mixed with 250 g of maize was more than twofold greater when consumed along with the CYS+ extract than with the CYS- (p less than 0.05). It is suggested that the enhancing effect of meat on nonheme iron absorption is due to cysteine, and that cysteine-containing peptides, rather than the free amino acid, are responsible for this effect.

The effect of desferrioxamine on the proliferative response of rat lymphocytes stimulated with various mitogens in vitro.

This paper reports experiments designed to evaluate the effect of desferrioxamine on lymphocytes obtained from the spleen of Sprague-Dawley rats. The results showed that desferrioxamine inhibits the proliferative response of lymphocytes stimulated by concanavalin A or pokeweed mitogen, the effect being proportional to the dose used, and being maximal at the end of the culture period. To exert its effect, desferrioxamine has to be present from the initiation of the cultures--its addition 24 h later markedly reduced its inhibitory effect. Studies of cell viability indicated that the inhibition was not due to toxicity. Lymphocytes obtained from young animals (4 to 12-week-old) were more sensitive to desferrioxamine than older animals (13 to 18-week-old). Saturation of desferrioxamine with iron abolished its effect, suggesting a direct relationship between chelating properties and inhibition, and reinforcing the hypothesis that iron plays an important role in the process of cell division.

Circulating immune complexes, immunoglobulins, complement, antibodies to dietary antigens, cholesterol and lipoproteins levels in patients with occlusive coronary lesions.

The presence of circulating immune complexes, serum immunoglobulins, C3 (the third component of complement), antibodies to alpha-lactalbumin, beta-lactoglobulin and bovine serum albumin was studied in 39 patients subjected to coronary arteriography. Total serum cholesterol and triglycerides and cholesterol in VLDL, LDL and HDL were also estimated. The results obtained in the group of patients found with occlusive lesions were compared with those found in the group without lesions. With one of the five assays used for the detection of immune complexes, higher and significant levels were found in the group with lesions. A negative and significant correlation was found between the number of vessels with lesions and the levels of serum C3.