A C-type lectin from Bothrops jararacussu venom reprograms endothelial cell biology.

Snake venoms are intricate mixtures of enzymes and bioactive factors that induce a range of detrimental effects in afflicted hosts. Certain Viperids, including Bothrops jararacussu, harbor C-type lectins (CTLs) known for their modulation of a variety of host cellular responses. In this study, we isolated and purified BjcuL, a CTL from B. jararacussu venom and investigated its impact on endothelial cell behavior, contrasting it with human galectin-1 (Gal-1), a prototype member of the galectin family with shared ß-galactoside-binding activity. We found that BjcuL binds to human dermal microvascular endothelial cells (HMECs) in a concentration- and carbohydrate-dependent fashion and reprograms the function of these cells, favoring a pro-inflammatory and pro-coagulant endothelial phenotype. In light of the quest for universal antagonists capable of mitigating the harmful consequences of snake venoms, BjcuL emerges as a promising target to be blocked in order to regulate pathological endothelial cell responses.

Peptidoglycan recognition protein-peptidoglycan complexes increase monocyte/macrophage activation and enhance the inflammatory response.

Peptidoglycan recognition proteins (PGRP) are pattern recognition receptors that can bind or hydrolyse peptidoglycan (PGN). Four human PGRP have been described: PGRP-S, PGRP-L, PGRP-Iα and PGRP-Iß. Mammalian PGRP-S has been implicated in intracellular destruction of bacteria by polymorphonuclear cells, PGRP-Iα and PGRP-Iß have been found in keratinocytes and epithelial cells, and PGRP-L is a serum protein that hydrolyses PGN. We have expressed recombinant human PGRP and observed that PGRP-S and PGRP-Iα exist as monomer and disulphide dimer proteins. The PGRP dimers maintain their biological functions. We detected the PGRP-S dimer in human serum and polymorphonuclear cells, from where it is secreted after degranulation; these cells being a possible source of serum PGRP-S. Recombinant PGRP do not act as bactericidal or bacteriostatic agents in the assayed conditions; however, PGRP-S and PGRP-Iα cause slight damage in the bacterial membrane. Monocytes/macrophages increase Staphylococcus aureus phagocytosis in the presence of PGRP-S, PGRP-Iα and PGRP-Iß. All PGRP bind to monocyte/macrophage membranes and are endocytosed by them. In addition, all PGRP protect cells from PGN-induced apoptosis. PGRP increase THP-1 cell proliferation and enhance activation by PGN. PGRP-S-PGN complexes increase the membrane expression of CD14, CD80 and CD86, and enhance secretion of interleukin-8, interleukin-12 and tumour necrosis factor-α, but reduce interleukin-10, clearly inducing an inflammatory profile.

Effect of Aspergillus niger prolyl endopeptidase in patients with celiac disease on a long-term gluten-free diet.

BACKGROUND: The gluten-free diet (GFD) has limitations, and there is intense research in the development of adjuvant therapies. AIM: To examine the effects of orally administered Aspergillus niger prolyl endopeptidase protease (AN-PEP) on inadvertent gluten exposure and symptom prevention in adult celiac disease (CeD) patients following their usual GFD. METHODS: This was an exploratory, double-blind, randomized, placebo-controlled trial that enrolled CeD patients on a long-term GFD. After a 4-wk run-in period, patients were randomized to 4 wk of two AN-PEP capsules (GliadinX; AVI Research, LLC, United States) at each of three meals per day or placebo. Outcome endpoints were: (1) Average weekly stool gluten immunogenic peptides (GIP) between the run-in and end of treatments and between AN-PEP and placebo; (2) celiac symptom index (CSI); (3) CeD-specific serology; and (4) quality of life. Stool samples were collected for GIP testing by ELISA every Tuesday and Friday during run-ins and treatments. RESULTS: Forty patients were randomized for the intention-to-treat analysis, and three were excluded from the per-protocol assessment. Overall, 628/640 (98.1%) stool samples were collected. GIP was undetectable (< 0.08 µg/g) in 65.6% of samples, and no differences between treatment arms were detected. Only 0.5% of samples had GIP concentrations sufficiently high (> 0.32 µg/g) to potentially cause mucosal damage. Median GIP concentration in the AN-PEP arm was 44.7% lower than in the run-in period. One-third of patients exhibiting GIP > 0.08 µg/g during run-in had lower or undetectable GIP after AN-PEP treatment. Compared with the run- in period, the proportion of symptomatic patients (CSI > 38) in the AN-PEP arm was significantly lower (P < 0.03). AN-PEP did not result in changes in specific serologies. CONCLUSION: This exploratory study conducted in a real-life setting revealed high adherence to the GFD. The AN-PEP treatment did not significantly reduce the overall GIP stool concentration. However, given the observation of a significantly lower prevalence of patients with severe symptoms in the AN-PEP arm, further clinical research is warranted.

Crystal structure of staphylococcal enterotoxin G (SEG) in complex with a mouse T-cell receptor {beta} chain.

Superantigens (SAgs) are bacterial or viral toxins that bind MHC class II (MHC-II) molecules and T-cell receptor (TCR) in a nonconventional manner, inducing T-cell activation that leads to inflammatory cytokine production, which may result in acute toxic shock. In addition, the emerging threat of purpura fulminans and community-associated meticillin-resistant Staphylococcus aureus emphasizes the importance of a better characterization of SAg binding to their natural ligands that may allow the development of reagents to neutralize their action. The three-dimensional structure of the complex between a mouse TCR ß chain (mVß8.2) and staphylococcal enterotoxin G (SEG) at 2.0 Å resolution revealed a binding site that does not conserve the "hot spots" present in mVß8.2-SEC2, mVß8.2-SEC3, mVß8.2-SEB, and mVß8.2-SPEA complexes. Analysis of the mVß8.2-SEG interface allowed us to explain the higher affinity of this complex compared with the others, which may account for the early activation of T-cells bearing mVß8.2 by SEG. This mode of interaction between SEG and mVß8.2 could be an adaptive advantage to bestow on the pathogen a faster rate of colonization of the host.

Cytokines expression from altered motor thalamus and behavior deficits following sublethal administration of Shiga toxin 2a involve the induction of the globotriaosylceramide receptor.

Encephalopathy associated with hemolytic uremic syndrome is produced by enterohemorrhagic E. coli (EHEC) infection, which releases the virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS). Neurological compromise is a poor prognosis and mortality factor of the disease, and the thalamus is one of the brain areas most frequently affected. We have previously demonstrated the effectiveness of anti-inflammatory drugs to ameliorate the deleterious effects of these toxins. However, the thalamic production of cytokines involved in pro-inflammatory processes has not yet been acknowledged. The aim of this work attempts to determine whether systemic sublethal Stx2a or co-administration of Stx2a with LPS are able to rise a proinflammatory profile accompanying alterations of the neurovascular unit in anterior and lateral ventral nuclei of the thalamus (VA-VL) and motor behavior in mice. After 4 days of treatment, Stx2a affected the lectin-bound microvasculature distribution while increasing the expression of GFAP in reactive astrocytes and producing aberrant NeuN distribution in degenerative neurons. In addition, increased swimming latency was observed in a motor behavioral test. All these alterations were heightened when Stx2a was co-administered with LPS. The expression of pro-inflammatory cytokines TNFα, INF-γ and IL-2 was detected in VA-VL. All these effects were concomitant with increased expression of the Stx receptor globotriaosylceramide (Gb3), which hints at receptor involvement in the neuroinflammatory process as a key finding of this study. In conclusion, Stx2a to Gb3 may be determinant in triggering a neuroinflammatory event, which may resemble clinical outcomes and should thus be considered in the development of preventive strategies.

Immunotherapeutic nanoparticles: From autoimmune disease control to the development of vaccines.

The development of nanoparticles (NPs) with potential therapeutic uses represents an area of vast interest in the scientific community during the last years. Recently, the pandemic caused by COVID-19 motivated a race for vaccines creation to overcome the crisis generated. This is a good demonstration that nanotechnology will most likely be the basis of future immunotherapy. Moreover, the number of publications based on nanosystems has significantly increased in recent years and it is expected that most of these developments can go on to experimentation in clinical stages soon. The therapeutic use of NPs to combat different diseases such as cancer, allergies or autoimmune diseases will depend on their characteristics, their targets, and the transported molecules. This review presents an in-depth analysis of recent advances that have been developed in order to obtain novel nanoparticulate based tools for the treatment of allergies, autoimmune diseases and for their use in vaccines. Moreover, it is highlighted that by providing targeted delivery an increase in the potential of vaccines to induce an immune response is expected in the future. Definitively, the here gathered analysis is a good demonstration that nanotechnology will be the basis of future immunotherapy.

South American snake venoms with abundant neurotoxic components. Composition and toxicological properties. A literature review.

In South America there are three snake genera with predominantly neurotoxic venoms: Crotalus, Micrurus and Hydrophis, which include nine species/subspecies, 97 species and a single marine species, respectively. Although accidents with neurotoxic venoms are less frequent than those with anticoagulant, cytotoxic or necrotic venoms (e.g. from Bothrops), they are of major public health importance. Venoms from genus Crotalus have been extensively studied, while data on the venoms from the other two genera are very limited, especially for Hydrophis. The venoms of North and South American Crotalus species show biochemical and physiopathological differences. The former species cause bothrops-like envenomation symptoms, while the latter mainly have neurotoxic and myotoxic effects, leading to respiratory paralysis and, occasionally, renal failure by myoglobinuria and death, often with no local lesions. Micrurus and Hydrophis also cause neurotoxic envenomations. Many studies have isolated, identified and characterized new enzymes and toxins, thus expanding the knowledge of snake venom composition. The present review summarizes the currently available information on neurotoxic venoms from South American snakes, with a focus on protein composition and toxicological properties. It also includes some comments concerning potential medical applications of elapid and crotalic toxins.

Surface chemistry modification of silica nanoparticles alters the activation of monocytes.

Aim: Nanoparticles (NPs) interaction with immune system is a growing topic of study. Materials & methods: Bare and amine grafted silica NPs effects on monocytes/macrophages cells were analyzed by flow cytometry, MTT test and LIVE/DEAD® viability/cytotoxicity assay. Results: Bare silica NPs inhibited proliferation and induced monocyte/macrophages activation (increasing CD40/CD80 expression besides pro-inflammatory cytokines and nitrite secretion). Furthermore, silica NPs increased cell membrane damage and reduced the number of living cells. In contrast, amine grafted silica NPs did not alter these parameters. Conclusion: Cell activation properties of bare silica NPs could be hindered after grafting with amine moieties. This strategy is useful to tune the immune system stimulation by NPs or to design NPs suitable to transport therapeutic molecules.

Physicochemical and biological characterization of nanovenoms, a new tool formed by silica nanoparticles and Crotalus durissus terrificus venom.

Nanoparticles (NPs) are being studied due to their potential use as therapeutic and immunomodulatory tools, including their ability to transport antigens with the aim to induce a specific immune response. The production of snake antivenoms (AV) involves several inoculations of venom (V) in the presence of adjuvants (ADJ) to improve the immune response of inoculated animals, causing a decrease in its quality and shelf life. Therefore, it is interesting to develop new strategies for reduce these side effects. For that reason, associating V to NPs to replace conventional ADJ could be a useful tool for future AV production. In this work, nanovenoms (NVs) were generated by the adsorption of Crotalus durissus terrificus (Cdt) V proteins over silica NPs (SiNPs) synthesized according to the Stöber method. Microphotographies obtained under Transmission Electron Microscopy (TEM) displayed a protein crown over NPs and Fourier Transform Infrared (FT-IR) presented the expected spectra for NVs resulting from the sum of those exhibited by Cdt V and SiNPs separately. SDS PAGE and immunoblotting assays confirmed the presence of proteins over SiNPs. Furthermore, the different enzymatic activities detected demonstrated that SiNPs were capable of binding V proteins preserving its activity and therefore would keep its native structure. In the same way, the NVs conserve the potential cytotoxic effects present in the V as we observed when culturing THP-1 cells with these complexes. This evidence allows us to infer that developed NVs could be used as a new platform for the production of antisera or for immunomodulatory therapies.

Nanoparticles and Immune Cells.

Nanoparticles have gained ground in several fields. However, it is important to consider their potentially hazardous effects on humans, flora, and fauna. Human exposure to nanomaterials can occur unintentionally in daily life or in industrial settings, and the continuous exposure of the biological components (cells, receptors, proteins, etc.) of the immune system to these particles can trigger an unwanted immune response (activation or suppression). Here, we present different studies that have been carried out to evaluate the response of immune cells in the presence of nanoparticles and their possible applications in the biomedical field.

Silicified collagen materials: Modulation of the in vitro and in vivo response.

Collagen derived materials offer distinct advantages over synthetic polymers, considering their natural inherited biocompatibility and mechanical properties. However, because of the extraction procedure, the latter frequently need to be enhanced through the use of different crosslinking methods. Aldehydes are often used for the stabilization of biomaterials but the introduction of crosslinkers slightly alters the protein's surface reactivity hence calling for new biocompatibility studies. At the same time, silicate modification of natural polymers has gained interest within the biomaterials field for their strengthening potential and ease of manipulation, giving rise to different surfaces and bulk materials. In the present work, collagen gels modified with glutaraldehyde (ColGA) or glutaraldehyde and an aminosilane (ColGASi) were evaluated in vitro and in vivo with the aim to obtain biomaterials for wound dressings. The results obtained were compared to those derived from unmodified collagen matrices (Col). In vitro assays focused on the interaction of the materials with elements present in the human blood whereas in vivo assays evaluated their ability to support cell proliferation and angiogenesis for a period of 30 days in a rodent model. Col gels induced an increase in platelet aggregation while ColGA gels decreased it. On the other hand, ColGASi had no effect on platelet aggregation but induced IL-1ß and nitric oxide platelet secretion. All gels induced lower IL-6 levels in PMN cells cultures when compared to controls. Col and ColGA gels decreased IL-1ß concentration whereas ColGASi induced high expression of TGF-ß in PMN cells. All gels decreased nitric oxide secretion but Col and ColGA gels increased IL-1ß production by monocytes. Definitely, all gels induced an anti and pro-inflammatory profile depending on the cell type with which they interact. In vivo, an increased cellular infiltration was observed along with new blood vessel formation in those matrices containing silicified collagen, while glutaraldehyde fixed collagen induced a foreign body reaction and appeared surrounded by a fibrous capsule after 30 days of subcutaneous implantation. Overall, the results obtained show that the silicification of collagen has advantages not only through the enhancement of its mechanical properties but also through the stimulation of the integration of the material with the surrounding tissue.

Dodecenylsuccinic anhydride modified collagen hydrogels loaded with simvastatin as skin wound dressings.

Skin wound healing presents a unique challenge because of its complex healing process. Herein, we developed a hydrophobic wound dressing to incorporate simvastatin, which has potential application in the treatment of ulcers and prevention of wound infection. For that matter, collagen hydrogels were grafted with dodecenylsuccinic anhydride (DDSA). The chemical modification was confirmed by FTIR and solid state 13 C-NMR spectroscopies while the ultrastructure was observed by scanning electron microscope (SEM) images. In contact angle measurements, a higher water droplet angle in DDSA-collagen gels was observed. This was consistent with the swelling assay, in which water absorption was 5.2 g/g for collagen and 1.9 g/g for DDSA-collagen. Additionally, viability and adhesion studies were performed. Cell adhesion decreased ~11% in DDSA-collagen and the number of viable cells showed a tendency to decrease as DDSA concentration increased but it was only significantly lower above concentrations of 12%. Modified gels were loaded with simvastatin showing higher adsorption capacity and lower release. Lastly, the antimicrobial and anti-inflammatory activity of DDSA-collagen materials were assessed. DDSA-collagen hydrogels, either unloaded or loaded with simvastatin showed sustained antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus for 72 hr probably due to the hydrophobic interaction of DDSA chains with bacterial cell walls. The antimicrobial activity was stronger against S. aureus. Collagen hydrogels also presented a prolonged antibacterial activity when they were loaded with simvastatin, confirming the antimicrobial properties of statins. Finally, it was observed that these materials can stimulate resident macrophages and promote an M2 profile which is desirable in wound healing processes.

egc Superantigens Impair Monocytes/Macrophages Inducing Cell Death and Inefficient Activation.

Bacterial superantigens (SAgs) are enterotoxins that bind to MHC-II and TCR molecules, activating as much as 20% of the T cell population and promoting a cytokine storm which enhances susceptibility to endotoxic shock, causing immunosuppression, and hindering the immune response against bacterial infection. Since monocytes/macrophages are one of the first cells SAgs find in infected host and considering the effect these cells have on directing the immune response, here, we investigated the effect of four non-classical SAgs of the staphylococcal egc operon, namely, SEG, SEI, SEO, and SEM on monocytic-macrophagic cells, in the absence of T cells. We also analyzed the molecular targets on APCs which could mediate SAg effects. We found that egc SAgs depleted the pool of innate immune effector cells and induced an inefficient activation of monocytic-macrophagic cells, driving the immune response to an impaired proinflammatory profile, which could be mediated directly or indirectly by interactions with MHC class II. In addition, performing surface plasmon resonance assays, we demonstrated that non-classical SAgs bind the gp130 molecule, which is also present in the monocytic cell surface, among other cells.

Antibody detection employing sol-gel immobilized parasites.

Immunofluorescence assay (IFA) and immunoperoxidase assay (IPA) are useful diagnostic techniques for specific antibody detection for different diseases. Both involve several alternatives for immobilization of cells, such as solvent or heat fixation. Non-covalent immobilization implies rigorous storage conditions at -20 degrees C to preserve the slides, and usually numerous cells are detached during the washing steps, which can lead to inconsistencies in the results. Sol-gel chemistry is usually used for coating different materials because of the mild conditions of the polymerization reaction and the ability to introduce functional groups to a wide variety of surfaces. We have developed a novel procedure for the attachment of Trypanosoma cruzi epimastigotes and Leishmania guyanensis promastigotes to a silicon oxide polymer covered glass surface. The film was prepared using standard microscope slides with tetraethoxysilane and 3-aminopropyl triethoxysilane as polymeric precursors. When acetone was used as the major coating solvent, the IFA showed the fluorescence of the attached parasites without matrix background interference. Similar results were observed when the IPA was evaluated. The sensitivity and specificity of the sol-gel immobilized parasite slides were comparable with the heat fixation technique. The performance of the coated slides was maintained for at least 2 months at 4 degrees C storage temperature. This immobilization method does not affect the molecular epitopes of the attached cells. Thus, homogeneous, ready to use, long lasting coated slides were obtained, which are appropriate for field conditions.

Cellular clot formation in a sipunculan worm: entrapment of foreign particles, cell death and identification of a PGRP-related protein.

Clotting in animals having open or closed circulatory system comprises humoral and cellular mechanisms. Sipunculans are a small phylum of non-segmented marine worms that do not have a true circulatory system. These worms can form a cellular clot without transforming cell-free coelomic fluid into an insoluble mass. The clot may also contribute to immune response by entrapping foreign particles. We evaluated the formation of a cellular clot ex vivo in the sipunculan Themiste petricola after activation through glass contact and sea water, the ability to entrap magnetic beads and non-pathogen cyanobacteria particles within the clot, and the presence of a peptidoglycan recognition protein S (PGRP-S) antigen in cells forming the clot. Our results showed that the clot was formed by homotypic aggregation of large granular leukocytes (LGLs), a subtype of cells found in the coelomic fluid. Aggregated LGLs served to entrap magnetic beads and non-pathogen cyanobacteria particles, and PGRP-S antigen was detected both in non-activated LGLs and in activated homotypic aggregates through immunofluorescence, Western blot and flow cytometry. Cellular clots were found to be positive to Annexin V-FITC labelling. Complete disintegration of cytoplasm with shedding of microparticles, nuclear isolation and degradation were also observed by light microscopy and flow cytometry. In conclusion, cellular clot formation in Themiste petricola may serve both haemostatic and immune functions entailing rapid activation changes in LGLs, entrapment of foreign particles within a homotypic aggregate, and further cell death.

Superantigen natural affinity maturation revealed by the crystal structure of staphylococcal enterotoxin G and its binding to T-cell receptor Vbeta8.2.

The illnesses associated with bacterial superantigens (SAgs) such as food poisoning and toxic shock syndrome, as well as the emerging threat of purpura fulminans and community-associated methicillin-resistant S. aureus producer of SAgs, emphasize the importance of a better characterization of SAg binding to their natural ligands, which would allow the development of drugs or biological reagents able to neutralize their action. SAgs are toxins that bind major histocompatibility complex class II molecules (MHC-II) and T-cell receptors (TCR), in a nonconventional manner, inducing T-cell activation that leads to production of cytokines such as tumor necrosis factor and interleukin-2, which may result in acute toxic shock. Previously, we cloned and expressed a new natural variant of staphylococcal enterotoxin G (SEG) and evaluated its ability to stimulate in vivo murine T-cell subpopulations. We found an early, strong, and widespread stimulation of mouse Vbeta8.2 T-cells when compared with other SAgs member of the SEB subfamily. In search for the reason of the strong mitogenic potency, we determined the SEG crystal structure by X-ray crystallography to 2.2 A resolution and analyzed SEG binding to mVbeta8.2 and MHC-II. Calorimetry and SPR analysis showed that SEG has an affinity for mVbeta8.2 40 to 100-fold higher than that reported for other members of SEB subfamily, and the highest reported for a wild type SAg-TCR couple. We also found that mutations introduced in mVbeta8.2 to produce a high affinity mutant for other members of the SEB subfamily do not greatly affect binding to SEG. Crystallographic analysis and docking into mVbeta8.2 in complex with SEB, SEC3, and SPEA showed that the deletions and substitution of key amino acids remodeled the putative surface of the mVbeta8.2 binding site without affecting the binding to MHC-II. This results in a SAg with improved binding to its natural ligands, which may confer a possible evolutionary advantage for bacterial strains expressing SEG.

Binding of natural variants of staphylococcal superantigens SEG and SEI to TCR and MHC class II molecule.

SEG and SEI are staphylococcal superantigens (SAgs) identified recently that belong to the egc operon and whose genes are in tandem orientation. Only a few allelic variants of SEG and SEI have been reported. Here we analyzed four Staphylococcus aureus strains with genotypic variation in both SAgs. However, both SAgs retain key residues in their putative TCR and MHC binding sites and, accordingly, their superantigenic properties. Thus, SEI significantly stimulates mouse T-cells bearing Vbeta3, 5 and 13, while SEG stimulates Vbeta7 and 9 in the draining node when inoculated in the footpad. As another member of the SEB subfamily, SEG also stimulates mouse Vbeta8.1+2. However, the increase in Vbeta8.1+2 T-cells observed at day 2 after inoculation reverts to normal values at day 4, whereas it remains high at day 4 following inoculation with SEC3 or SSA. T-cell stimulation assays in the mouse and analysis of the putative Vbeta8.2 binding site on SEG, which includes three non-conserved residues, suggest a possibly unique interaction between Vbeta8.2 and SEG. We also analyzed biochemical and biophysical characteristics of SEI and SEG binding to their cognate human beta chains by surface plasmon resonance, and binding to the HLA-DR1 MHC class II molecule by gel filtration. SEI binds human Vbeta5.2 and Vbeta1 with apparent K(D)'s of 23 and 118 microM, respectively; SEG binds Vbeta13.6 with a K(D) of 5 microM. As suggested by sequence homology, SEI requires Zn2+ for strong binding to DR1, which goes undetected in the presence of EDTA. SEG and SEI have characteristics such as co-expression, different interaction with MHC class II and stimulation of completely different subsets of human and mouse T-cells, which indicate complementary superantigenic activity and suggest an important advantage to staphylococcal strains in producing them both.

Evidence of size-dependent effect of silica micro- and nano-particles on basal and specialized monocyte functions.

AIM: To analyze the effect of silica particles on monocyte/macrophage functions. MATERIALS & METHODS: Silica micro- and nanoparticles were obtained by the Stöber method. Their effect on monocyte/macrophage proliferation, activation, membrane integrity and metabolic activity were determined. RESULTS: Silica particles inhibit cell proliferation while 10 nm nanoparticles (NPs) did not affect it. Similarly, silica particles induced strong cell activation. However, 10 nm NPs do not alter IL-12 or nitrite levels. Furthermore, bigger NPs and microparticles increase cell membrane damage and reduce the number of living cells but smallest NPs (10 and 240 nm) did not. CONCLUSION: Cell activation properties of silica particles could be useful tools for immune stimulation therapy, while 10 nm NPs would be suitable for molecule transportation.

Rabbit IgG antibodies against phospholipase A2 from Crotalus durissus terrificus neutralize the lethal activity of the venom.

Crotalus durissus terrificus (C.d.t.) (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. Crotoxin contains a basic phospholipase A2 (PLA2) and a non toxic acidic protein, crotapotin. We have produced and investigated the ability of IgG antibodies raised in rabbits against PLA2 to neutralize the lethality of the whole venom. PLA2 was isolated by gel filtration chromatography (Sephadex G-75). Specific antibodies were obtained by subcutaneous and intramuscular inoculation of PLA2 (700 microg) with Freund adjuvant. Groups of six mice (20 + 2 g) were inoculated with 0.5 ml i.p. of C. d t. venom (4 microg) or a mixture of venom that had been preincubated with the desired volume of IgG antibodies. Mortality, recorded 24 and 48 h after inoculation, showed that IgG anti-PLA2 were more effective than anticrotalic serum in neutralizing the lethal activity. These results demonstrate that it could be possible to obtain an anti-venom made by specific antibodies with a high level of protection against the lethal component of C.d.t. venom, and/or the inclusion of these antibodies as a supplement in heterologous anti-venoms.