Electrophoresis and lectin Western blot.

Samples of N. annulifera venom (15 μg) were separated by SDS-PAGE on 8–16% or 12% gels [43], under reducing or non-reducing conditions, and were silver stained [44] or blotted on nitrocellulose membranes [45]. After the transfer, the membranes were blocked with 5% BSA in Phosphate Buffered Saline (PBS) (8.1 mM sodium phosphate, 1.5 mM potassium phosphate, 137 mM sodium chloride and 2.7 potassium chloride, p.H. 7.2) and then incubated with peroxidase labeled lectins, ConA (1:1000 dilution) or WGA (1:2000 dilution) to detect residues of Mannose and N-acetylglucosamine [46]. Glycosylated proteins were detected using a solution that contained 0.1% hydrogen peroxide plus 0.5 mg/mL DAB.

Mass spectrometric protein identification.

For the proteomic analysis, N. annulifera venom was submitted to trypsin digestion as described by Kinter and Sherman [47]. Briefly, 200 μg venom (three replicates) were denatured with 6 M urea in 100 mM Tris-HCl, pH 8, and reduced with dithiothreitol (DTT) 10 mM for 1 h at room temperature. Cysteine carbamidomethylation was performed by incubation with iodoacetamide 40 mM for 1 h at room temperature, followed by addition of DTT 40 mM to consume excess of alkylating agent. Protein samples were diluted with water until the concentration of urea decreased to about 0.7 M and trypsin was added using an enzyme: substrate ratio of 1:50 (w/w) at 37°C for 18 h. The reaction was stopped with 20% TFA until pH 3 and desalting was carried out by loading individual samples to p-1000 StageTip SDB-XC [48]. After drying using a vacuum centrifuge, peptide mixtures were dissolved in 100 μL of 0.1% formic acid (solution A) and peptide concentration was estimated. For that, a peptide standard curve was prepared using a mixture of trypsin-digested bovine serum albumin (MassPREP BSA Digestion Standard; Waters) ranging from 0.2 to 1.2 μg/μL (in solution A). The absorbance was measured at 214 nm using a NanoDrop spectrophotometer (Thermo Scientific), and peptide sample concentration was determined using the standard curve. For analysis, each peptide sample (5 μg) was automatically injected using an EASY Nano LCII system (Thermo Scientific) into a 5 cm of 10 μm Jupiter C-18 trap column (100 μm I.D. × 360 μm O.D.) coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). Chromatographic separation of tryptic peptides was performed on a 15-cm long column (75 μm I.D. x 360 μm O.D.) packed in-house with 3 μm ReproSil-Pur C-18 beads (Dr. Maisch, Germany). Peptides were eluted with a linear gradient of acetonitrile in 0.1% formic acid (solution B) at 200 nL/min: the linear step used for the elution was 5–35% in 75 min. Spray voltage was set at 2.2 kV and the mass spectrometer was operated in data dependent mode, in which one full MS scan was acquired in the m/z range of 300–1,600 followed by MS/MS acquisition using collision induced dissociation of the ten most intense ions from the MS scan. MS spectra were acquired in the Orbitrap analyzer at 30,000 resolution (at 400 m/z) whereas the MS/MS scans were acquired in the linear ion trap. The minimum signal threshold to trigger fragmentation event, isolation window, activation time and normalized collision energy were set to, respectively, 1,000 cps, 2 m/z, 10 ms and 35%. A dynamic peak exclusion (list size of 500) was applied to avoid the same m/z of being selected for the next 20 seconds. Two independent LC-MS/MS runs were performed for each sample.

Database search.

Protein identification was performed using the MaxQuant software suite (v 1.5.3.12) [49] using the theoretical trypsin fragments produced from the database restricted to the taxonomy “Serpentes” (Uniprot release September 14, 2016; 63,114 sequences). The database search considered the precursor mass tolerance of 20 ppm, in the first search, and the nonlinear mass recalibration was made in the masses of first pass identifications. The second search considered the mass tolerance of 4.5 ppm and tolerance for fragments was set to 0.5 Da. The following search parameters were used: iodoacetamide derivatives of cysteine were considered as fixed modification, oxidation of methionine was specified as variable modification, and up to two missed cleavages by trypsin were accepted. Assignments for peptides and proteins were accepted at a false discovery rate < 1%. For the identification, only proteins identified in at least two experimental replicates, with at least 1 unique peptide and posterior error probability ≤ 0.01 were accepted. “Match between runs” feature was enabled. For each protein group in the MaxQuant’s ‘proteinGroups.txt’ file, the first protein entry was selected as representative. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org), via the PRIDE partner repository [50], with the dataset identifier: PXD010962 (Username: reviewer27828@ebi.ac.uk; Password: BNxFCLRG).

Gelatinolytic activity.

The gelatinolytic activity of the N. annulifera snake venom was evaluated with zymography using gelatin as the substrate [51]. Samples of the venom (30 μg), prepared under non-reducing conditions, were separated via 10% SDS-PAGE with 1 mg/ml gelatin. The gels were washed for 2 hours at room temperature in 2.5% Triton X-100 solution and incubated overnight at 37°C in substrate buffer (50 mM Tris-HCl, 200 mM NaCl, 10 mM CaCl₂, 0.05% Brij-35, p.H. 8.3). After this period, the gels were stained with Coomassie Brilliant Blue solution (40% methanol, 10% acetic acid and 0.1% Coomassie Brilliant Blue).

Fibrinogen cleavage.

Human fibrinogen samples (30 μg) were incubated with 5 μg of N. annulifera venom or saline for 1 hour at 37°C under constant agitation. In parallel, venom samples were pre-incubated with 1.10 Phe (10 mM) or PMSF (10 mM) at room temperature for 30 minutes. After incubation, the mixtures were submitted to SDS-PAGE using a 8–16% gradient gel under reducing conditions and stained with Coomassie Brilliant Blue solution. The positive control of the reaction was human Thrombin (1U).

Hyaluronidase activity.

Hyaluronidase activity was analyzed according to the methodology described by Purkrittayakamee et al. [52] with slight modifications. Briefly, samples of N. annulifera venom (20 μg) were incubated with a mixture that contained hyaluronic acid (1 mg/mL) (100 μL) and acetate buffer (200 mM sodium acetate and 0.15 M of NaCl, p.H. 6.0) (400 μL) at 37°C for 15 minutes. After incubation, the reactions were stopped with 1 mL of CTAB (2.5% CTAB and 2% NaOH). The absorbances were measured at λ405 nm in a spectrophotometer (Multiskan EX, Labsystems, Finland) against a blank that contained hyaluronic acid, acetate buffer and CTAB. The results are expressed as units of turbidity reduction (UTR) per mg of venom. T. serrulatus scorpion venom (20 μg) was used as a positive control.

PLA₂ activity.

PLA₂ activity was evaluated using the EnzChek^TM Phospholipase A2 Assay Kit according to the manufacturer’s recommendations. Briefly, samples of N. annulifera venom (0.5 μg) was incubated with a phospholipid mix that contained 10 mM Dioleoylphosphatidylcholine and 10 mM Dioleoylphosphatidylglycerol in 96-well microtiter plates. Increased fluorescence was evaluated using a spectrometer FLUOstar Omega (BMG Labtech, Ortenberg, Germany) at λ_EM 460 and λ_EX 515 nm and 37°C for 10 minutes. Specific activity was expressed as Units of Fluorescence (UF) per minute per microgram of venom. C. d. terrificus (0.5 μg) venom was used as positive control.

Cytotoxic activity.

The HaCat human keratinocyte cell lineage was cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO₂. The action of N. annulifera venom on the viability of human keratinocytes was evaluated using the MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) method [53] with minor modifications. Keratinocytes (5×10⁴ cells/well) were grown in 96-well plates that contained supplemented DMEM medium and were then maintained for 24 hours in DMEM medium without SFB. After this period, cells were incubated for 72 hours in increasing venom concentrations at 37°C and 5% CO₂. The supernatant from the wells was aspirated and 60 μl of the MTT solution (0.83 μg/μL) in incomplete DMEM medium was added to the wells. The plates were then incubated for 30 minutes at 37° C and 5% CO₂. Thereafter, the supernatant was aspirated, 100 μL/well of DMSO was added, and the plates were allowed to stand at room temperature for five minutes. The spectrophotometric analysis of the reactions (Multiskan-EX, Labsystems, Helsinki, Finland) was performed at λ 540 nm. Cell viability was calculated as: [O.D. experimental sample _(540nm)*100]/[O.D. control sample_(540nm)]. The results are expressed as cell viability (%).

Additionally, the action of N. annulifera venom on the viability of human keratinocytes was evaluated by Lactate Dehydrogenase (LDH) release assay. Supernatants of keratinocyte cultures, treated for 72 hours with increasing venom concentrations, were centrifuged at 405× g for 10 min, and tested for the presence of LDH, using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit, according to the manufacturer's instructions.

Activated Partial Thromboplastin Time (APTT).

PPP samples (50 μL) were treated with saline or increasing concentrations of venom and incubated for 3 minutes at 37°C with 50 μL of Cephalin. PPP was then recalcified with CaCl₂ (0.025 M), and the coagulation time was measured over 240 seconds in a Stago Start System (Asnières-sur-Seine, France). The results are expressed in seconds and R-time, which was obtained from the experimental sample/control sample. Disorders in coagulation time were considered significant when the R-time was over 1.3 [54].

Prothrombin Time (PT).

PPP samples (50 μL) were treated with saline or increasing concentrations of venom and incubated for 1 minute at 37°C. PPP was then incubated with CaCl₂ (0.025 M) that contained thromboplastin (100 μL), and the coagulation time was followed for 60 seconds using the Stago Start System (Asnières-sur-Seine, France). The results are expressed in seconds and R-time, which was obtained from the experimental sample/control sample. Abnormalities in coagulation time were considered significant when the R-time was over 1.3 [54].

Lethal Activity of the venom (LD₅₀).

Balb/c mice groups (n = 6) were intraperitoneally (i.p) inoculated with increasing concentrations of venom (20, 40, 60, 80, 100 and 120 μg) [55] or sterile saline. After inoculation, animals were monitored for 72 hours and LD₅₀ was calculated using the Probit transformation [56]. The results are expressed as μg of venom per mouse.

Local reactions: Edematogenic activity and pharmacological modulation.

The venom edematogenic activity was assessed via the method previously published by Yamakawa et al. [57] with some modifications. Balb/c mice (n = 6) were inoculated with 10 μg of N. annulifera venom at a volume of 50 μL (sterile saline) in the subcutaneous (s.c) tissue of the plantar region of the animal’s left hindpaw. The contralateral hindpaw (control) was inoculated with 50 μL of sterile saline. To assess edematogenic activity, the thickness of the hindpaw was evaluated with a caliper rule (Mitutoyo, Suzano-SP, Brazil; sensibility of 0.01 mm) at different time points before (T0) and over the 24 h, following either venom or saline inoculation (Te). Increases in paw volume are expressed in percentage form (%), calculated with the following formula: (Te-T0)/T0*100.

To evaluate the contribution of mast cells and different lipid mediators to the hindpaw edema induced by N. annulifera venom, mice (n = 6 per drug) were pretreated with different anti-inflammatory compounds: a) Sodium Cromoglycate, a mast cell degranulation inhibitor (10 mg/kg), administered i.p. for 3 days before edema induction [58]; b) dexamethasone, an indirect cPLA₂ inhibitor, was given to mice (2 mg/kg) i.p. 2 h before venom inoculation [59]; c) the non-selective cyclooxygenase inhibitor Indomethacin, was administered (10 mg/kg) i.p. 30 minutes [60] before venom; d) MK-886, a 5-lipoxigenase-activating protein inhibitor, was administered (5 mg/kg) i.p. 30 minutes [61] before edema induction; and e) WEB-2086, a Platelet Activating Factor Receptor antagonist, was administered (5 mg/kg) s.c. 1 hour before venom administration [62, 63]. Dexamethasone and Cromolyn were diluted in saline, while Indomethacin, MK-886, WEB-2086 were diluted in DMSO. Following these pretreatments, edema was induced and monitored for 24 h.

Systemic reactions.

To analyze the possible systemic reactions induced by N. annulifera venom, two experimental groups were established. The first measured systemic alterations over time. LD₅₀ assays were performed to define the sublethal dosing level that was able to induce the deleterious effects of the venom, including an increase in inflammatory parameters, but not kill the animals. LD₅₀ values that varied between 50 and 75% were tested, and the selected dose for the inflammatory assays was 60% of LD₅₀. Balb/c mice (n = 6) were then inoculated with 60% of LD₅₀ or sterile saline i.p. and euthanized at different time points to obtain blood and collect the organs.

In another experimental set, it was assessed whether death promoted by venom was preceded by systemic reactions. Animal groups (n = 6) were inoculated with saline or 2 LD₅₀ of N. annulifera venom i.p. Immediately after animals’ death, their blood and organs were collected. Blood samples were obtained by cardiac puncture and were immediately incubated with EDTA (2.5 mg/mL). Aliquots of these samples were used to measure systemic leukocyte changes, while other blood samples were centrifuged at 2800× g at 4°C for 10 minutes to isolate plasma. Plasma samples were stored at -80°C and used to measure inflammatory mediators.

Leukocyte alterations.

To analyze changes in the total number of leukocytes, blood samples were diluted 1:10 in Turk’s solution (0.1% crystal violet dye in 2% acetic acid), and the cells were counted in a Neubauer chamber. Blood samples were also submitted for blood smear and stained with a Fast Panoptic stain kit. Differential cell counts were performed by analyzing 100 cells that were identified as lymphocytes, neutrophils or monocytes based on morphologic criteria.

Detection of inflammatory mediators.

IL-6, TNF-α, MCP-1, IL-12, IFN-γ and IL-10 detection was performed using the BD Cytometric Bead Array (CBA) Mouse Inflammation Kit (BD Bioscience). IL-1β and IL-17 quantification was performed using the Mouse IL-1β ELISA (BD Bioscience) and Mouse IL-17 Duo3.2.43 ELISA (R&D Systems) kits, respectively. All assays were performed according to the manufacturer’s instructions. The results are expressed as pg/mL.

Histopathologic analysis.

To analyze systemic histopathologic changes, Balb/c mice (n = 6) were inoculated i.p. with a sublethal dose or 2DL₅₀ of N. annulifera venom. After euthanasia or death, animals were exsanguinated, and their brains, lungs, hearts, kidneys, spleens and livers were collected. To analyze local histopathological changes, Balb/c mice (n = 3) were injected subcutaneously with 10 μg of N. annulifera venom at a volume of 50 μL (sterile saline) in the plantar region of their left hindpaws. The contralateral hindpaws (control) were inoculated with 50 μL of sterile saline. Animals were euthanized at different time points (0, 20, 60, 240 and 1440 minutes) and had their hindpaws removed. The hindpaws and organs were fixed in a 10% formaldehyde solution for 24 hours, submitted for routine histology and stained with hematoxylin and eosin (HE). All samples were analyzed with a light microscope and were examined for the presence of necrosis, edema, inflammatory infiltrates or hemorrhagic foci.

Immunogenicity.

H_III mice (n = 12) were immunized subcutaneously with 10 μg of N. annulifera venom diluted 1:25 on Al(OH)₃ to achieve a final volume of 200 μL. These animals received three booster doses (5 μg) at 20-day intervals. Control animals were inoculated with sterile saline. Bleeding was performed 10 days after each venom inoculation. Blood was allowed to clot at room temperature for 15 minutes and was then left at 4° C for 6 hours. After centrifugation at 252× g and 4° C, the sera were collected and immediately frozen at -20° C.

Sera antibody titers.

To evaluate the sera antibody titers of immunized animals, ELISA plates (Costar) were coated with 100 μL of N. annulifera venom (10 μg/mL) and incubated overnight at 4° C. The plates were then blocked with 5% BSA in PBS for 2 hours at 37° C and incubated with crescent dilutions of non-immune or experimental sera samples for 1 hour at room temperature. After incubation, the plates were washed with PBS-Tween 20 0.05%, and incubated with the IgG-HRPO (1:5000 diluted) secondary antibody in 1% BSA/PBS for 1 hour at 37° C. The plates were then washed, and the reactions were developed with OPD substrate according to the manufacturer’s instructions. The optical densities were obtained using spectrophotometry (Multiskan EX, Labsystems, Finland) at λ492 nm.

Recognition of venom components using experimental antivenom.

Samples of N. annulifera venom (15 μg) were separated using 8–16% gradient SDS-PAGE [43] and blotted on nitrocellulose membranes [45]. The membranes were blocked for 2 hours with 5% BSA in PBS, washed three times with PBS, incubated with normal or experimental sera pools, and diluted 1:5000 in 0.01% PBS/BSA for 1 hour at room temperature. After incubation, the membranes were washed with PBS-0.05% Tween 20, incubated with IgG-AP secondary antibodies, and diluted 1:5000 in 1% BSA/PBS for 1 hour at room temperature. After incubation, the membranes were washed three times in PBS-0.05% Tween 20. Antigenic reactivity was measured by the addition of NBT and BCIP substrates according to the manufacturer’s recommendations.

Serum neutralization of the venom’s lethal activity.

To determine the neutralizing potential of the experimental antivenom, samples of the venom, equivalent to 2LD₅₀ (188.28 μg), were incubated with saline or crescent dilutions of the experimental serum for 30 minutes at 37° C. These mixtures were then i.p. administered to Balb/c mice (n = 6) (200 μL). Animals were observed for 72 hours and the death/survival ratio recorded. The neutralizing potential was calculated by Probit analysis and the results expressed as potency (mg of venom neutralized by mL of experimental serum) [56].

Local reactions.

To evaluate the edematogenic activity of N. annulifera venom, Balb/c mice were inoculated with 10 μg of venom into their left hind footpads, and their paws were measured at different time points. The venom was able to induce a significant rapid onset of edema, with its maximum peak (110.13% of increase on paw volume) reached 20 minutes after inoculation. It returned to basal level 24 hours after venom inoculation (Fig 3A).

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Fig 3. Local alterations induced by N. annulifera venom.

[A] Edema: samples of venom (10 μg/50 μL) or sterile saline were injected into the left or right hind footpad, respectively, of Balb/c mice (n = 6). Paw edema was assessed by measuring the paw thickness using a caliper rule before (T0) and after inoculation with venom or saline (Te). The increased paw volume was expressed as percentage (%) and was calculated with the following formula: (Te-T0)/T0*100. Statistical analysis was performed using two-way ANOVA, followed by the Bonferroni multiple comparison test (***p≤ 0.05) ± SD. [B] The effect of inhibitors on the paw edema induced by N. annulifera venom: groups of Balb/c mice (n = 6) were treated with different inhibitors before induction of edema with venom: Cromolyn, a mast cell degranulation inhibitor (10 mg/kg, three days consecutively before the edema, i.p. route); dexamethasone (Dexa), a cPLA₂ inhibitor (2 mg/kg, 2 hours before the edema, i.p. route); indomethacin (Indo), a COX isoform inhibitor (10 mg/kg, 30 minutes before the edema, i.p. route); MK886, a 5-lipoxygenase-activating protein inhibitor (5 mg/kg, 30 minutes before the edema, i.p. route); and WEB2086, a PAFR antagonist (5 mg/kg, 1 hour before the edema, s.c. route). After treatment, edema was induced, and the paws were measured with a caliper rule at several time points. The increased paw volume was expressed as a percentage. Statistical analysis was performed using two-way ANOVA, followed by the Bonferroni multiple comparison test (***p≤ 0.05) ± SD. [C] Histopathological changes promoted by N. annulifera venom: samples of N. annulifera venom (10 μg/50 μL) were injected into the left hind paw of Balb/c mice (n = 6). The contralateral paws were injected with sterile saline (50 μL) to serve as controls. The animals were euthanized at different time points, and their paws were removed and submitted for histopathological analysis. Control group [Panel 1]: epidermis (Ep), dermis (De), sweat glands (Sg) and muscle tissue (Mt). Experimental group [Panel 2]: arrows highlight vascular congestion and a perivascular inflammatory infiltrate; asterisks indicate areas of edema. The rectangle indicates areas of neutrophil infiltration [Panel 3] and myonecrosis [Panels 4, 5]. The arrow and asterisk in Panel 5 indicate dead and living cells, respectively. Scale = 10μm.

https://doi.org/10.1371/journal.pntd.0007017.g003

In addition to the evaluation of edematogenic activity, the contribution of mast cell and lipid mediators to edema development was also assessed. It was found that mast cell degranulation is important for the earliest development (10–30 minutes) of edema as treatment with Cromolyn, a mast cell degranulation inhibitor, was able to reduce swelling (S1A Fig). Dexamethasone, a synthetic corticosteroid compound with a potent anti-inflammatory activity, had a strong effect on the paw edema induced by N. annulifera venom. It was able to decrease hindpaw thickness throughout the duration of edema, mainly during the early stages (10–30 minutes), suggesting that lipid mediators participate in the development of edema (S1B Fig). Indomethacin, a non-selective COX inhibitor, was also able to reduce the degree of edema (10 to 120 minutes), suggesting that prostaglandins and thromboxanes may contribute to the local reaction to the venom. However, treatment with Indomethacin was not able to control swelling during the late phase (180 to 1440 minutes) (S1C Fig). A similar effect was observed in animals treated with a FLAP inhibitor (S1D Fig). The PAFR blocker WEB-2086 demonstrated inhibitory action only in the earliest phases of the edema (20–30 minutes) (S1E Fig). Although all inhibitors showed different modulation profiles, they were able to significantly decrease the edema at its peak [Fig 3B].

Histopathologic analyses showed that the venom promotes several changes, ranging from mild to intense, at all time periods [Fig 3C]. After inoculation with the venom, it was observed that it promotes an acute inflammatory response at the inoculation site, with a disorganized dermal collagen matrix, subcutaneous edema, vascular congestion [Panel 2], mild erythrocyte extravasation and neutrophil migration to the dermis and muscle [Panels 3, 4, 5]. Myonecrosis was also observed [Panels 4, 5] along with polymorphonuclear cell infiltration. These alterations persisted for several hours. However, after 24 hours of inoculation, a decrease in the subcutaneous edema was observed, along with the absence of vascular congestion and few remaining neutrophils in the tissue. By contrast, an increased number of mononuclear cells and reorganization of the dermal collagen matrix was observed [Panel 6].