Effect of pregnancy and placental factors on the quality of humoral immune response.

Asymmetrical IgG molecules are characterised by the presence of a mannose-rich oligosaccharide group in only one of the two Fab fragments, which impairs the corresponding paratope, causing such molecules to behave as univalent antibodies and therefore as antigen blockers [1-3]. During human and murine pregnancy, an increase has been detected in asymmetrical IgG molecules in serum and those bound to the placenta, which normally releases factors capable of modulating the immune response. It thus seemed of interest to investigate the effect of placental culture supernatants (PCS) on in vivo and in vitro synthesis of rat immunoglobulin IgG1, IgG2a, IgG2b and IgG2C, particularly the ratio of symmetrical and asymmetrical molecules in each isotype. The effect of PCS was determined in vivo by means of passive transfer to virgin females and in vitro by analysing the supernatants of spleen cells cultured in the presence of PCS. The results showed that neither pregnancy status nor PCS were capable of modifying serum levels of IgG2a, IgG2b or IgG2c, whereas the level of IgG1 was reduced. When PCS were added to the spleen cells cultures, an in vitro increase was observed in IgG2a, IgG2b and IgG2c production. The separation of symmetrical from asymmetrical IgG molecules was performed by affinity chromatography in Concanavalin A-Sepharose, as such lectin binds high mannose sugars present only in asymmetrical IgG molecules. It is shown that pregnancy and PCS induce an increase in IgG1 and IgG2 molecules asymmetrically glycosylated, capable of binding to ConA-Sepharose. Therefore, the placenta is capable of releasing factors which can regulate the relative proportion of asymmetrical IgG molecules and induce quantitative and qualitative modifications of the in vitro and in vivo produced antibodies.

A study on the venom yield of venomous snake species from Argentina.

A study on the venom yield of snakes from Argentina over a three year period was carried out on adult specimens of Bothrops alternatus (n = 74); Bothrops neuwiedii (n = 127); Bothrops ammodytoides (n = 30); Bothrops moojeni (n = 14); Bothrops jararaca (n = 14); B. jararacussu (n = 6); Crotalus durissus terrificus (n = 120) and Micrurus spp. (n = 6) as well as with 12 specimens of newborn C. d. terrificus kept in captivity. While for each species there was a positive correlation between venom yield and number of snakes milked, the correlation with the snake's body weights after individual milkings was even better, suggesting that the size of the snakes is more important in determining the venom yield than the number of snakes milked or the specimen's sex. Individual milkings indicated that, in addition to the snake size, when the amount of venom is normalized per 100 g body weight there is a species specific difference in venom yield. It follows the order B. jararacussu > B. moojeni approximately = B. jararaca approximately = B. alternatus > B. neuwiedii> Micrurus spp approximately = B. ammodytoides> C. d. terrificus. Although the venom yield per 100 g body weight of newborn C. d. terrificus specimens is 2-fold higher than that of adults, no correlation was observed between venom yield and body weight.

A comparison of different methods to assess the hemorrhagic activity of Bothrops venoms.

The hemorrhagic activity of Bothrops (B.) alternatus, B. ammodytoides, B. jararaca, B. jararacussu, B. moojeni and B. neuwiedii venoms from specimens captured in Argentina was assayed after i.d. injection to mice. The hemorrhagic haloes produced by each venom had different color intensities, although no significant differences were observed by measurement of the average diameters or the weight of the excised hemorrhagic haloes. Conversely, important differences were found by measuring the amount of hemoglobin extracted from excised hemorrhagic haloes of similar size produced by different venoms. The relationship between the amount of hemoglobin extracted and the weight of the excised hemorrhagic haloes was linear, with a slope (hemoglobin released per gram of hemorrhagic halo) characteristic for each venom, and proportional to the potency. On this basis, the activity of B. alternatus, B. ammodytoides and B. jararaca is similar, about 1.5 times higher than that of B. jararacussu and B. moojeni venoms and threefold higher than that of B. neuwiedii venom. Thus, measurement of the of hemoglobin released provides additional information in comparative studies, and may be used to assess the antihemorrhagic potency of antivenoms.

Some toxic and enzymatic activities of Bothrops ammodytoides (yarará ñata) venom.

Bothrops ammodytoides, the smallest representative of this genus, is found only in Argentina. Venom was extracted from thirty adult specimens (35-70 cm in length, 90-300 g in weight) captured in the Province of Buenos Aires and kept in captivity. Venom yield was 3-30 mg. SDS-PAGE showed strong bands at 14.0; 23-25; 45; 54 and 63 kDa and weak bands at 17.0; 30.0; 40.0 and 85.0 kDa. Toxic activities were: LD50 (intravenous, mice) 0.5+/-0.2 microg/g; minimal procoagulant dose on human plasma (MPD-P) 35+/-2 mg/l; and minimal defibrinogenating dose (MDD, mice) 6-12 microg. Hemorrhagic and/or necrotic activities appear to play a major role in lethality; minimal hemorrhagic dose (MHD, mice) is 10+/-2 microg/g and minimal necrotizing dose (MND, mice) is 38+/-5 microg. The LD50, MPD-P and MND are among the lowest in venoms from Bothrops species found in Argentina. B. ammodytoides venom exhibited high proteolytic and phospholipase A2 activities. Most of the B. ammodytoides venom components cross-react with Bivalent Bothropic antivenom (Instituto Nacional de Producción de Biológicos ANLIS Dr. G. Malbrin, against B. alternatus and B. neuwiedii venoms). One ml of antivenom neutralizes 1.2 mg of B. ammodytoides venom.

IgG precipitating and non-precipitating antibodies in rabbits repeatedly injected with soluble and particulate antigens.

The immune response of precipitating antibodies and non-precipitating antibodies of high affinity (co-precipitating) of the IgG class was analyzed in rabbits repeatedly injected with egg albumin (as a soluble antigen) B. abortus-egg albumin and polymerized egg albumin (as particulate antigens). The results showed that the levels of anti-egg albumin non-precipitating antibodies induced by the soluble antigen were never higher than 10-15% of total antibodies throughout the experimental time. When particulate antigens were injected, the levels of non-precipitating antibodies increased up to 30-70% of the total antibody levels. This phenomenon is related to the way in which the antigen is available to the immune system (particle or aggregated), and is independent of the response induced by the particulate carrier. Components from the cell wall or bacterial membrane that could act as coadjuvants do not participate in this phenomenon. The results obtained seem to indicate that possibly there was a suppression of the synthesis of precipitating antibodies, and this would produce a relative increase in the non-precipitating antibodies.

[Cross neutralization of bothrops jararacussu venom by heterologous antivenoms]. / Neutralización cruzada de veneno de Bothrops jararacussu por sueros antiofidicos heterologos.

We have studied the immunochemical cross-reactivity and cross-neutralization of the lethal potency, hemorrhagic, necrotizing, procoagulant and (indirect) hemolytic activities of Bothrops jararacussu venom by the standard antivenoms produced in Argentina. These antivenoms are horse immunoglobulin F (ab')2 fragments from animals immunized with 1) Crotalus durissus terrificus venom (Monovalent Anticrotalic antivenom); 2) Bothrops alternatus and B. neuwiedii venoms (Bivalent Botropic antivenom); 3) B. alternatus, B. neuwiedii, B. jararaca and B. jararacussu venoms (Tetravalent Bothropic, or "Misiones" antivenom) and 4) B. alternatus, B. neuwiedii and C. d. terrificus venoms (Trivalent Botropic-Crotalic antivenom). In preincubation experiments, all the heterologous antivenoms neutralized the toxic and biological activities of B. jararacussu venom with a potency at least as high as the Tetravalent Botropic (i.e. the only homologous) antivenom, in which B. jararacussu venom was included as immunogen. These results suggest the possibility of using heterologous antibothropic antivenoms for the treatment of snake bites by B. jararacussu.

[Production of coprecipitating antibody in rabbits immunized repeatedly with ovalbumin in different physical states]. / Producción de anticuerpo coprecipitante en conejos inmunizados a repetición con ovoalbumina en diferente estado físico.

A comparative study has been made of rabbit precipitating and coprecipitating anti-egg albumin antibody in rabbits repeatedly injected with soluble and particulate antigens. Four different antigens were used. a) Soluble egg albumin. b) Polymerized egg albumin. c) Egg albumin linked Brucella abortus 19 strain. d) Brucella abortus 19 strain. Rabbits were subcutaneously injected every 2 weeks during 29 weeks. Before each inoculation they were bled and the concentration of serum precipitating and coprecipitating antibodies was determined. In animals inoculated with the soluble antigen, coprecipitating antibodies were present during the whole course of the immune response and they constituted 10% of the total antibody population. In rabbits injected with particulate antigen (egg albumin linked Brucella abortus) coprecipitating antibodies were also present during the whole course of the immune response but their serum concentration was not the same in different periods of time. An increase in coprecipitating antibodies began at the 13th week reaching 50% of the total antibodies at the 28th week. Similar results had been obtained in rabbits injected with Brucella s. p. In animals inoculated with polymerized egg albumin, coprecipitating antibodies increased to high levels from the 1st to the 23rd week; during this period it constituted 50% of the total antibody population, after which, the animals became tolerogenic. On the basis of these results, the increase in coprecipitating antibodies in rabbit sera would be related to: 1) antigen physicochemical characteristics (soluble o particulate); 2) the number of epitopes in the particle, and 3) the size of the antigen particles.

Incidence of rat-soluble placental factors on IgE and IgG2a synthesis.

PROBLEM: The in vivo effect of soluble factors present in placental culture supernatants (PCSs) on the synthesis of rat immunoglobulin E (IgE) and IgG2a isotypes was investigated. METHOD OF STUDY: Batches of Wistar SPF rats immunized with a 10-microgram dose of ovalbumin and Al(OH)3 were used: group I, consisted of virgin rats; group II, virgin females injected simultaneously with PCSs; and group III, pregnant females. As controls, nonimmunized batches were included. Serum samples were collected at days 0 (basal) and 10 after antigen challenge, determining levels of total and specific antiovalbumin of both IgE and IgG2a by enzyme-linked immunoadsorbent assay (ELISA). RESULTS: In vivo and at least at the doses administered, PCSs exert an inhibitory effect on the synthesis of specific and total anti-ovalbumin IgE during the course of immune response to such challenge. However, PCSs did not modify serum values of total and specific IgG2a. CONCLUSIONS: These results suggest that PCSs exert selective influence on the synthesis of diverse immunoglobulin isotypes during immune response, through the balance of cytokines synthesized by placental cells.

Asymmetric non-precipitating antibodies in commercial hyperimmune gamma-globulin for therapeutic use.

The presence of asymmetric (non-precipitating or co-precipitating) antibodies has been studied in three commercial preparations of antitetanus gamma-globulin. It was found that 27% of the specific antibodies are of asymmetric type, a value two to three times higher than that found in normal IgG. In toxicity tests in mice with tetanus toxin, the asymmetric antibodies were 2.7 times less effective than the symmetric ones. It is concluded that the protection capacity of the tetanus gamma-globulin preparations is dependent on the ratio of symmetric to asymmetric antibodies. The high content of asymmetric antibodies is probably due to the fact that immunization is performed with the antigen in a particulate form.

A possible explanation for the discrepancy between ELISA and neutralising antibodies to tetanus toxin.

The structure and protective activity of tetanus antibodies elicited in rabbits after whole-cell pertussis diphtheria-tetanus vaccine (DTPw) vaccination was studied. ELISA antibody levels and toxin neutralisation activity (TNT) were measured in individual serum samples. The ratio of symmetric and asymmetric (functionally monovalent) IgG molecules was determined by concanavalin A (Con A) chromatography. This test is based on the fact that the carbohydrate group responsible for the molecular asymmetry has high affinity for the lectin Con A. Asymmetric molecule ratio was observed to increase with immunisation time, as well as differences between TNT and ELISA levels. All serum samples were overestimated by ELISA as compared to TNT assay, in line with the markedly higher proportion of asymmetric molecules which have lower toxin neutralising activity. Protective levels could not be predicted reasonably from ELISA results below 0. 222 IU/ml, because this methodology fails to discriminate between both types of antibodies and only an in vivo serum neutralisation procedure (TNT) reflects the true neutralising serum activity.

Relationship between structure and neutralizing activity of rabbit tetanus antibodies elicited by acellular and whole-cell pertussis DTP vaccines.

Symmetric and asymmetric IgGs having different neutralizing capacity are synthesized in variable proportions by the same clones during the course of immune response. The neutralizing activity of tetanus antibodies was studied in rabbits vaccinated with acellular (DTPa) or whole-cell pertussis (DTPw) vaccines. Symmetric and asymmetric F(ab)'2 fragments from the IgG fraction of the peak serum pools from each group of rabbits were purified by concanavalin A chromatography and measured by ELISA. After the third vaccine dose the asymmetric antibody percentage for DTPw (40%) was twice that for DTPa (20%). The neutralizing activity of asymmetric antibodies was roughly sixfold lower than symmetric ones. When antibody values titrated by ELISA approach minimal protective level, the proportion of symmetric antibodies with high toxin neutralizing activity acquires crucial importance.

Structure and protective capacity of tetanus and diphtheria antibodies produced during human pregnancy and transferred to new-born.

PROBLEM: The structure and protective activity of antibodies against tetanus (anti-T) and diphtheria (anti-D), produced during human pregnancy and transferred to new-born, was studied. METHOD: Antibody levels were measured by ELISA in non-pregnant women (control group), primiparae, and multiparae, and in their children. The proportion of symmetric and asymmetric IgG molecules was determined and their respective protective capacity evaluated. RESULTS: The quantity of asymmetric anti-T and anti-D antibodies in mothers at the time of delivery was roughly four- and three-fold that of the control group, respectively, dropping significantly 1 month later. A similar proportion of these antibodies was observed in the new-born. The lower neutralizing capacity of asymmetric molecules was demonstrated in vivo. CONCLUSION: Results show that during pregnancy there is a modulation of the immune response with an increase in the production of asymmetric molecules of lower protective capacity.

Interleukin regulation of asymmetric antibody synthesized by isolated placental B cells.

PROBLEM: Protecting antibodies against trophoblast surface molecules were previously described. Here we analysed the synthesis of asymmetric IgG by placental B-lymphocytes. METHOD OF STUDY: B cells were isolated from human term placenta and cord blood, stimulated with anti-CD40 IgG and cocultured with transfected Fcgamma R-expressing mice Ltk-fibroblast. Interleukin-4, IL-6, IL-10, IL-11 and IL-13 were added to cultures for 14 days. Asymmetric IgG were assessed in culture supernatants by concanavalin A (Con A) fixation and enzyme-linked immunosorbent assay. RESULTS: When IL-6 was added to the cultures, the percentages of asymmetric IgG synthesized by placental B cells were: IL-6: 29 +/- 10; IL-6 + IL-10: 24 +/- 7; IL-4 + IL-10 + IL-6: 38 +/- 9. The last combination induced the highest increase in the asymmetric IgG synthesis as compared with control (19 +/- 10%, P < 0.05). Additionally, placental B cells synthesized more asymmetric IgG than umbilical cord blood B-lymphocytes (P = 0.0015). CONCLUSIONS: Isolated placental B-lymphocytes synthesized asymmetric IgG in response to Th2 interleukins, more notably IL-6 in combination with IL-4 and IL-10. The in vitro increase of protective asymmetric IgG synthesis in response to Th2-cytokines support the hypothesis that a local Th2-switch is beneficial for pregnancy outcome.

The immunochemical reactivity and neutralizing capacity of polyvalent Vipera (European) antivenom on enzymatic and toxic activities in the venoms of Crotalids from Argentina

The immunochemical reactivity and neutralizing capacity of polyvalent Vipera antivenom (Vipera ammodytes, Vipera aspis, Vipera berus, Vipera lebetina, and Vipera xanthina) were tested on the enzymatic and biological activities of Crotalus durissus terrificus and the following Bothrops venoms from Argentina (Bothrops alternatus, Bothrops ammodytoides, Bothrops neuwiedii, Bothrops jararaca, Bothrops jararacussu, and Bothrops moojeni). The Vipera antivenom reacted weakly when tested by double immunoprecipitation (DIP) and reacted with all the venoms when tested by ELISA. Several components in all the venoms studied were recognized in Western blots. Vipera antivenom deactivated to different degrees in vitro procoagulant, (indirect) hemolytic, and proteolytic activities in all the venoms studied. Preincubation of Bothrops alternatus venom with Vipera antivenom neutralized a lethal potency of 4.5 LD50 in mice with an ED50 of 1.25 ñ 0.25 µl per µg of venom, and with 1.0 µl/µg inhibited 54 per cent of the hemorragic activity and 48 per cent of necrotic activity. Vipera antivenom (2.0 µl per µg toxin) inhibited the phospholipase A2 activity of purified crotoxin and decreased its lethal potency by 60 per cent, while the neutralizing capacity on the lethal potency of crude Crotalus durissus terrificus venom was poor even at a level of 5.0 µl/µg of venom.

Neutralizacion cruzada de veneno de bothrops jararacussu por sueros antiofidicos heterologo / Cross neutralization of bothrops jararacussu venom by heterologous antivenoms

En la Argentina se utilizan tres tipos de sueros antiofídicos frete a la mordedura de crotálidos, el Anticrotálico Monovalente, contra el veneno de Crotalus durissus terrifucus ("víbora de cascabel"), el Botrópico Bivalente y el Botrópico Tetravalente ("Misiones") contra los venenos de las diferentes especies de Bothrops que se encuentran en la Argentina (las diferentes "y"rará")" Además, existe un suero Botrópico-Crotálico (Trivalente) el cual cubriria el mismo espectro que el Bivalente más el Anticrotálico. En este trabajo estudiamos la reactividad inmunoquímica de los sueros antibotrópicos Bivalente y Tetravalente, del Botrópico Crotálico (Trivalente) y del Anticrotálico Monovalente con el veneno de Bothrops jararacussu ("yararacuzú", "tapete dourado"). Además, se estudió la capacidad neutralizante de estos antivenenos sobre la potencia letal, y las actividades hemorrágica, necrotizante, procoagulante y hemolítica indirecta del veneno de B. jararacussu. La potencia neutralizante sobre las actividades biológicas y tóxicas de este veneno por todos los antivenenos utilizados es similar a la obtenida con el suero antibotrópico tetravalente (homólogo). Estos resultados sugieren la posibilidad de emplear sueros antibotrópicos heterólogos en el tratamiento de los accidentes por B. jararacussu.