RESUMO
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.
Assuntos
Alelos , Frequência do Gene , Antígenos de Histocompatibilidade Classe II/genética , Hanseníase/genética , Adulto , Brasil , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Cadeias HLA-DRB1 , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
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.
Assuntos
Animais , Camundongos , Antivenenos/imunologia , Mordeduras de Serpentes , Venenos de Serpentes/classificação , ImunoterapiaRESUMO
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.
Assuntos
Transtornos da Coagulação Sanguínea/induzido quimicamente , Fragmentos Fab das Imunoglobulinas/imunologia , Peçonhas/toxicidade , Animais , Formação de Anticorpos , Eletroforese em Gel de Poliacrilamida , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos BALB C , Mariposas , Testes de Neutralização , Distribuição Tecidual , Peçonhas/imunologiaRESUMO
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.
Assuntos
Antivenenos/imunologia , Bothrops , Venenos de Crotalídeos/imunologia , Venenos de Crotalídeos/toxicidade , Crotalus , Venenos de Serpentes/imunologia , Animais , Formação de Anticorpos , Antivenenos/biossíntese , Galinhas/imunologia , Gema de Ovo , Ensaio de Imunoadsorção Enzimática , Imunoglobulinas/análise , Imunoglobulinas/imunologiaRESUMO
The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement (C) dependent haemolysis. The aim of this study was to characterise the toxins in the venom responsible for the different biological effects. We have previously shown that a 35 kDa protein, named F35, purified from Loxosceles intermedia venom, incorporates into the membranes of human erythrocytes and renders them susceptible to the alternative pathway of autologous C. Here we have further purified the F35 protein which was resolved by reversed phase chromatography into three tightly contiguous peaks termed P1, P2, and P3. P1 and P2 were shown to be homogeneous by SDS-PAGE and N-terminal aminoacid analysis, while P3 consisted of two highly homologous proteins. N-terminal sequencing of all four proteins showed a high degree of homology, which was confirmed by cross-reactivity of antisera raised against the individual purified proteins. Functional characterisation of P1 and P2 indicated the presence of sphingomyelinase activity and either protein in isolation was capable of inducing all the in vivo effects seen with whole spider venom, including C-dependent haemolysis and dermonecrosis. In all assays, P2 was more active than P1, while P3 was completely inactive. These data show that different biological effects of L. intermedia venom can be assigned to the sphingomyelinase activity of two highly homologous proteins, P1 and P2. Identification of these proteins as inducers of the principal pathological effects induced by whole venom will aid studies of the mechanism of action of the venom and the development of a effective therapy.
Assuntos
Dermotoxinas/farmacologia , Hemólise/efeitos dos fármacos , Esfingomielina Fosfodiesterase/farmacologia , Venenos de Aranha/farmacologia , Sequência de Aminoácidos , Animais , Ensaio de Atividade Hemolítica de Complemento , Reações Cruzadas , Dermotoxinas/sangue , Ensaio de Imunoadsorção Enzimática , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Necrose , Fragmentos de Peptídeos/isolamento & purificação , Coelhos , Alinhamento de Sequência , Esfingomielina Fosfodiesterase/sangue , Esfingomielina Fosfodiesterase/química , Venenos de Aranha/sangue , Venenos de Aranha/enzimologiaRESUMO
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.
Assuntos
Antivenenos/uso terapêutico , Bothrops , Venenos de Crotalídeos/imunologia , Imunoglobulina G/uso terapêutico , Mordeduras de Serpentes/terapia , Adolescente , Adulto , Idoso , Animais , Antivenenos/efeitos adversos , Antivenenos/metabolismo , Criança , Pré-Escolar , Colômbia , Venenos de Crotalídeos/sangue , Método Duplo-Cego , Fibrinogênio/análise , Humanos , Imunoglobulina G/efeitos adversos , Pessoa de Meia-Idade , Pepsina A/metabolismo , Mordeduras de Serpentes/fisiopatologiaRESUMO
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.
Assuntos
Antivenenos/farmacologia , Animais , Formação de Anticorpos , Especificidade de Anticorpos , Antivenenos/química , Antivenenos/isolamento & purificação , Antivenenos/metabolismo , Venenos de Artrópodes , Coagulação Sanguínea/efeitos dos fármacos , Desenho de Fármacos , Hemorragia/tratamento farmacológico , Hemorragia/prevenção & controle , Hemostasia , Cavalos , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Mariposas , Ratos , VacinaçãoRESUMO
Cutaneous inoculation of Loxosceles spp. spider venoms produces local necrosis, occasionally accompanied by systemic intravascular clotting and hemolysis. In this work, we analyzed the role of the C system on the lysis of human erythrocytes (Eh) induced by Loxosceles venoms in vitro. Eh were treated with whole venom of Loxosceles laeta, Loxosceles gaucho, or Loxosceles intermedia, or with purified venom proteins, and incubated with C-sufficient (Cs-NHS) or C9-depleted autologous (C9d-NHS) serum. Hemolysis was determined spectrophotometrically, and deposition of C components or removal of C regulatory proteins was analyzed by FACS. Eh suspensions exposed to venoms or to a purified 35-kDa protein from L. intermedia were lysed after incubation with Cs-NHS, but not with C9d-NHS. Lysis was blocked by heating the serum at 50 degrees C or Ca2+/Mg2+ chelation by EDTA, but not by Ca2+ chelation with EGTA. Deposition of C1, C2, C3, C4, C5, and factor B on the venom-treated Eh occurred during activation of autologous C. Regulatory proteins decay-accelerating factor (DAF) and CD59 were not altered significantly. Conversion of C-resistant Eh into C-susceptible Eh by the L. intermedia venom was accompanied by incorporation of a 35-kDa venom protein onto the cell surface. Thirty-five-kilodalton-related proteins were detected in the two other Loxosceles venoms by ELISA, using rabbit antiserum against the L. intermedia 35-kDa protein. These data suggest that the C system mediates the lysis of human erythrocytes and, by extension, of other cell types able to incorporate the lytic factor of Loxosceles venoms on their cell surfaces.
Assuntos
Via Alternativa do Complemento/efeitos dos fármacos , Eritrócitos/imunologia , Venenos de Aranha/sangue , Venenos de Aranha/farmacologia , Animais , Antígenos CD55/sangue , Fracionamento Químico , Cromatografia em Gel , Eritrócitos/química , Eritrócitos/efeitos dos fármacos , Proteínas Hemolisinas/isolamento & purificação , Proteínas Hemolisinas/farmacologia , Hemólise/efeitos dos fármacos , Hemólise/imunologia , Humanos , Proteínas de Membrana/sangue , Proteínas de Membrana/farmacologia , Peso Molecular , CoelhosRESUMO
Saponin has been described to contain adjuvant activity in vaccination protocols, in protection against disease, and on humoral immune response. In this paper we describe the effect of a pure saponin from Quillaja saponaria (molina) on the immune response elicited in mice by two antigens, BSA and Crotalus durissus terrificus (South American rattlesnake) venom. Antibody production as measured by ELISA shows that saponin was able to increase antibody synthesis to both antigens. Moreover, mice immunized with verom plus saponin were completely protected against the lethal effects of the venom. The effect of saponin was also evaluated for cytokine production. Tumour necrosis factor activity about 2.9 times higher than in control mice was detectable in sera from animals immunized with saponin. Interferon-gamma was produced only when BSA and saponin were injected together into the mice.
Assuntos
Adjuvantes Imunológicos/farmacologia , Formação de Anticorpos/efeitos dos fármacos , Interferon gama/biossíntese , Saponinas/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Animais , Venenos de Crotalídeos/imunologia , Venenos de Crotalídeos/toxicidade , Ensaio de Imunoadsorção Enzimática , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Soroalbumina Bovina/imunologiaRESUMO
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.