• View in gallery
    Figure 1.

    Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) Loxosceles spp. venoms and analysis of serum cross-reactivity. Samples of L. gaucho, L. intermedia, and L. laeta venoms (10 μg) were subjected to electrophoresis on a 12% SDS-PAGE gel under non-reducing conditions and stained with silver (left panel) or subjected to Western blotting (middle and right panels). Blots were probed with anti-arachnidic (middle panel) or anti-SMase D (right panel) horse sera diluted 1:2,000 and goat anti-horse IgG labeled with alkaline phosphatase diluted 1:3,000. Reactions were developed using 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tet-razolium.

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    Figure 2.

    Cross-reactivity of horse sera. A, Enzyme-linked im-munosorbent assay (ELISA) plates were coated with 1 μg of Loxosceles spp. venoms and incubated with different dilutions of normal, anti-arachnidic and anti-sphingomyelinase D (SMase) D sera and goat anti-horse IgG labeled with alkaline phosphatase (1:3,000). Absorbance of samples was determined at 492 nm. B, ELISA plates were coated with 1 μg of mouse IgG anti-horse IgGT and incubated with different dilutions of normal, anti-arachnidic and anti-SMase D sera and goat anti-mouse IgG labeled with horseradish peroxidase diluted 1:3,000. Absorbance of samples was determined at 492 nm.

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    Figure 3.

    Neutralization of the dermonecrotic effect of Loxosceles spp. venoms. A, Dermonecrosis reaction: adult rabbits were injected intradermally with 3 μg of Loxosceles spp. venoms and the size of the lesions was measured for 72 hours. B–D, Serum neutralization: adult rabbits were injected intradermally with 3 μg of Loxosceles spp. venoms and intravenously with 1 mL of anti-arachnidic (○), anti-sphingomyelinase D (•), or control horse sera. Size of the lesions was measured for 72 hours and results are expressed as percentage reduction ± SD of size of the dermonecrotic lesion of three experiments.

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    Figure 4.

    Neutralization of hemolytic activity of Loxosceles spp. venoms. A, Hemolysis: erythrocytes pretreated with veronal-buffered saline (VBS++) or different amounts of Loxosceles spp. venoms were incubated with normal human serum. After incubation for one hour at 37°C, unlysed cells were centrifuged and the absorbance of supernatants was measured at 414 nm and expressed as percentage of lysis. B–D, Serum neutralization: erythrocytes were treated with venoms from Loxosceles spp. or with VBS++ buffer in the presence or absence of anti-arachnidic (□) or anti-sphingomyelinase D (▪) sera and analyzed for expression GPC by flow cytometry. Results are representative for three experiments and expressed as percentage reduction of fluorescence ± SD of duplicate samples.

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    Figure 5.

    Neutralization of sphingomyelinase activity of Loxosceles spp. venoms. A, Sphingomyelinase (SMase) activity: SMAse (50 μg) was incubated with buffer or with increasing amounts of Loxosceles spp. venoms. After incubation at 37C for 20 minutes, the choline formed was oxidized to betaine and measured fluorimetrically. B–D, Serum neutralization: SMase was incubated with 1 μg of Loxosceles spp. venoms or veronal-buffered saline in the presence or absence the anti-arachnidic (□) or anti-SMase D (▪) sera for 30 minutes at 37°C. Samples were centrifuged and supernatants were analyzed for SMase D activity as described above. Results are representative for three experiments and expressed as percentage reduction ± SD of fluorescence of duplicate samples.

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A New Anti-loxoscelic Serum Produced Against Recombinant Sphingomyelinase D: Results of Preclinical Trials

Daniel Manzoni de AlmeidaLaboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil; Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Norte, Natal, Brazil; Divisão Bioindustrial e Centro de Biotecnologia, Instituto Butantan, Natal, Brazil; Cardiff University, Wales College of Medicine, Cardiff, United Kingdom

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Matheus de F. Fernandes-PedrosaLaboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil; Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Norte, Natal, Brazil; Divisão Bioindustrial e Centro de Biotecnologia, Instituto Butantan, Natal, Brazil; Cardiff University, Wales College of Medicine, Cardiff, United Kingdom

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Rute M. Gonçalves de AndradeLaboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil; Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Norte, Natal, Brazil; Divisão Bioindustrial e Centro de Biotecnologia, Instituto Butantan, Natal, Brazil; Cardiff University, Wales College of Medicine, Cardiff, United Kingdom

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José Roberto MarcelinoLaboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil; Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Norte, Natal, Brazil; Divisão Bioindustrial e Centro de Biotecnologia, Instituto Butantan, Natal, Brazil; Cardiff University, Wales College of Medicine, Cardiff, United Kingdom

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Hisako Gondo-HigashiLaboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil; Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Norte, Natal, Brazil; Divisão Bioindustrial e Centro de Biotecnologia, Instituto Butantan, Natal, Brazil; Cardiff University, Wales College of Medicine, Cardiff, United Kingdom

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Inácio de L. M. Junqueira de AzevedoLaboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil; Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Norte, Natal, Brazil; Divisão Bioindustrial e Centro de Biotecnologia, Instituto Butantan, Natal, Brazil; Cardiff University, Wales College of Medicine, Cardiff, United Kingdom

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Paulo Lee HoLaboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil; Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Norte, Natal, Brazil; Divisão Bioindustrial e Centro de Biotecnologia, Instituto Butantan, Natal, Brazil; Cardiff University, Wales College of Medicine, Cardiff, United Kingdom

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Carmen van den BergLaboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil; Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Norte, Natal, Brazil; Divisão Bioindustrial e Centro de Biotecnologia, Instituto Butantan, Natal, Brazil; Cardiff University, Wales College of Medicine, Cardiff, United Kingdom

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Denise V. TambourgiLaboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil; Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Norte, Natal, Brazil; Divisão Bioindustrial e Centro de Biotecnologia, Instituto Butantan, Natal, Brazil; Cardiff University, Wales College of Medicine, Cardiff, United Kingdom

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Envenomation by Loxosceles species (brown spider) can lead to local dermonecrosis and to serious systemic effects. The main toxic component in the venom of these spiders is sphingomyelinase D (SMase D) and various isoforms of this toxin are present in Loxosceles venoms. We have produced a new anti-loxoscelic serum by immunizing horses with recombinant SMase D. In the present study, we compared the neutralization efficacy of the new anti-loxoscelic serum and anti-arachnidic serum (the latter serum is used for therapy for loxoscelism in Brazil) against the toxic effects of venoms from spiders of the genus Loxosceles. Neutralization tests showed that anti-SMase D serum has a higher activity against toxic effects of L. intermedia and L. laeta venoms and similar or slightly weaker activity against toxic effects of L. gaucho than that of Arachnidic serum. These results demonstrate that recombinant SMase D can replace venom for anti-venom production and therapy.

INTRODUCTION

Envenomation by Loxosceles spp. spiders is a public health problem in Brazil, and loxoscelism is considered the most dangerous form of araneism in this country. Systemic reactions, including shock, hemolysis, renal insufficiency, and disseminated intravascular coagulation, are rare. In contrast, bite reactions ranging from small areas of erythema to large areas of ulceration and necrosis are frequently observed.1 The bite is relatively painless and patients often are unaware that they have been bitten. Mild to severe pain, beginning 2–8 hours after envenomation, is probably caused by ischemia. Erythema with itching, swelling, and mild to severe tenderness is also observed. At this point, the patient might consult a physician.1

At least three Loxosceles species of medical importance are known in Brazil (L. intermedia, L. gaucho, and L. laeta) and more than 3,000 cases of envenomation by L. intermedia are reported each year. In North America, several Loxosceles species, including L. reclusa (brown recluse), L. apachea, L. arizonica, L. unicolor, L. deserta, and L. bonetti, are the principal cause of numerous incidents of envenomation.25 In South Africa, L. parrami and L. spinulosa are responsible for cutaneous loxoscelism6 and in Australia, a cosmopolitan species, L. rufescens, is capable of causing ulceration in humans.

A variety of treatments such as systemic steroids, anti-venom, phentolamine, heparin, chloroprofenpyridamine maleate, dapsone, hyperbaric oxygen, and other substances have been used for therapy, with little or no benefit and, in some cases, with undesirable collateral effects.720 Most treatments have attempted to reduce infiltration of polymorphonuclear (PMN) leukocytes, the hallmark of cutaneous loxoscelism. PMN leukocyte infiltration, in part recruited by indirect activation of the complement system, is a major contributor to tissue damage.2125 Treatments to reduce PMN leukocyte infiltration are fraught with side effects and, in some cases, can increase tissue injury.22

In Brazil, serum therapy combined with corticosteroids constitute the most common intervention for loxoscelism, and the Brazilian Ministry of Health recommends its use in moderate and severe cases with systemic illness and to reduce severity of the reaction and healing time.26 The antiserum most commonly used for treatment of loxoscelism in Brazil is anti-arachnidic serum. This serum is produced by the Instituto Butantan (São Paulo, Brazil) by hyperimmunization of horses with venoms of the spiders L. gaucho and Phoneutria nigriventer and the scorpion Tityus serrulatus. Several studies have indicated that sphingomyelinase D (SMase D) in venom of Loxosceles spp. spiders is the main component responsible for local and systemic effects observed in loxoscelism.25,2735

The difficulty in obtaining large amounts of venom and purified venom components is one of the limiting factors in studying the mechanisms involved in loxoscelism and raising an effective therapeutic antiserum. We have recently cloned and expressed one of the sphingomyelinases from L. laeta venom (SMase I), which displayed all biological activities endowed by whole venom.35 An antiserum raised in rabbits against this recombinant protein effectively neutralized L. laeta venom toxicity. We have also cloned and expressed two functional isoforms of SMase (P1 and P2) from L. intermedia and shown that the recombinant proteins display all functional characteristics of whole venom, e.g., dermonecrotic and complement-dependent hemolytic activities and ability to hydrolyze sphingomyelin (SM). We have also compared the cross-reactivities of antisera raised in rabbits against SMase D from different Loxosceles species and showed that the cross-reactivity is high when toxins are from the same species but lower when the toxins are from different species (L. intermedia versus L. laeta).32

These data suggest that to obtain a suitable neutralizing antiserum using recombinant SMase D as immunogen, a mixture of the recombinant toxins from the different species is a requirement. On the basis of these data, we have developed a new anti-loxoscelic serum by horse immunization with a mixture of recombinant sphingomyelinases (P1, P2, and SMaseI), by a patented process (patent no. 0404765-6–020040006198; 3/11/2004, Brazilian National Institute of Industrial Property [INPI], 2005). In the present study, we have analyzed the neutralization potential of the new serum with that of anti-arachnidic serum in preclinical tests using the venoms of L. intermedia, L. gaucho, and L. laeta.

MATERIALS AND METHODS

Chemicals, reagents, and buffers

Tween 20, bovine serum albumin (BSA), paraformaldehyde, SM, choline oxidase, horseradish peroxidase (HRP), and 3-(4-hydroxy-phenyl)propionic acid were obtained from Sigma (St. Louis, MO). 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitroblue tetrazolium (NBT) were obtained from Promega (Madison, WI). Buffers used were veronal-buffered saline (VBS++), pH 7.4 (10 mM sodium barbitone, 0.15 mM CaCl2, and 0.5 mM MgCl2; phosphate-buffered saline (PBS), pH 7.2 (10 mM sodium phosphate, 150 mM NaCl); HEPES-buffered saline (HBS), pH 7.4, 10 mM HEPES (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2); fluorescence-activated cell sorting (FACS) buffer (PBS, 1% BSA, 0.01% sodium azide); and Alsever’s old solution (114 mM sodium citrate, 27 mM glucose, 72 mM NaCl, pH 6.1).

Antibodies

Monoclonal antibody against glycophorin C (GPC) (Bric4) was obtained from the International Blood Group Reference Laboratory (Bristol, United Kingdom). Rabbit anti-mouse IgG labeled with fluorescein isothiocyanate (FITC) was obtained from Amersham Pharmacia Biotech GE (Little Chalfont, Buckinghamshire, United Kingdom). Goat anti-horse IgG labeled with alkaline phosphatase (AP), HRP, or FITC was obtained from Sigma. Goat anti-horse IgGT was obtained from Bethyl Laboratories Inc. (Montgomery, TX).

Animals

Adult New Zealand white rabbits weighing approximately 3 kg were obtained from Biotério de Criação de Animais do Instituto Butantan (São Paulo, SP, Brazil). All procedures involving animals were in accordance with the ethical principles in animal research adopted by the Brazilian College of Animal Experimentation.

Spiders and venoms

The spiders L. intermedia, L. laeta, and L. gaucho were bred in the Biotério de Criação e Manutenção de Aranhas Loxosceles do Laboratório de Imunoquímica do Instituto Butantan. Venoms were obtained by electrostimulation using the method of Bucherl36 with slight modifications. Briefly, 15–20V electrical stimuli were repeatedly applied to the spider sternum and the venom drops were collected with a micropipette, vacuum dried, and stored at −20°C. Stock solutions were prepared in PBS at a concentration of 1.0 mg/mL. The protein content of the samples was evaluated by the method of Smith and others37 by using the BCA Protein Assay kit (Pierce Biotechnology, Woburn, MA).

Expression of recombinant protein

Production of recombinant mature SMases I from L. laeta (accession no. AY093599) and P1 and P2 from L. intermedia (accession nos. AY304471 and AY304472, respectively) was performed as described.32,35

Antisera

Commercial anti-arachnidic serum (batch no 0506118), produced by immunizing horses with a mixture of venoms from L. gaucho (21.5%), P. nigriventer (21.5%), and T. serrulatus (57%) and experimental anti-SMase D serum, produced by immunizing horses with a mixture of recombinant SMases D from Loxosceles spp. spiders (P1 and P2 from L. intermedia32 and SMase I from L. laeta35), were obtained from the BioIndustrial Division, Instituto Butantan. Production and purification of anti-SMase D serum are patented processes (patent no. 0404765-6, INPI). Normal horse serum was obtained from non-immunized animals.

Normal human serum and erythrocytes

Human blood was obtained from healthy donors. Blood samples used for serum were collected without anticoagulant and allowed to clot for two hours at room temperature; normal human serum was stored at −80°C. Blood samples used for obtaining erythrocytes for subsequent use as target cells were collected in Alsever’s old solution.

Electrophoresis and Western blotting

Samples of venoms and recombinant proteins were solubilized in reducing sample buffer and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gels38 and silver staining. Alternatively, gels were blotted onto nitrocellulose membranes.39 After transfer, membranes were blocked with PBS containing 5% BSA and incubated with anti-arachnidic or anti-SMase D horse sera (diluted 1:2,000) for one hour at room temperature. Membranes were washed three times (10 minutes/wash) with PBS/0.05% Tween 20 and incubated with goat anti-horse IgG labeled with AP (diluted 1:3,000) in PBS/1% BSA for one hour at room temperature. The membranes were then washed three times (10 minutes/wash) with PBS/0.05% Tween 20 and blots were developed using NBT/BCIP according to the manufacturer’s instructions (Promega).

Enzyme-linked immunosorbent assay (ELISA)

Microtiter plates were coated with 100 μL of Loxosceles spp. venom or recombinant proteins (10 μg/mL) overnight at 4°C. Plates were blocked with 5% BSA in PBS and dilutions of anti-arachnidic or anti-SMase D horse sera in PBS were added. After incubation for one hour at room temperature, plates were washed with PBS/0.1% Tween 20 and incubated with goat anti-human IgG labeled with HRP (diluted 1:1,000) for one hour at room temperature. Plates were washed and developed with o-phenylenediamine dihydrochloride (OPD) substrate according to the manufacturer’s (Sigma) recommendations. Alternatively, titers of IgGT from anti-arachnidic and anti-SMase D horse sera were determined by coating microtiter plates with 100 μL of goat anti-horse IgG against horse IgGT (250 μg/mL) overnight at 4°C. Plates were blocked with 5% BSA in PBS and dilutions of anti-arachnidic and anti-SMase D sera made in PBS were added. After incubation for one hour at room temperature, plates were washed with PBS/0.1% Tween 20 and incubated with goat anti-horse IgG labeled with HRP RP (diluted 1:3,000) for one hour at room temperature. Plates were washed and developed with OPD substrate according to the manufacturer’s (Sigma) recommendations. Titers were established as the highest antiserum dilution in which an absorbance was five times greater than that for normal serum.

Neutralization of dermonecrotic activity by sera

The ability of the anti-arachnidic and anti-SMase D sera to neutralize dermonecrotic activity was measured by an in vivo method.40 Two hundred microliters of L. gaucho, L. intermedia, or L. laeta venoms in PBS (15 μg/mL) were injected intradermally into shaved backs of adult rabbits. Simultaneously, 1 mL of normal or test sera was injected intravenously. The size of the lesions was measured over a period of 72 hours after injection. Neutralization was expressed as percentage reduction in size of the dermonecrotic lesion and calculated using as reference the extent of lesions developed by animals injected with the venoms and treated with normal serum.

Neutralization of hemolytic activity by sera

The ability of the anti-arachnidic and anti-SMase D sera to block hemolytic activity induced by Loxosceles spp. venoms was assessed by analyzing inhibition of GPC cleavage on the erythrocyte cell membrane as described.30 Human erythrocytes were washed and resuspended at a concentration of 2% in VBS++ and incubated with Loxosceles spp. venom for 30 minutes at 37°C. Control samples were incubated with VBS++. Cells were washed three times, resuspended in their original volume in VBS++, and analyzed in a hemolysis assay.

For this assay, 100 μL of 2% erythrocytes pre-treated with Loxosceles spp. spider venom or VBS++ were mixed with 100 μL of normal human serum (1:2 in VBS++). Background or total cell lysis was evaluated by incubation of erythrocytes with VBS++ or water, respectively. After incubation for one hour at 37°C, unlysed cells were centrifuged and the absorbance of the supernatant was measured at 414 nm and expressed as percentage of lysis. Because high concentrations of serum were used in the assays, the absorbance of serum without erythrocytes was subtracted from all samples. Percentage lysis was calculated as (absorbancesample − absorbance serum)/(absorbancewater − absorbanceVBS++) × 100. Mean and standard deviations were determined from duplicate samples. Erythrocytes and normal human serum were always obtained from the same donor.

Once the venom concentration to be used in the neutralization assays was determined (3 μg/mL), 25 μL of 2% human erythrocytes were incubated with Loxosceles spp. venoms in the presence or absence of normal, anti-arachnidic, or anti-SMase D sera for 30 minutes at 37°C. Control samples were incubated with VBS++. Cells were washed three times, resuspended in their original volume in VBS++, and prepared for flow cytometry analysis. Cells were incubated for 30 minutes with 25 μL of the primary monoclonal antibody (MAb Bric4: anti-GPC; 1 μg/mL) in FACS buffer. After washing, cells were incubated with FITC-labeled secondary antibodies for 30 minutes. Cells were washed and fixed in FACS buffer containing 1% paraformaldehyde and analyzed by flow cytometry (FACScalibur; Becton Dickinson, San Jose, CA).

Neutralization of SMase activity by sera

The SMase D activity in Loxosceles spp. venoms was measured as described.41 Briefly, samples containing increasing concentrations of the Loxosceles spp. venoms were incubated with 50 μg of SM diluted in 1 mL of HBS for 20 minutes at 37°C. One unit/mL of choline oxidase, 0.06 units/mL of HRP, and 50 μM of 3-(4-hydroxy-phenyl)propionic acid in HBS was added and incubated for 10 minutes. The choline liberated was oxidized to betaine and H2O2. The oxidation products were measured by fluorimetry at λem = 405 nm and λex = 320 nm by using 96-well microtiter plates in a spectrofluorimeter (Victor 3™; Perkin-Elmer, Waltham, MA). Once the venom concentration used in neutralization assays (1 μg) was determined, venom samples were incubated for 30 minutes at 37°C with increasing dilutions of normal, anti-arachnidic, or anti-SMase D sera. Samples were then centrifuged for 20 minutes at 10,000 × g and supernatants were analyzed for SMase D activity in the samples as described above.

Statistical analysis

Data were analyzed by using the Student’s t-test. A P value < 0.05 was considered significant.

RESULTS

Immunochemical cross-reactivity and serum titers

Analysis of venoms from L. gaucho, L. laeta, and L. intermedia by SDS-PAGE and silver staining showed differences in composition, number and intensity of bands. However, all venoms contained components with molecular masses of 32–35 kD, which includes the main toxic components of Loxosceles spp. venoms, the SMases D (Figure 1). Western blot analysis showed that anti-arachnidic serum recognized most of the components in venoms and that antibodies to SMase D identified only components with molecular masses of 32–35 kD in L. laeta and L. gaucho venom (Figure 1). In L. intermedia venom, antibodies to SMase D reacted with 32–35-kD SMase D proteins and with proteins with molecular masses twice that of the 32–35-kD proteins, which may indicate the presence of SMase D dimers.

Cross-reactivities of the two sera were tested by ELISA using L. gaucho, L. intermedia, and L. laeta venoms as antigens. As shown in Figure 2A, both sera recognized all tested antigens. Although anti-arachnidic serum contained higher levels of antibodies against venom of L. gaucho than against L. intermedia and L. laeta venom, antibodies to SMase D contained the highest level of antibodies against L. intermedia venom. The antibody titer against L. gaucho was much lower in the anti-SMase D serum than that in the anti-arachnidic serum. Both antisera had the same antibody titer against L. laeta venom. Because horse IgGT is considered the most important antibody isotype in the neutralization of toxins,42 the anti-arachnid and anti-SMase D sera were compared by ELISA for their contents of IgGT. Figure 2B shows that the anti-SMase D serum had much higher titers of IgGT than the anti-arachnidic serum.

Analysis of neutralization potential of anti-arachnidic and anti-SMase D sera

To determine whether toxins responsible for the main toxic effects of Loxosceles spp. venoms could be neutralized by the anti-arachnidic and anti-SMase D sera, in vivo and in vitro-in vivo neutralization experiments were performed.

Inhibition of dermonecrotic activity of Loxosceles spp. venoms by sera.

The ability of Loxosceles spp. venoms to induce dermonecrotic lesions was investigated by injecting rabbits with different venoms. Typical loxoscelic lesions developed in the skin area injected with all venoms within a few hours of injection, as shown by the presence of edema, erythema, and mild tenderness. Approximately 24-hours post-injection, necrosis at the inoculation site was observed. A more potent dermonecrotic activity was observed with L. laeta and L. gaucho venoms than with L. intermedia venom (Figure 3A) (P < 0.05).

Neutralizing ability of the two antisera on Loxosceles spp. venom dermonecrotic activity was determined by an in vivo test. Loxosceles gaucho, L. intermedia, or L. laeta venoms were injected intradermally into the backs of adult rabbits and, simultaneously, antisera were injected intravenously. Size of the lesions was measured during a 72-hour after injection. Groups of animals were injected intradermally with venoms and intravenously with normal horse serum were used as controls. Figure 3B–D shows that anti-SMase D serum was more active than anti-arachnidic serum in neutralizing the dermonecrotic activity of L. intermedia and L. laeta (P < 0.05) venoms, and the neutralization activity of the two antisera for the L. gaucho venom was the same.

Inhibition of hemolytic activity of Loxosceles spp. venoms by sera.

We previously showed that venoms/sphingomyelinases from L. intermedia, L. gaucho, and L. laeta spiders transform human erythrocytes into activators of the complement system.28,43 We subsequently elucidated the mechanism of complement susceptibility and showed that the toxins facilitate activation of the alternative pathway of complement on human erythrocytes by removal of GLPs as a consequence of activation of an endogenous metalloproteinase30 and activation of the classic pathway of complement possibly by alteration of the membrane asymmetry with exposure of phosphatidylserine.31,44

Figure 4A shows that all Loxosceles spp. venoms tested induced autologous complement lysis of human erythrocytes in a dose-dependent manner. To determine if toxins responsible for induction of the removal of GPC and autologous complement hemolysis could be neutralized by the antisera, the venoms from Loxosceles spp. were mixed with human erythrocytes in the presence or absence of control serum or experimental sera. Figure 4B shows that anti-arachnidic serum was more efficient in neutralizing removal of GPC induced by L. gaucho venom than the anti-SMase D (P < 0.05). For L. intermedia venom and L. laeta venom, anti-SMas D serum (Figure 4C and D) inhibited induction of removal of GPC more efficiently than anti-arachnidic serum (P < 0.05). Under the same experimental conditions, the control serum had no effect.

Inhibition of SMase activity of Loxosceles spp. venoms by sera.

We previously showed that all local and systemic effects induced by whole Loxosceles spp. venoms were associated with the action of SMase D.2832 Figure 5A shows that all Loxosceles venoms hydrolyzed SM in a dose-dependent manner. To verify if antisera could inhibit the hydrolytic activity of sphingomyelinases, venoms were assayed for their ability to cleave SM in the presence or absence of control serum and experimental antisera. Figure 5B shows that the anti-arachnidic serum was more efficient at neutralizing sphingomyelinase activity of L. gaucho venom than anti-SMase D at dilutions of 1:1 to 1:75 (P < 0.05). At the highest dilutions, both sera showed similar neutralization values. For L. intermedia venom, the anti-arachnidic serum was more efficient in neutralization than anti-SMase D at dilutions of 1:1 to 1:15 (P < 0.05). The two sera showed similar neutralization at dilutions of 1:30 to 1:75. At dilutions of 1:90 and 1:120 (P < 0.05) the anti-SMase D serum was more effective. For L. laeta, anti-SMase D serum was more efficient in neutralization than anti-arachnidic serum, mainly at the highest serum dilutions (≥ 1:30; P < 0.05). Under the same experimental conditions, the control serum was not able to inhibit the sphingomyelinase activity of the venoms.

DISCUSSION

Envenomation by Loxosceles spp. spiders is a well-documented cause of necrotic skin lesions in humans. Although systemic loxoscelism is less common than the cutaneous form, it is the main cause of death associated with Loxosceles spp. envenomation. Most deaths occur in children and are related to the South American species L. laeta.1 Loxosceles spp. are the most poisonous spider in Brazil and more than 3,000 cases of envenomation by L. intermedia are reported each year. Sphingomyelinase D is the primary enzyme in Loxosceles spp. venom responsible for its major clinical effects in humans.

It is difficult to obtain large quantities of Loxosceles spp. venom. In addition, only venom from L. gaucho is used for the anti-arachnid serum and success of toxin neutralization by an antivenom depends on abundance of specific antibodies against the principal lethal toxin of the venom and its binding affinity for the toxin.45,46 Because we have recently cloned and expressed the main toxic components of L. intermedia and L. laeta (both considered to cause more severe pathologic effects than L. gaucho), we have produced a new anti-loxoscelic serum by immunizing horses with a mixture of recombinant SMase D from venoms of L. intermedia and L. laeta, which are the main agents of loxoscelism in Brazil and South America, respectively. In the present study, we have analyzed the neutralization potential of this new anti-loxoscelic serum against the toxic effects of venoms from spiders of the genus Loxosceles of medical importance in Brazil and compared it with the anti-arachnidic serum used for human therapy in this country.

Analysis of venoms from Loxosceles spp. by SDS-PAGE showed differences in composition, number, and intensity of bands. Western blotting demonstrated that although anti-arachnidic serum was developed against venom from L. gaucho, this serum recognized most components in the three venoms. As expected, antibodies to SMase D recognized only components with molecular masses of 32–35 kD, which corresponded to SMase D in these venoms, although with less intensity than with these components in L. gaucho venom. In L. intermedia venom, antibodies to SMase D also reacted with some proteins with double the molecular of SMase D, which suggested the presence of SMase D dimers.

Results of ELISA also showed cross-reactivity of the anti-arachnidic serum, but reactivity was highest within the same species. For antibodies to anti-SMase D, the highest titer was obtained against L. intermedia venom because in this horse serum preparation two SMases D, P1 and P2, were used in the immunization pool. Antibody titers against venoms in anti-arachnidic serum and anti-SMase D serum indicate that anti-arachnidic serum was raised against whole venom. Thus, antibody titers against active toxins (SMase D in venom) may be much lower; this result was confirmed by Western blotting (Figure 1), which showed that anti-arachnidic serum recognizes not only SMase D but other proteins in venom.

We have shown that the amount of total IgGT, which is found in all antibodies to toxin,4750 was higher in antibodies to SMase D. This result indicated a higher neutralization potential for this serum than for anti-arachnidic serum. To confirm this hypothesis, in vivo and in vivoin vitro neutralization assays were performed. Results showed that, as previously suggested,32,35 the new anti-loxoscelic serum, which was raised against only the toxic component (SMase D), is able to control the toxic action of Loxosceles spp. venoms. Comparative analysis indicated that the anti-arachnidic serum, although able to neutralize heterologous venoms, such as those from L. intermedia and L. laeta, is less efficient than anti-SMas D. Conversely, for L. gaucho, anti-arachnidic serum, as expected, showed a better neutralization potential because its formulation venom was from L. gaucho and was used in the immunization pool.

In conclusion, using recombinant Loxosceles spp. toxins of the SMase D family, we have generated an effective anti-Loxosceles serum with higher in vivo neutralizing capacity against L. intermedia and L. laeta venoms than the most widely used anti-arachnid antiserum. This development represents an important proof of the concept that recombinant SMases D can replace whole venom for anti-venom production and therapy. The fact that antibodies against a single toxin type are able to neutralize the major deleterious activities of a venom containing many other toxic components is not surprising because of the amount of SMase D in Loxosceles spp. venoms. A recent catalog of expressed sequence tags from L. laeta51 showed large amounts of SMase D expressed in venom glands, representing 16% of all mRNAs and orders of magnitude higher than most other toxins. The remaining components may be important for other biological needs of the spider and may not play a role in the pathophysiologic effects of venom in humans.

If one considers that L. intermedia and L. laeta cause most cases of envenomation in Brazil and South America, respectively, antiserum used in therapy is effective against these venoms. Therefore, we recommend use of anti-SMase D serum. Local knowledge of the distribution of different Loxosceles spp. may also help in the choice of antiserum used. Inclusion of SMase D isoforms from L. gaucho venom in the immunization formulation to obtain a fully neutralizing horse antiserum against the three predominant Loxosceles spp. spiders that cause envenomation in Brazil would be beneficial.

Figure 1.
Figure 1.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) Loxosceles spp. venoms and analysis of serum cross-reactivity. Samples of L. gaucho, L. intermedia, and L. laeta venoms (10 μg) were subjected to electrophoresis on a 12% SDS-PAGE gel under non-reducing conditions and stained with silver (left panel) or subjected to Western blotting (middle and right panels). Blots were probed with anti-arachnidic (middle panel) or anti-SMase D (right panel) horse sera diluted 1:2,000 and goat anti-horse IgG labeled with alkaline phosphatase diluted 1:3,000. Reactions were developed using 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tet-razolium.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 3; 10.4269/ajtmh.2008.79.463

Figure 2.
Figure 2.

Cross-reactivity of horse sera. A, Enzyme-linked im-munosorbent assay (ELISA) plates were coated with 1 μg of Loxosceles spp. venoms and incubated with different dilutions of normal, anti-arachnidic and anti-sphingomyelinase D (SMase) D sera and goat anti-horse IgG labeled with alkaline phosphatase (1:3,000). Absorbance of samples was determined at 492 nm. B, ELISA plates were coated with 1 μg of mouse IgG anti-horse IgGT and incubated with different dilutions of normal, anti-arachnidic and anti-SMase D sera and goat anti-mouse IgG labeled with horseradish peroxidase diluted 1:3,000. Absorbance of samples was determined at 492 nm.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 3; 10.4269/ajtmh.2008.79.463

Figure 3.
Figure 3.

Neutralization of the dermonecrotic effect of Loxosceles spp. venoms. A, Dermonecrosis reaction: adult rabbits were injected intradermally with 3 μg of Loxosceles spp. venoms and the size of the lesions was measured for 72 hours. B–D, Serum neutralization: adult rabbits were injected intradermally with 3 μg of Loxosceles spp. venoms and intravenously with 1 mL of anti-arachnidic (○), anti-sphingomyelinase D (•), or control horse sera. Size of the lesions was measured for 72 hours and results are expressed as percentage reduction ± SD of size of the dermonecrotic lesion of three experiments.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 3; 10.4269/ajtmh.2008.79.463

Figure 4.
Figure 4.

Neutralization of hemolytic activity of Loxosceles spp. venoms. A, Hemolysis: erythrocytes pretreated with veronal-buffered saline (VBS++) or different amounts of Loxosceles spp. venoms were incubated with normal human serum. After incubation for one hour at 37°C, unlysed cells were centrifuged and the absorbance of supernatants was measured at 414 nm and expressed as percentage of lysis. B–D, Serum neutralization: erythrocytes were treated with venoms from Loxosceles spp. or with VBS++ buffer in the presence or absence of anti-arachnidic (□) or anti-sphingomyelinase D (▪) sera and analyzed for expression GPC by flow cytometry. Results are representative for three experiments and expressed as percentage reduction of fluorescence ± SD of duplicate samples.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 3; 10.4269/ajtmh.2008.79.463

Figure 5.
Figure 5.

Neutralization of sphingomyelinase activity of Loxosceles spp. venoms. A, Sphingomyelinase (SMase) activity: SMAse (50 μg) was incubated with buffer or with increasing amounts of Loxosceles spp. venoms. After incubation at 37C for 20 minutes, the choline formed was oxidized to betaine and measured fluorimetrically. B–D, Serum neutralization: SMase was incubated with 1 μg of Loxosceles spp. venoms or veronal-buffered saline in the presence or absence the anti-arachnidic (□) or anti-SMase D (▪) sera for 30 minutes at 37°C. Samples were centrifuged and supernatants were analyzed for SMase D activity as described above. Results are representative for three experiments and expressed as percentage reduction ± SD of fluorescence of duplicate samples.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 3; 10.4269/ajtmh.2008.79.463

*

Address correspondence to Denise V. Tambourgi, Laboratório de Imunoquímica, Instituto Butantan, Av. Prof. Vital Brazil, 1500, CEP 05503-900, São Paulo, São Paulo, Brazil. E-mail: dvtambourgi@butantan.gov.br

Authors’ addresses: Daniel Manzoni de Almeida, Rute M. Gonçalves de Andrade, and Denise V. Tambourgi, Laboratório de Imunoquímica, Instituto Butantan, Av. Prof. Vital Brazil, 1500, CEP 05503-900, São Paulo, São Paulo, Brazil, E-mails: daniel@butantan.gov.br, rutemgdeandrade@butantan.gov.br, and dvtambourgi@butantan.gov.br. Matheus de F. Fernandes-Pedrosa, Laboratório de Imunoquímica, Instituto Butantan, Av. Prof. Vital Brazil, 1500, CEP 05503-900, São Paulo, São Paulo, Brazil and Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal do Rio Grande do Norte, Natal, Rio Grande do Norte, Brazil, E-mail: mpedrosa@ufrnet.br. José Roberto Marcelino and Hisako Gondo-Higashi, Di-visão Bioindustrial, Av. Prof. Vital Brazil, 1500, CEP 05503-900, São Paulo, São Paulo, Brazil, E-mails: marcelino@butantan.gov.br and hisa@butantan.gov.br. Inácio de L. M. Junqueira de Azevedo and Paulo Lee Ho, Centro de Biotecnologia, Av. Prof. Vital Brazil, 1500, CEP 05503-900, São Paulo, São Paulo, Brazil, E-mails: ijuncaze@butantan.gov.br and hoplee@butantan.gov.br. Carmen van den Berg, Cardiff University, Wales College of Medicine, Cardiff CF10 3XQ, United Kingdom, E-mail: vandenbergcw@yahoo.com.

Financial support: This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo, the Conselho Nacional de Desenvolvimento Científico e Tecnológico, and the Butantan Foundation.

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