RESUMO
BACKGROUND: Rhipicephalus (Boophilus) microplus is the main ectoparasite affecting livestock worldwide. For a successful parasitism, ticks need to evade several immune responses of their hosts, including the activation of the complement system. In spite of the importance of R. microplus, previous work only identified one salivary molecule that blocks the complement system. The current study describes complement inhibitory activities induced by R. microplus salivary components and mechanisms elicited by putative salivary proteins on both classical and alternative complement pathways. RESULTS: We found that R. microplus saliva from fully- and partially engorged females was able to inhibit both pathways. Saliva acts strongly at the initial steps of both complement activation pathways. In the classical pathway, the saliva blocked C4 cleavage, and hence, deposition of C4b on the activation surface, suggesting that the inhibition occurs at some point between C1q and C4. In the alternative pathway, saliva acts by binding to initial components of the cascade (C3b and properdin) thereby preventing the C3 convertase formation and reducing C3b production and deposition as well as cleavage of factor B. Saliva has no effect on formation or decay of the C6 to C8 components of the membrane attack complex. CONCLUSION: The saliva of R. microplus is able to inhibit the early steps of classical and alternative pathways of the complement system. Saliva acts by blocking C4 cleavage and deposition of C4b on the classical pathway activation surface and, in the alternative pathway, saliva bind to initial components of the cascade (C3b and properdin) thereby preventing the C3 convertase formation and the production and deposition of additional C3b.
Assuntos
Proteínas Inativadoras do Complemento/metabolismo , Via Alternativa do Complemento/efeitos dos fármacos , Via Clássica do Complemento/efeitos dos fármacos , Rhipicephalus/imunologia , Saliva/metabolismo , Animais , Evasão da Resposta Imune , Tolerância ImunológicaRESUMO
Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.
Assuntos
Inativadores do Complemento/farmacologia , Via Clássica do Complemento/efeitos dos fármacos , Proteínas de Insetos/farmacologia , Psychodidae/imunologia , Psychodidae/metabolismo , Saliva/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Ativação do Complemento/efeitos dos fármacos , Complemento C1/antagonistas & inibidores , Complemento C1/imunologia , Complemento C1/metabolismo , Complemento C4/antagonistas & inibidores , Complemento C4/imunologia , Complemento C4/metabolismo , Humanos , Proteínas Recombinantes/farmacologiaRESUMO
Envenomation by poisonous animals is a neglected condition according to the World Health Organization (WHO). Antivenoms are included in the WHO Essential Medicines List. It has been assumed that immunoglobulin G (IgG) antivenoms could activate the complement system through Fc and induce early adverse reactions (EARs). However, data in the literature indicate that F(ab')2 fragments can also activate the complement system. Herein, we show that several batches of IgG and F(ab')2 antivenoms from the Butantan, Vital Brazil, and Clodomiro Picado Institutes activated the complement classical pathway and induced the production of C3a; however, only those antivenoms from Clodomiro Picado generated C5a. Different protein profiles (IgG heavy chain, protein contaminants, and aggregates) were observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses. Our results show that various antivenoms from different producers are able to activate the classical pathway of the complement system and generate anaphylatoxins, and these findings suggest that factors, such as composition, contaminant proteins, and aggregates, may influence the anticomplementary activity of antivenoms in vitro. Therefore, there is a need to further improve antivenom production methods to reduce their anticomplementary activity and potential to cause EARs.
Assuntos
Antivenenos/farmacologia , Via Clássica do Complemento/efeitos dos fármacos , Fragmentos Fab das Imunoglobulinas/farmacologia , Imunoglobulina G/farmacologia , Fatores Imunológicos/farmacologia , Anafilatoxinas , Animais , Western Blotting , Ativação do Complemento/efeitos dos fármacos , Complemento C3a/biossíntese , Complemento C3a/efeitos dos fármacos , Complemento C5a/biossíntese , Complemento C5a/efeitos dos fármacos , Venenos de Crotalídeos , Eletroforese em Gel de Poliacrilamida , Cavalos , Humanos , Testes de Neutralização , Coelhos , Venenos de Escorpião , OvinosRESUMO
INTRODUCTION: Low-molecular-weight heparin is used clinically for the prevention of pregnancy complications associated with prothrombotic disorders, particularly anti-phospholipid syndrome. Nevertheless, recent studies have suggested that heparin may exert direct effects on the placental trophoblast, independently of its anticoagulant activity. In addition, heparin prevents complement activation in vivo and protects mice from pregnancy complications. MATERIALS AND METHODS: The inhibition of the classical complement activation pathway by heparin was analyzed by means of in vitro assays and in pregnant women receiving prophylaxis with therapeutic doses (40 mg/day) of subcutaneous low molecular weight heparin by haemolysis of antibody-sensitized sheep erythrocytes (CH(50) assay). RESULTS: The specific interaction between low-molecular-weight heparin and the C1q subunit of the C1 complex of the complement cascade allowed the isolation of a small subpopulation of heparin ( 8.03+/-1.20 microg %), with an anti-activated factor X activity more than four times greater than the starting material. This subpopulation could be responsible for the in vitro inhibition of the classical complement activation pathway evaluated by the total haemolysis of antibody-sensitized sheep erythrocytes. About 60 microg/ml of low molecular weight heparin was needed to achieve 50% of haemolysis. The detection of the classical complement pathway inhibition in pregnant women treated with heparin required a first activation with aggregated human IgG. CONCLUSIONS: We concluded that the interaction between low-molecular-weight heparin and C1q could be relevant not only in the complement-dependent, but also in the complement-independent inflammation mechanisms responsible for the prevention of pregnancy loss.
Assuntos
Via Clássica do Complemento/efeitos dos fármacos , Via Clássica do Complemento/imunologia , Heparina de Baixo Peso Molecular/administração & dosagem , Adolescente , Adulto , Relação Dose-Resposta a Droga , Feminino , Humanos , Gravidez , Adulto JovemRESUMO
Complement, an important effector mechanism of the immune system, is an enzymatic cascade of approx. 30 serum proteins leading to the amplification of a specific humoral response. It can be activated through the classical or alternative pathways, or through the mannose-binding lectin pathway. The activation of the classical pathway is initiated by the binding of the C1 component to antigen-bound antibodies, known as immunocomplexes. C1 is a complex of one molecule of C1q, two molecules of C1r and two molecules of C1s. C1q contains three copies of a Y-shaped fundamental unit with globular heads included in its structure, which play a major role in the interaction with the Fc portion of immunoglobulins. Deficient or exacerbated activation of the complement system leads to diseases of variable severity, and pharmacological inhibition of the complement system is considered as a therapeutic strategy to ameliorate the inflammatory effects of exacerbated complement activation. Bilirubin is a product of haem degradation by the concerted action of haem oxygenase, which converts haem into biliverdin, and biliverdin reductase, which reduces biliverdin to UCB (unconjugated bilirubin). UCB exerts both cytoprotective and cytotoxic effects in a variety of tissues and cells, acting either as an antioxidant at low concentrations or as an oxidant at high concentrations. In the present review, we describe in detail the anti-complement properties of bilirubin, occurring at levels above the UCB concentrations found in normal human serum, as a beneficial effect of potential clinical relevance. We provide evidence that UCB interferes with the interaction between C1q and immunoglobulins, thus inhibiting the initial step in the activation of complement through the classical pathway. A molecular model is proposed for the interaction between UCB and C1q.
Assuntos
Via Clássica do Complemento/imunologia , Hiperbilirrubinemia/imunologia , Inflamação/prevenção & controle , Antioxidantes/farmacologia , Bilirrubina/farmacologia , Bilirrubina/fisiologia , Complemento C1q/metabolismo , Inativadores do Complemento/farmacologia , Via Clássica do Complemento/efeitos dos fármacos , Citoproteção/fisiologia , Humanos , Inflamação/imunologia , Estresse Oxidativo/imunologiaRESUMO
This work evaluated a crude hydroalcoholic extract (ExT) from the pulp of the tamarind (Tamarindus indica) fruit as a source of compounds active on the complement system (CS) in vitro. ExT, previously characterized by other authors, had time and concentration dependent effects on the lytic activity of the CS. The activity of 0.8 mg/mL of the extract on the classical/lectin pathways (CP/LP) increased after 30 min of pre-incubation, while that of the alternative pathway (AP) decreased after 15 min at 1mg/mL. Since the CS is a mediator of inflammation, studies were also made in vivo, taking advantage of a model of hypercholesterolemia in hamsters to investigate the role of CS in the phase preceding the inflammatory process of atherosclerosis. Hamsters submitted to a diet rich in cholesterol showed increased lytic activity of the CP/LP and AP after 45 days. The activity levels of C2 and factor B components on respectively, classical/lectin and alternative pathways of the CS also increased. Early events cooperating to trigger the process of atherosclerotic lesions are not completely understood, and these alterations of complement may participate in these events. When treatment with a diet rich in cholesterol was associated to the furnishing of ExT, evaluation of complement components and complement lytic activity showed values similar to those of the controls, showing that treatment with ExT blocked the increase of complement activity caused by the cholesterol-rich diet. By itself, ExT had no effect on the complement system in vivo. ExT activity on the CS may be of interest for therapy and research purposes.
Assuntos
Complemento C2/imunologia , Fator B do Complemento/imunologia , Via Alternativa do Complemento/efeitos dos fármacos , Via Clássica do Complemento/efeitos dos fármacos , Hiperlipidemias/imunologia , Extratos Vegetais/farmacologia , Tamarindus/química , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Colesterol/sangue , Complemento C2/metabolismo , Fator B do Complemento/metabolismo , Ensaio de Atividade Hemolítica de Complemento , Cricetinae , Frutas/química , Hiperlipidemias/tratamento farmacológico , Masculino , Mesocricetus , Estatísticas não Paramétricas , Triglicerídeos/sangueRESUMO
Six 3H-spiro[benzofuran-2,1'-cyclohexane] derivatives were synthesized from naturally occurring filifolinol, and their classical complement pathway inhibitory activity was determined. IC(50) values of the most potent compounds were comparable to the activity of the natural complement inhibitor K76-COOH and some synthetic tricyclic analogs of it.
Assuntos
Via Clássica do Complemento/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/síntese química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Extratos Vegetais/química , Animais , Benzofuranos/síntese química , Benzofuranos/farmacologia , Cicloexanos/síntese química , Cicloexanos/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Extratos Vegetais/farmacologia , Sesquiterpenos , Ovinos , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Relação Estrutura-Atividade , TrítioRESUMO
The scorpion Tityus serrulatus is considered one of the most dangerous species in Brazil. Its venom evokes an inflammatory response, although the exact mechanism of this effect is still unknown. The aim of the present study was to investigate the effect of Tityus serrulatus venom (TsV) on the complement system (CS) and on leukocyte recruitment. Complement consumption by TsV was evaluated using in vitro hemolytic assays, immunoelectrophoresis and two-dimensional immunoelectrophoresis of complement components (factor B and C3). In order to evaluate neutrophil migration induced in normal human serum (NHS) in the presence of TsV, in vitro chemotaxis assays were performed using the Boyden chamber model. In vitro TsV induced a concentration- and time-dependent reduction in hemolytic activity of the classical/lectin and alternative complement pathways, with samples of 43.0 microg and 43.4 microg, respectively, inhibiting 50% of the lytic activity. Alterations in C3 and factor B electrophoretic mobility after incubation of NHS with TsV, were identical to those obtained with zymosan (positive control). Incubation of NHS with TsV induced neutrophil chemotaxis similar to that observed with zymosan-activated serum. Our results show that TsV activates the CS, leading to factor B and C3 cleavage, to reduction of serum lytic activity and generation of complement chemotactic factors. Therefore, CS may play an important role in the inflammatory response observed upon scorpion envenomation.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Venenos de Escorpião/farmacologia , Animais , Quimiotaxia de Leucócito/efeitos dos fármacos , Complemento C3/metabolismo , Fator B do Complemento/metabolismo , Ensaio de Atividade Hemolítica de Complemento , Via Alternativa do Complemento/efeitos dos fármacos , Via Clássica do Complemento/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Imunoeletroforese Bidimensional , Técnicas In Vitro , Lectinas , Masculino , Coelhos , OvinosRESUMO
The effects of Tityus serrulatus venom and TsTX-I (Ts1 or gamma-toxin) on the lytic activity of the complement system (CS) were investigated in vivo. Serum classical pathway (CP) and alternative pathway (AP) activities were determined in sera of rats (200+/-10 g) injected i.p. with soluble venom (150 microg/kg), TsTX-I (150 microg/kg) or saline (control). The animals were sacrificed 0.5, 1, 2, 4, 24 and 48 h after injection. The results showed an increase in serum lytic activity of animals injected with venom, reaching values up to 70% above controls in CP activity and 120% in AP activity. These effects were biphasic with maximum values 1 and 24 h after venom injection. Similar effects were obtained for TsTX-I, but with lower intensity. Hematocrit values of all tested animals were determined to evaluate the effect of hemoconcentration on the lytic activity of the CS. It was observed that the maximum hematocrit value was obtained 1 h after injection and returned to normal values within 24 h. These data indicate that hemoconcentration can play a relevant role in the first peak of complement activity, but we cannot discard a direct action of the venom on the system during this period, since the serum venom concentration is maximal 15-30 min after envenomation. The high lytic activity of the serum observed after 24 h, period in which the hematocrit values are normal and no venom can be detected, may be consequence of the inflammatory process induced by the venom or toxin. The lytic activity of the serum of rats injected with venom, TsTX-I or saline was abolished when the serum was previously adsorbed on zymosan. These data confirm that the increase of the lytic activity of the serum induced by the venom or toxin is dependent on CS. These results show that CS is involved in the inflammatory process induced by the venom or toxin and consequently in the lung edema, hemolysis, leukocytosis, among other clinical manifestations of severe envenomation.
Assuntos
Via Alternativa do Complemento/efeitos dos fármacos , Via Clássica do Complemento/efeitos dos fármacos , Citotoxinas/farmacologia , Venenos de Escorpião/farmacologia , Escorpiões , Animais , Eritrócitos , Feminino , Hemólise/efeitos dos fármacos , Masculino , Coelhos , Ratos , Ratos Wistar , Venenos de Escorpião/química , Ovinos , Fatores de TempoRESUMO
A water-soluble polysaccharide extracted from leaves of the Meliaceae Cedrela tubiflora was separated into neutral and acidic polysaccharide fractions. The best anticomplementary activity was exhibited by the neutral product which was further purified by means of gelpermeation chromatography. The composition and methylation analysis of the purified product were determined.