Isotype of antibodies responsible for immune lysis in Trypanosoma cruzi infected mice.

Serum obtained from mice infected with Trypanosoma cruzi have antibodies able to induce immune lysis in presence of complement. Gel filtration in Sephadex G-200 and affinity chromatography with rabbit anti-mouse IgG and protein A-Sepharose showed that the antibodies responsible for the immune serum lytic activity are exclusively located in Ig of IgG isotype, including IgG1, IgG2a, IgG2b and probably IgG3. It is suggested that antibodies responsible for protection against T. cruzi infection and antibodies responsible for immune lysis are the same.

Role of the antibody Fc in the immune clearance of Trypanosoma cruzi.

Passive transfer of immune serum obtained from mice chronically infected with Trypanosoma cruzi to mice containing circulating bloodstream trypomastigotes induces a very fast clearance of the parasites. Comparison of trypomastigotes clearance in normocomplementemic and C5-deficient mice showed no difference. IgG fraction obtained from immune serum was very efficient at inducing complement-mediated lysis and immune clearance of bloodstream trypomastigotes, whereas its Fc-missing F (ab') 2 fragments, although able to induce lysis, were unable to induce clearance. It is suggested that the immune clearance of bloodstream trypomastigotes is dependent on the antibody Fc region and that complement-mediated lysis is not a prerequisite for elimination of the parasites from circulation.

Characterization of antibody isotype responsible for immune clearance in mice infected with Trypanosoma cruzi.

Humans and mice chronically infected with Trypanosoma cruzi present a strong humoral immune response mediated by specific antibodies. Passive transfer of homologous immune serum to normal mice containing circulating bloodstream trypomastigotes (Bts) induces a very fast clearance of the parasites. In order to find out the role of the different immunoglobulin classes in the clearance, mice containing a known number of these parasite forms in circulation were injected with total immune serum, IgG-free serum, IgG1, or IgG2 fractions and the speed of removal of the parasites from circulation was determined. The results of these experiments suggest that the immune clearance of T. cruzi is due to antibodies located in the IgG isotype, particularly in the IgG2 subclass.

A comparative study of anti-Trypanosoma cruzi serum obtained in acute and chronic phase of infection in mice.

The IgG antibody content, specificity, lytic activity, clearance capacity and protective ability of mouse anti-Trypanosoma cruzi serum was determined during the course of infection. The IgG antibody content increased during the course of infection, reaching its highest level in the serum collected in the chronic phase of the infection. The T. cruzi antigens recognized by antibodies using the protein transfer technique also increased with time of infection. Antibodies present in day 22 post-infection (p.i.) serum were already able to recognize all the antigens detected by antibodies present in serum from the chronic phase. The lytic and clearance ability were not detected on day 7 p.i., but appeared on day 14 p.i. and reached their highest level on day 45 p.i. The protective ability was present in serum of the chronic phase, but was absent from the acute serum. The IgG antibody content of the acute serum was four times less than that of the chronic serum. When the IgG antibody concentration of the acute serum was equalized to that of the chronic serum, the acute serum was as able to protect the infected animals as the chronic serum. It is suggested that the disagreement between the protective ability of anti-T. cruzi antisera collected in the acute or in the chronic phase of the infection is due to a quantitative rather than a qualitative difference.

Trypanosoma cruzi: advantages of isolating bloodstream trypomastigotes by the carboxy methyl cellulose method.

Bloodstream trypomastigotes were isolated from blood of A/Sn mice 7 d after infection with 10(5) Trypanosoma cruzi Y strain. Red blood cells were removed by centrifugation and hypotonic shock and platelets and leucocytes by passage through a carboxy methyl cellulose column. Binding of trypomastigotes to the resin was prevented by including 10% normal mouse serum in the eluting buffer. In such conditions, more than 90% of the parasites applied to the column were recovered, free of white blood cells and platelets. A comparative study of the pre- and post-separation trypomastigotes showed that both had the same infecting capacity, ability to evade destruction by the complement system, and antigenic profile.

Isolation of IgGT from hyperimmune horse anti-snake venom serum: its protective ability.

Hyperimmune horse anti-bothropic serum, used in serum therapy, was analyzed for its IgGT content and protective ability. IgGT was isolated through a combination of salt-mediated hydrophobic chromatography and protein A affinity chromatography. The chromatographic fractions obtained were analyzed with regard to their isotype content and protective ability. The results suggest that the protective ability of hyperimmune anti-venom serum is located mainly in the IgGT subclass.

Efficacy of bothropic antivenom and its IgG(T) fraction in restoring fibrinogen levels of Bothrops jararaca envenomed mice.

Bothropic antivenom and its IgG(T) fraction, administered 4 h after experimental envenoming by Bothrops jararaca in Swiss mice, were compared for their abilities to restore fibrinogen 24 or 48 h after treatment. IgG(T) was able to normalise fibrinogen levels as efficiently as conventional antivenom. As IgG(T) also neutralises most anti-toxic activities of Bothrops venom, our results suggest that IgG(T) could be a better alternative treatment for envenoming due to the reduced amount of extraneous proteins, which may facilitate the induction of early adverse reactions.

Neutralization of Thalassophryne nattereri (niquim) fish venom by an experimental antivenom.

T. nattereri (niquim) is a venomous fish involved in many human accidents in Brazil. The clinical picture includes mild local erythema, severe edema, intense pain and rapid progression to necrosis. The present therapy with anti-inflammatory and analgesic drugs is ineffective and, therefore, we decided to assess serum therapy as an alternative treatment using an experimental antivenom. The antivenom used was raised in rabbits showing an ELISA antibody titer of 1:8,192,000 and its ability to neutralize lethality, necrosis, nociception and edema was evaluated both by pre-incubating the venom with antivenom before injection into mice or by independent injections of venom and antivenom. Lethality was completely neutralized by pre-incubation (ED(50)=141.5 microl/mg) while necrosis and nociception were neutralized by pre-incubation or the independent injection of antivenom. Edema was only partially prevented even when large amounts of antivenom were used. These data suggest that antivenom may be a promising treatment for patients stung by T. nattereri and suggest the viability of producing a horse antivenom for use in clinical trials.

Neutralization of bothropic and crotalic venom toxic activities by IgG(T) and IgGa subclasses isolated from immune horse serum.

IgG(T) and IgGa isotypes were isolated from horse hyperimmune anti-bothropic and anti-crotalic sera using a combination of two affinity chromatographic processes. IgG(T) and IgGa isotypes were isolated from these sera by chromatography on protein A-Sepharose followed by separation of the two isotypes by chromatography on a column of anti-IgG(T)-Sepharose. LO-HoGT-1, a rat anti-horse IgG(T) monoclonal antibody, was used. A comparative study of the efficiency of these isotypes in neutralizing the main toxic activities of the homologous venoms was carried out. It was found that IgG(T) was about three-fold and seven-fold more protective than IgGa for neutralization of the lethal activity of B. jararaca and C. d. terrificus venoms, respectively. IgG(T) was also more effective than IgGa for the neutralization of the haemorrhagic activity induced by B. jararaca venom, while both isotypes neutralized equally well the blood incoagulability induced by this venom. The results suggest that IgG(T) is the most protective isotype present in both anti-bothropic and anti-crotalic sera, followed by IgGa. Owing to their very low concentration in the serum, other IgG isotypes are not likely to be important in neutralizing the venoms' toxic activities.

Tolerance to the antinociceptive effect of Crotalus durissus terrificus snake venom in mice is mediated by pharmacodynamic mechanisms.

Crotalus durissus terrificus venom exerts central and peripheral antinociceptive effect mediated by opioid receptors. The present work investigated the tolerance to the antinociceptive effect of the venom and characterised the mechanisms involved in this phenomenon. The hot plate test, applied in mice, was used for pain threshold determination. The venom (200 microg/kg) was administered by oral route, daily, for 14 days, and the nociceptive test was applied before and on days 1, 7 and 14 of the treatment. Prolonged treatment with venom lead to the development of tolerance to the antinociceptive effect. Tolerant animals exhibited increased sodium pentobarbital-induced sleeping time, although total hepatic microsomal cytochrome P450 was not altered. The antinociceptive effect of a single dose of venom (200 microg/kg) is mediated by kappa opioid receptors. Mice long-term-treated with venom showed cross-tolerance to U-TRANS, an agonist of kappa-opioid receptor, but not to morphine or DAMGO, two mu-opioid receptor agonists. Prolonged administration of venom did not cause symptoms of abstinence syndrome. These data indicate that prolonged treatment with C. durissus terrificus venom induces tolerance to the antinociceptive effect and that pharmacodynamic mechanisms are involved in the genesis of this phenomenon.

Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica.

Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica. Toxicon 000-000. This work compared the specificity, ELISA titers and IgG subclass content of the polyvalent antivenom (anti-Bothrops asper, Crotalus durissus durissus and Lachesis muta stenophrys) of Instituto Clodomiro Picado (Costa Rica) and the bothropic antivenom (anti-Bothrops jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. alternatus) of Instituto Butantan (Brazil). The role of IgG(T) and IgGa subclasses in neutralization of some venom toxic activities and the cross neutralization of the antivenoms against B. jararaca and B. asper venoms were also evaluated. Both antivenoms were able to recognize B. asper and B. jararaca venoms by immunoblotting and presented similar antibody titers when assayed by ELISA. IgG(T) was highest, followed by IgGa, IgGb and IgGc. IgGa and IgG(T) isotypes isolated from both antivenoms by affinity chromatography were tested for neutralization of lethal, hemorrhagic, coagulant and phospholipase A2 activities of the homologous venoms. In both antivenoms, IgG(T) was the major isotype responsible for neutralization of all the tested activities, followed by IgGa. These results suggest that Instituto Butantan and Instituto Clodomiro Picado antivenoms have the same IgG profile and their neutralizing ability is due mostly to the IgG(T) isotype. Also, they neutralize lethality in mice induced by homologous and heterologous venoms, the bothropic antivenom of Instituto Butantan being more effective.

The possible mechanism of action of IgG antibodies and platelets protecting against Trypanosoma cruzi infection.

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 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.

Isolation of horse IgG with protein A.

Horse immunoglobulins were obtained from normal serum 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 degrees 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%. It is suggested that isolated IgG, instead of whole Igs, be used in serum therapy, reducing the amount of Igs and diminishing serum-related reactions.

A rapid and efficient purification method for horse IgG(T) using a rat monoclonal antibody.

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 ELISA 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 10(10) M-1. 3. Ascites was induced 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-1a 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).

Comparison between the antigenic composition of bloodstream and cell culture-derived trypomastigotes of Trypanosoma cruzi.

Antigens of bloodstream and cell culture-derived trypomastigotes of T. cruzi were compared by western blotting using sera of chronic chagasic patients as a source of antibodies. 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.

Detection and neutralization of B. jararaca venom in mice.

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 i.m. or i.d. 2. The results showed that even 10 min after i.m. 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 i.d., 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 i.d. or i.v. routes only if the antivenom was given a short time after venom injection, even when antivenom is administered in high concentration. 4. In contrast, experiments performed in mice receiving venom i.d. and treated by i.d. or i.v. 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 i.d. could be given as a first aid measure after a snake bite accident. However, further experimental studies using the i.d. route for antivenom administration are essential to confirm this possibility.

Antigenic cross-reactivity among components of Brazilian Elapidae snake venoms.

Snake venoms from M. corallinus (LD50 = 7.1 +/- 0.83 micrograms), M. frontalis (LD50 = 19.3 +/- 3.13 micrograms), M. ibiboboca (LD50 = 19.8 +/- 2.07 micrograms) and M. spiixi (LD50 = 6.7 +/- 1.25 micrograms) (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 antisera 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 bands 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.

Differences in the antigenic profile of bloodstream and cell culture derived trypomastigotes of Trypanosoma cruzi.

A comparative study of the antigenic profile of bloodstream and cell culture derived trypomastigotes showed many differences in their components. Using mouse anti-T. cruzi antibodies the differences were located mostly in the 120 kDa band, whereas using chagasic patient sera the differences were located in the 85 and 52 kDa bands. These findings might explain known physiological differences between trypomastigotes obtained from cell culture and from infected blood. A brief report of this work has already been published.

Immunomodulatory effects of crotoxin isolated from Crotalus durissus terrificus venom in mice immunised with human serum albumin.

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 soluble factors of the immune system are responsible for the elimination of pathogenic molecules to ensure the specific protection against subsequent antigen contact. Accordingly, we evaluated the immunomodulatory effects of CTX on the immune response of mice that had been previously primed by immunisation with human serum albumin (HSA). CTX inoculation after HSA immunisation, along with complete Freund's adjuvant (CFA) or Aluminium hydroxide (Alum) immunisation, was able to suppress anti-HSA IgG1 and IgG2a antibody production. 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 lymphocytes from mice immunised with HSA/CFA or HSA/Alum that received the toxin was observed in comparison to the mice that were only immunised. In conclusion, CTX is able to exert potent inhibitory effects on humoral and cellular responses induced by HSA immunisation, even when injected after an innate immune response has been initiated.

Immunochemical and biological characterization of monoclonal antibodies against BaP1, a metalloproteinase from Bothrops asper snake venom.

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 (K(d)), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The K(d)s 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 structure-function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.