Distribution of the HLA class II frequency alleles in patients with leprosy from the mid-west of Brazil.

In an attempt to clarify the issue of genetic predisposition to leprosy, we examined the distribution of class II human leucocyte antigen variants (DR and DQ) in 70 patients from around the city of Goiânia, Brazil. Only two of the patients presented the tuberculoid form of the disease, whereas 17 fell into the lepromatous category; 51 were intermediate. The allele frequencies found were compared with those in a group of 77 healthy controls. We found an increased frequency of the HLA-DRB1*11 allele in patients with lepromatous leprosy compared with healthy controls (P=0.0132; RR=4.130, 95% Cl: 1.338 to 12.747). These results suggest that the DRB1*11 allele could be related with susceptibility to lepromatous leprosy in Brazil.

Complement as a mediator of inflammation. 3. Purification of the activity with anaphylatoxin properties generated by interaction of the first four components of complement and its identification as a cleavage product of C'3.

Purified preparations of human C'1 esterase, C'4, C'2, C'3, and C'5 were labeled with (125)I. Reaction mixtures were prepared containing a single labeled component and other unlabled components. After incubation at 37 degrees C for 10 min at pH 7.4 in the presence of 5 x 10(-4)M Mg(2+), they were adjusted to pH 3.5 and subjected to sucrose density gradient ultracentrifugation and gel filtration at pH 3.5. In all cases, an activity capable of contracting guinea pig ileum with tachyphylaxis was obtained in low molecular weight fractions. However, these fractions were labeled only when (125)I-C'3 was employed, indicating that biological activity was associated with a cleavage product of C'3. This fragment has been designated F(a)C'3 in a nomenclature consistent with that of immunoglobulin degradation products. The much larger, residual portion of the C'3 molecule has been designated F(b)C'3. The biochemical characteristics of generation of F(a)C'3 were consistent with a mechanism involving action of C'1 esterase on C'4 and C'2, activation of C'2, and cleavage of C'3. F(a)C'3 had a molecular weight by gel filtration techniques of 6800 or less. It was thermostable and susceptible to inactivation by endo- and exopeptidases. The isolated fragment possessed all of the biological properties of unfractionated mixtures of C'1 esterase, C'4, C'2, and C'3. In addition to contraction of guinea pig ileum, these included failure to contract rat uterus, enhancement of vascular permeability in guinea pig skin, degranulation of mast cells in guinea pig mesentery, and release of histamine from rat peritoneal mast cells. F(a)C'3 did not cross-desensitize guinea pig ileum to rat agar anaphylatoxin and vice versa. The existence of different protein fragments with anaphylatoxin properties has been discussed. Distinctive characteristics of F(a)C'3 from classical anaphylatoxin generated by treatment of fresh rat serum with agar have been indicated.

Pro-inflammatory activities in elapid snake venoms.

1. Snake venoms from the genera Micrurus (M. ibiboboca and M. spixii) and Naja (N. naja, N. melanoleuca and N. nigricollis) were analysed, using biological and immunochemical methods, to detect pro-inflammatory activities, cobra venom factor (COF), proteolytic enzymes, thrombin-like substances, haemorrhagic and oedema-producing substances. 2. The venoms of the five snake species activate the complement system (C) in normal human serum (NHS) in a dose-related fashion, at concentrations ranging from 5 micrograms to 200 micrograms ml-1 serum. Electrophoretic conversion of C3 was observed with all venoms in NHS containing normal concentrations of Ca2+ and Mg2+, but only by venoms from N. naja and N. melanoleuca when Ca2+ was chelated by adding Mg(2+)-EGTA. 3. Purified human C3 was electrophoretically converted, in the absence of other C components, by the venoms from N. naja, N. nigricollis and M. ibiboboca. However, only the venoms from N. naja and N. melanoleuca contained a 144 kDa protein revealed in Western blot with sera against COF or human C3. 4. All venoms, at minimum concentrations of 30 ng ml-1, were capable of lysing sheep red blood cells, also in a dose-related fashion, when incubated with these cells in presence of egg yolk as a source of lecithin. Although the venoms from M. spixii and N. nigricollis showed detectable thrombin-like activity, these and the other venoms were free of proteolytic activity when fibrin, gelatin and casein, were used as substrates. 5. When tested on mice skin, all five venoms were capable of inducing an increase in vascular permeability and oedema, but were devoid of haemorrhagic producing substances (haemorrhagins). 6. These data provide evidence indicating that Elapidae venoms contain various pro-inflammatory factors which may be important in the spreading of neurotoxins throughout the tissues of the prey or human victim.

Complement-mediated adherence of cells to microfilariae of Brugia malayi.

Immune sera promote the adherence of human buffy coat cells to microfilariae of Brugia malayi. This effect is augmented by the simultaneous addition of fresh human or guinea pig serum. The heat-labile, adherence-promoting factor present in nonimmune sera appears to be complement. In the absence of antibody, microfilariae activate complement via the alternative pathway and C3 is deposited on the microfilarial sheath.

Use of enzyme immunoassays to compare the effect and assess the dosage regimens of three Brazilian Bothrops antivenoms. The Butantan Institute Antivenom Study Group (BIASG).

The effect of the three main Brazilian polyspecific antivenoms on venom clearance was assessed in 118 moderately envenomed victims of bites by Bothrops species (mainly B. jararaca) in Sao Paulo State, Brazil. Serum samples taken from patients at intervals during their stay in the hospital and at followup approximately four weeks later were tested by enzyme immunoassay for the presence of whole venom and therapeutic antivenom. Results indicated that in patients treated with the standard regimen of either four (40 ml) or eight (80 ml) ampules of each antivenom, venom was cleared from the circulation within four days of antivenom administration. However, high concentrations of antivenom persisted for approximately 10 days and remained detectable until 30-50 days after administration. This suggests that patients may be being treated with excessive amounts of antivenom in Brazil. This practice increases the national cost of antivenom therapy and may contribute to the high frequency of antivenom reactions. Clinically, there was no obvious difference in the efficacy between the three antivenoms.

Specific heterologous F(ab')2 antibodies revert blood incoagulability resulting from envenoming by Lonomia obliqua caterpillars.

Contact with Lonomia obliqua caterpillars results in a bleeding syndrome characterized by hemorrhage and blood coagulation disturbances. Conventional therapy using antifibrinolytics or cryoprecipitates has been unable to treat pathophysiologic alterations. As antivenoms are effective therapy for treatment of victims of venomous animals, a process of manufacturing a specific antilonomic serum by immunizing horses with Lonomia caterpillar bristle extracts (LBE) was developed. Lonomia caterpillar bristle extracts exhibited several protein bands on SDS-PAGE, induced blood coagulation abnormalities and lethality in mice, and stimulated specific antibody production in horses. Sera obtained from immunized horses were rich in anti-LBE specific antibodies distributed among the horse IgG isotypes. These antibodies had the ability to recognize various LBE antigens as well as to neutralize their coagulopathy-inducing activity. The antivenom manufactured by the developed process was composed of purified and sterilized F(ab')2 with ED50 = 38.61 microl, potency = 0.29 mg/ml, and 95% confidence limit of potency 0.20-1.36.

A randomized, blinded, comparative trial of one pepsin-digested and two whole IgG antivenoms for Bothrops snake bites in Uraba, Colombia. The Regional Group on Antivenom Therapy Research (REGATHER).

The therapeutic efficacy and the incidence of early antivenom reactions (EARs) were compared in a clinical trial performed in 79 patients bitten by Bothrops sp. in Urabá, Colombia. Patients were randomized into three groups according to the antivenom administered: A (n = 30, Butantan polyspecific, pepsin-digested Bothrops antivenom); B (n = 27, Butantan polyspecific, whole IgG Bothrops antivenom); and C (n = 22, Colombian commercial, monovalent, whole IgG Bothrops antivenom). The groups were comparable in all clinical and epidemiologic aspects; 33 patients had mild, 22 moderate, and 24 severe envenoming. At the doses used (two, four, and six vials [10 ml/vial] for mild, moderate, and severe envenomings, respectively) there were no differences between the antivenoms in restoring normal hemostatic parameters within 24 hr. The evolution of local envenoming was comparable in the three groups. Serum venom/antivenom kinetics determined by ELISA showed a complete clearance of venom levels 1 hr after treatment in mild/moderate envenomings. In severe cases, venom levels remained detectable up to 24 hr and recurrence of antigenemia was observed in some cases. Antivenom concentrations remained at high levels up to 24 hr of treatment. The incidence of EARs was significantly different in the groups: A (36.7%), B (11.1.%), and C (81.8%). There were no life-threatening anaphylactic reactions. We conclude that the efficacy of the three antivenoms was similar in neutralizing human Bothrops envenomings and that the production of whole IgG antivenoms by caprylic acid fractionation is a good alternative for reducing the incidence of EARs.

[Characterization of the biological activities of the 'yellow' and 'white' venoms from Crotalus durissus ruruima compared with the Crotalus durissus terrificus venom. Neutralizing activity of Crotalus durissus ruruima antivenins]. / Caracterizacion de las actividades biologicas de los venenos 'amarillo' y 'blanco' de Crotalus durissus ruruima comparados con el veneno de Crotalus durissus terrificus. Poder neutralizante de los antivenenos frente a los venenos de Crotalus durissus ruruima.

The biological activities of 'yellow' and 'white' venom of a rattlesnake Crotalus durissus ruruima Hoge, 1965, found in the savanna-like vegetation (cerrado) of northern Brazil (Roraima) and Venezuela have been studied, and compared to the reference Crotalus durissus terrificus venom. The lethal activity of venoms depended on the inoculation route. The most toxic venom was the white one. The venoms of C. d. terrificus and the yellow of C. d. ruruima had similar lethalities. The yellow venom of C. d. ruruima showed a caseinolytic activity three times higher than that obtained with either the venom of C. d. terrificus or the white one of the C. d. ruruima. Hemorrhagic and necrotic activities were found only in the yellow venom. White and yellow venoms from C. d. ruruima showed a similar action on fibrinogen; this thrombin-like action was greater with C. d. terrificus venom. On histopathological sections local and pulmonary hemorrhage was found only with the yellow venom, but myonecrotic activity was observed with both venoms of C. d. ruruima. Among all antivenoms studied, the anti-bothropic-crotalic was the best at neutralizing hemorrhagic and hemolytic activities. These results suggest that antivenom bothropic-crotalic should be used in the treatment of patients with snakebite by C. d. ruruima: besides its neutralization on lethal activity, it also neutralizes the hemorrhagic activity present in some venoms.

Comparison of the potency of three Brazilian Bothrops antivenoms using in vivo rodent and in vitro assays. BIASG (Butantan Institute Antivenom Study Group).

Three Brazilian polyspecific Bothrops antivenoms were compared using standard W.H.O. rodent in vivo and in vitro assays of their ability to neutralize the principal venom activities of pooled whole Bothrops jararaca venom. On a volume basis, the antivenoms were equally effective in neutralizing lethal activity in mice, and there were only minor differences in their ability to neutralize venom-induced haemorrhage, necrosis and procoagulant activity. Antivenom efficacy in neutralizing defibrinogenation varied. However, when equal amounts of antivenom IgG were compared, it was found that the FUNED antivenom best neutralized lethality, haemorrhage, necrosis and fibrinogen clotting activity. Vital Brazil and FUNED antivenoms were equally effective in neutralizing plasma coagulant activity but Vital Brazil antivenom was the more effective in neutralizing defibrinogenation.

Susceptibility of different strains of mice to South American rattlesnake (Crotalus durissus terrificus) venom: correlation between lethal effect and creatine kinase release.

In the present work we report that susceptibility to Crotalus durissus terrificus venom: varies according to the strain of inbred mouse used. The s.c. LD50 for Balb/c and C57BI/6 mice were 193 micrograms/kg and 171 micrograms/kg, whereas for A/J and DBA/J they were 78 micrograms/kg and 74 micrograms/kg, respectively. In addition, a direct correlation between susceptibility to C. d. terrificus venom and creatine kinase serum levels (CK) was observed.

Snake antivenoms from hyperimmunized horses: comparison of the antivenom activity and biological properties of their whole IgG and F(ab')2 fragments.

IgG and F(ab')2 fragments were prepared from horse plasma rich in specific antibodies against Brazilian Bothrops or Crotalus venoms. Both preparations, free of gross contamination with non-immunoglobulin proteins, were able to combine in vitro with their respective antigens, forming immune complexes at antigen excess, equivalence or antibody excess, and activating the C system, through either the classical or the alternative pathways. The IgG preparation was more effective in neutralizing the lethal factors in Bothrops or Crotalus venoms, compared with the F(ab')2 fragments. In contrast, IgG and F(ab')2 anti-Bothrops venom were almost equipotent in neutralizing the haemorrhagic and defibrinating activities in the venom. The method used to purify IgG, precipitation of most non-immunoglobulin plasma proteins with caprylic acid, produced antivenoms richer in specific antibodies, with higher specific activity, recovery and yield, compared with the method commonly used to prepare antivenoms containing F(ab')2.

Development of an antivenom against toxins of Lonomia obliqua caterpillars.

Skin contact with caterpillars of Lonomia moths causes haemostatic disorders that may evolve into a haemorrhagic syndrome. Replacement therapy has been shown to exacerbate the clinical symptoms of this envenoming. In this study it is shown that horses immunized with a bristle extract of L. obliqua caterpillars produced IgG antibodies that completely neutralized, in vitro, the toxin(s) responsible for the blood incoagulability observed in rats. This antivenom offers the possibility of specific treatment for envenoming caused by contact with caterpillars of Lonomia moths.

Antigenic cross-reactivity among the venoms from several species of Brazilian scorpions.

The venoms of seven species of scorpions living in different regions of Brazil were analysed with regard to their lethality, antigenic cross-reactivity and ability to induce antibody production. In mice, the tested scorpion venoms can be grouped as: (a) highly toxic: Tityus stigmurus Thorell (LD50 = 0.773 mg/kg), Tityus bahiensis (Perty) (LD50 = 1.062 mg/kg), Tityus serrulatus Lutz and Mello (LD50 = 1.160 mg/kg), and Tityus costatus (Karsch) (LD50 = 1.590 mg/kg); (b) moderately toxic: Tityus cambridgei Pocock (LD50 = 12.136 mg/kg); and (c) practically nontoxic: Rhopalurus agamemnon (Koch) (LD50 = 36.363 mg/kg), and Brotheas amazonicus Lourenço (LD50 = 90.909 mg/kg). On electrophoresis the venoms showed many protein bands displayed along the chromatogram, most of them cross-reacting in immunoelectrophoresis and immunoblotting using horse anti-T. serrulatus, anti-T. bahiensis or anti-T. serrulatus+T. bahiensis sera as probes. The antibodies present in these antivenoms combine with venom components as measured in vitro by the ELISA assay, and neutralize their lethal effects in vivo. These results indicate that horse anti-venoms against a mixture of T. serrulatus and T. bahiensis venoms or only against T. serrulatus venom yield an antibody population able to neutralize the toxic effects found in all venoms studied.

Antigenic cross-reactivity of venoms obtained from snakes of genus Bothrops.

Antigenic cross-reactivity was studied among the components of venoms from nine species of the genus Bothrops using species-specific antivenoms. Sera titration by DOT-ELISA detected similar levels of antibody when either homologous or heterologous antigens were used. Transblotted antigens, after SDS-PAGE fractionation, were also revealed by homologous and heterologous antivenoms. Antigens with mol. wt greater than 30,000 seemed to be the most cross-reactive. Antigens of about 24,000 mol. wt were poorly immunogenic. Antigens between 14-18,000 mol. wt cross-reacted only with B. moojeni, B. jararacussu, B. neuwiedi and B. pradoi venoms. Neutralization of the lethality of B. jararaca venom was observed by homologous and heterologous antivenoms.

Evasion of Trypanosoma cruzi from complement lysis.

This review outlines: a) the main biochemical and biological properties of the complement system (C) components; b) the manner through which they interact in the two distinct routes of C activation, the classical and the alternative pathways, to generate the enzymes C3 and C5 convertases responsible for release of the peptides C4a, C3a and C5a endowed with the properties of mediating the early events of the inflammatory process or the potentially cytolytic complex C5b-C9; c) the main features of control of these activation processes; d) the identification of cell surface components present in the trypomastigote forms of Trypanosoma cruzi possibly involved in the mechanisms developed by this parasite to evade C lysis; e) the inactivation or removal of these cell surface components by enzymatic (trypsin or papain), chemical (periodate) or physical (heating at 45 degrees C) treatments; f) isolation of these components by chromatographic methods; and, g) demonstration that some of these cell surface components interfere with C3 convertase formation or action in a manner similar to the decay accelerating factor (DAF).

Transformation of Trypanosoma cruzi trypomastigote bloodstream forms by immune IgM and its Fab mu fragment into activators of the alternative complement pathway.

1. We report that purified immune IgM obtained from Chagasic patients during the chronic stage of the disease and also immune IgM and its Fab mu fragments obtained from chronically T. cruzi-infected mice are capable of preparing trypomastigote forms of T. cruzi to be lysed by complement. 2. Mouse strains susceptible, moderately susceptible and resistant to T. cruzi infection are equally capable of producing antibodies with lytic activity as well as antibodies detectable by immunofluorescence and by immunoenzymatic assay. 3. The epitopes recognized by polyclonal lytic antibodies are present on the surface of trypomastigotes from many different strains and stocks of T. cruzi.

Development of snake antivenom antibodies in chickens and their purification from yolk.

Adult white leghorn hens hyperimmunised with Brazilian snake venoms of the genus Bothrops and/or Crotalus produced antibodies capable of recognising, combining with and neutralising the toxic and lethal components of the venoms. The antibodies were first detected by an enzyme-linked immunosorbent assay two weeks after starting the immunisation schedule, reached the highest titres by the third week and remained high for at least 24 weeks. These antibodies are transferred to the egg yolk from which they were isolated as enriched IgY preparations by a combination of methods using positive and negative precipitation with sodium sulphate and/or caprylic acid. The yolk-derived IgY preparations contained antibodies which blocked the phospholipase A2-dependent haemolytic activity of both venoms and the haemorrhagic activity of Bothrops venom, and neutralised the toxic lethal activities of the venoms with good efficacy. The median effective dose (ED50) of the IgY anti-Bothrops venom was 592.5 microliters/2LD50 and, 1.0 ml neutralised 0.0675 mg of venom. The ED50 of the IgY anti-Crotalus venom was 457.5 microliters/3LD50 and 1.0 ml neutralised 0.075 mg of venom.

Polyvalent horse F(Ab`)2 snake antivenom: Development of process to produce polyvalent horse F(Ab`)2 antibodies anti-african snake venom

A method to obtain polyvalent anti-Bitis and polyvalent-anti-Naja antibodies was developed by immunizing horses with B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca and N. mossambicacrude venoms. Antibody production was followed by the ELISA method during the immunization procedure. Once the desired anti-venom antibody titers were attained, horses were bled and theimmunoglobulins were separated from the sera by (NH4)2SO4 precipitation, cleaved with pepsin and filtered through a 30 kDa ultrafiltration membrane. F(ab´)2 fragments were further purified by Q-Fast Flow chromatography, concentrated by molecular ultrafiltration and sterilized by filtration through 0.22 m membranes. The resulting F(ab´)2 preparations were rich in intact L and in pieces of H IgG(T) chains, as demonstrated by electrophoresis and Western blot and exhibited high antibody titers, as assayed bythe ELISA method. In addition, the preparations possess a significant capacity to neutralize the lethalityof venoms, as estimated by ED50 determination in mouse assay and are free of toxic substances, pyrogen and bacterial or fungal contaminations.

Activation of human complement system Paracoccidioides brasiliensis and its deposition on the yeast form cell surface.

The yeast form of Paracoccidioides brasiliensis strain Pb18 was able to activate C3 of normal human serum diluted in phosphate-buffered saline or EGTA-MgCl2 in vitro. C3 convertase was also permissive when Pb18 cells were pre-treated with a pool of immune serum from patients with paracoccidioidomycosis and incubated in serum diluted in EDTA-CaCl2. The components C3, and fragments C3c, C3d, C3g, factor H, factor B, C4 and C5b-9 were demonstrated on the Pb18 cell surface by immunofluorescence although no effect was seen on fungal viability.