ABSTRACT
Crotalus durissus terrificus venom and its main component, crotoxin (CTX), have the ability to down-modulate the immune system. Certain mechanisms mediated by cells and solublefactors of the immune system are responsible for the elimination of pathogenic molecules to ensure the specific protection against subsequent antigen contact. Accordingly, weevaluated the immunomodulatory effects of CTX on the immune response of mice that had been previously primed by immunisation with human serum albumin (HSA). CTX inoculationafter HSA immunisation, along with complete Freunds adjuvant (CFA) or Aluminium hydroxide (Alum) immunisation, was able to suppress anti-HSA IgG1 and IgG2a antibodyproduction. We showed that the inhibitory effects of this toxin are not mediated by necrosis or apoptosis of any lymphoid cell population. Lower proliferation of T lymphocytesfrom mice immunised with HSA/CFA or HSA/Alum that received the toxin wasobserved in comparison to the mice that were only immunised. In conclusion, CTX is able to exert potent inhibitory effects on humoural and cellular responses induced by HSAimmunisation, even when injected after an innate immune response has been initiated.
Subject(s)
Mice , Freund's Adjuvant/therapeutic use , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/therapeutic use , Crotoxin/antagonists & inhibitors , Crotoxin/immunology , Snake Venoms/analysis , Snake Venoms/immunology , Snake Venoms/therapeutic use , Immunity, Cellular , Immunity, Cellular/immunology , Immunity, HumoralABSTRACT
BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (Kd), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The Kds of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structurefunction relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.
Subject(s)
Animals , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/immunology , Antivenins/immunology , Bothrops/classification , Metalloproteases/classification , Metalloproteases/toxicity , Snake Venoms/immunology , Antibodies, Neutralizing , Colubridae , Elapidae , ViperidaeABSTRACT
Snake venoms from M. corallinus (LD5=7.1 + or - 0.83 µg), M.frontalis (LD50=19.3 + or - 3.13 µg), M. ibiboboca (LD50=19.8 + or - 2.07 µg) and M. spiixi (LD50=6.7 + or - 1.25 µg) (family Elapidae, genus Micrurus) injected into horses alone or in combination (M. corallinus with M. frontalis) elicit antibody production, as indicated in vivo by neutralization of venom lethality and in vitro by enzyme-linked immunosorbent assay (ELISA), immunoelectrophoresis (IE) and Western blotting (WB). Venom lethality was efficiently neutralized by the antisera, with the monovalent antivenoms being more efficient than the bivalent antivenom. Antibodies against venom components were detected by all artisera at different titers by ELISA. Upon IE, antisera against M. spiixi and M. frontalis venoms cross-reacted with the four types of venoms studied and recognized several molecular components, the precipitin lines obtained had distinct intensities and electrophoretic motilities, whereas the antivenom against M. corallinus only recognized components of its venom but not of the others. All antivenoms cross-reacted with all the elapid venoms in WB revealing several blands with distinct MWs in M. corallinus and M. spiixi venoms, two very sharp and separate bands in M. corallinus venom and a very sharp band of high MW together with several other smaller and faint bands in M. frontalis venom. The data indicate that snake venoms of the genus Micrurus are good immunogens that contain many cross-reactive molecules, and that their toxic components are neutralized more effectively by monovalent rather than by bivalent antivenom
Subject(s)
Animals , Antivenins/biosynthesis , Cross Reactions , Elapid Venoms/immunology , Blotting, Western , Brazil , Enzyme-Linked Immunosorbent Assay , Horses , Immunoelectrophoresis , Lethal Dose 50ABSTRACT
1. Louvain rats (IgK-1a) were immunized with horse IgG(T). To generate mAb to IgG(T), popliteal lymph node cells taken from the immunized animals were fused to a non-secreting LOU/C immunocytoma (IR983F). The hybridomas were cultured in HAT -containing medium and cloned under limiting dilution conditions. Supernatants from the growing hybrids were screened by ELISE using plates coated with horse IgG(T) or IgGa+b+c. 2. The anti-IgG(T) mAb obtained was named LO-HoGT-1 (LOU anti-horse IgG(T)). It is an IgG2a rat antibody whose light chain allotype is IgK-1a, and with an affinity constant of 2.9 x 1010 M-1. 3. Ascites was isnduced in LOU (IgK-1b) rats by injecting the hybridoma cells and incomplete Freund's adjuvant ip. To obtain purified mAb, ascitic fluid was applied to a Sepharose anti-rat LOU IgK-1 a chain column. 4. The purified mAb was then coupled to Sepharose. Immunoelectrophoretically pure IgG(T) was obtained by passage of horse serum through this column. The entire procedure took less than 30 min and resulted in a highly purified IgG(T)
Subject(s)
Animals , Female , Male , Rats , Antibodies, Monoclonal/isolation & purification , Horses/immunology , Immunization , Immunoglobulin G/isolation & purification , Antibodies, Monoclonal/immunology , Cell Line , Chromatography, Affinity , Chromatography, Agarose , Enzyme-Linked Immunosorbent Assay , Hybridomas/immunology , Immunoelectrophoresis , Immunoglobulin G/immunologyABSTRACT
1. Bothrops jararaca venom was detected by ELISA at different times in the skin, muscle, blood, liver, lung, heart, kidney and spleen of mice injected with venom im or id. 2. The results showed that even 10 min after im injection the venom is detected mostly in skin rather than in the muscle of the venom injection site. A small amount of venom was detected in the kidney up to 12 h after im venom injection, and none was detected in tissues of lung, heart, liver or spleen. 3. However, in mice injected id, the venom could be detected in the skin up to 24 h after injection. Local necrosis and haemorrhage could be neutralized by antivenom injected by the id or iv routes only if the antivenom was given a short time after venom injection, even when antivenom is adminsitered in high concentration. 4. In contrast, experiments performed in mice receiving venom id and treated by id or iv routes with antivenom injected at different times after envenoming showed that the effect of venom on blood coagulation could be counteracted by antivenom administered by either route up to 2 h after venom injection 5. We suggest that a feasible amount of antivenom administered id could be given as a first aid measure after a snake bite accident. However, further experimental studies using the id route for antivenom administration are essential to confirm this possibility
Subject(s)
Animals , Male , Mice , Crotalid Venoms/administration & dosage , Antivenins/analysis , Bothrops , Injections, Intravenous , Injections, Intradermal , Kidney/chemistry , Skin/chemistry , Time Factors , Crotalid Venoms/adverse effects , Crotalid Venoms/immunologyABSTRACT
1. The role of IgG antibody and platelets in the mechanism of defense against Trypanosoma cruzi infection is reviewed. 2. Experimental data showing the participation of the different IgG subclasses in the immune lysis and immune clearance of the parasites are discussed. 3. The involvement of the platelets in the removal of the parasites from the circulation is considered. 4. It is suggested that IgG anti-T. cruzi antibodies interact with circulating parasites leading to formation of microaggregates, activation of C3 and deposition of C3 and deposition of C3b on the immune aggregates followed by adherence of platelets through C3b receptors. The immune aggregates would then be taken up by cells of the mononuclear phagocytic system
Subject(s)
Humans , Animals , Mice , Antibodies, Anti-Idiotypic/physiology , Blood Platelets/physiology , Chagas Disease/immunology , Immunoglobulin G/immunology , Complement Activation , Complement C3b/physiology , Complement C3/physiology , ImmunizationABSTRACT
Horse immunoglobulins were obtained from normal defatted with dextran sulfate and precipitated with ammonium sulfate. Eight mg of this preparation was submitted to affinity chromatography with protein A-Sepharose CL-4B. Low temperature (4-C) and a starting buffer at pH 8.0 were conditions required for all IgG subclasses to bind to protein A, even those with low affinity. The IgGs bound to protein A were eluted with glycine buffer at pH 2.8. The yield was about 90%. Its suggested that isolated IgG, instead of whole Igs, be used in serum therapy, reducing the amount of Igs and diminishing serum-related reactions
Subject(s)
Animals , Immunoglobulin G/isolation & purification , Staphylococcal Protein A/metabolism , Chromatography, Affinity , Horses , Immunoglobulin G/metabolismABSTRACT
Antigens of bloodstream and cell culture-derived trypomastigotes of T. cruzi were compared by western blotting using sera of chronic chagasic patient as a source of antiobodies. The immunoblots demonstrated that the two forms display extensive homology except for the 85 - and 52 - kDa bands. These antigens were more strongly stained in culture - derived trypomastigotes. Although the reported differences are not related to major antigens, these results might offer an explanation for previous studies showing that culture - derived trypomastigotes are more antigenic and infective in vitro than bloodstream trypomastigotes