Crotoxin modulates macrophage phenotypic reprogramming

Macrophage plasticity is a fundamental feature of the immune response since it favors the rapid and adequate change of the functional phenotype in response to the pathogen or the microenvironment. Several studies have shown that Crotoxin (CTX), the major toxin of the Crotalus durissus terrificus snake venom, has a long-lasting antitumor effect both in experimental models and in clinical trials. In this study, we show the CTX effect on the phenotypic reprogramming of macrophages in the mesenchymal tumor microenvironment or those obtained from the peritoneal cavity of healthy animals. CTX (0.9 or 5 μg/animal subcutaneously) administered concomitantly with intraperitoneal inoculation of tumor cells (1 × 107/0.5 mL, injected intraperitoneally) of Ehrlich Ascitic Tumor (EAT) modulated the macrophages phenotype (M1), accompanied by increased NO• production by cells from ascites, and was evaluated after 13 days. On the other hand, in healthy animals, the phenotypic profile of macrophages was modulated in a dose-dependent way at 0.9 μg/animal: M1 and at 5.0 μg/animal: M2; this was accompanied by increased NO• production by peritoneal macrophages only for the dose of 0.9 μg/animal of CTX. This study shows that a single administration of CTX interferes with the phenotypic reprogramming of macrophages, as well as with the secretory state of cells from ascites, influencing events involved with mesenchymal tumor progression. These findings may favor the selection of new therapeutic targets to correct compromised immunity in different systems.

A new experimental model to study shrimp allergy

Shrimp is among the most sensitizing food allergens and has been associated with many anaphylaxis reactions. However, there is still a shortage of studies that enable a systematic understanding of this disease and the investigation of new therapeutic approaches. This study aimed to develop a new experimental model of shrimp allergy that could enable the evaluation of new prophylactic treatments. BALB/c mice were subcutaneously sensitized with 100 μg of shrimp proteins of Litopenaeus vannamei adsorbed in 1 mg of aluminum hydroxide on day 0, and a booster (100 µg of shrimp proteins only) on day 14. The oral challenge protocol was based on the addition of 5 mg/ml of shrimp proteins to water from day 21 to day 35. Analysis of shrimp extract content detected at least 4 of the major allergens reported to L. vannamei. In response to the sensitization, allergic mice showed significantly enhanced IL-4 and IL-10 production in restimulated cervical draining lymph node cells. High detection of serum anti-shrimp IgE and IgG1 suggested the development of allergies to shrimp while Passive Cutaneous Anaphylaxis assay revealed an IgE-mediated response. Immunoblotting analysis revealed that Allergic mice developed antibodies to multiple antigens present in the shrimp extract. These observations were supported by the detection of anti-shrimp IgA production in intestinal lavage samples and morphometric intestinal mucosal changes. Therefore, this experimental protocol can be a tool to evaluate prophylactic and therapeutic approaches.

Cationic and Biocompatible Polymer/Lipid Nanoparticles as Immunoadjuvants.

Nanostructures have been of paramount importance for developing immunoadjuvants. They must be cationic and non-cytotoxic, easily assembling with usually oppositely charged antigens such as proteins, haptens or nucleic acids for use in vaccines. We obtained optimal hybrid nanoparticles (NPs) from the biocompatible polymer poly(methyl methacrylate) (PMMA) and the cationic lipid dioctadecyl dimethyl ammonium bromide (DODAB) by emulsion polymerization of methyl methacrylate (MMA) in the presence of DODAB. NPs adsorbed ovalbumin (OVA) as a model antigen and we determined their adjuvant properties. Interestingly, they elicited high double immune responses of the cellular and humoral types overcoming the poor biocompatibility of DODAB-based adjuvants of the bilayer type. The results suggested that the novel adjuvant would be possibly of use in a variety of vaccines.

Recombinant production and characterization of a new toxin from cryptops iheringi centipede venom revealed by proteome and transcriptome analysis

Among the Chilopoda class of centipede, the Cryptops genus is one of the most associated with envenomation in humans in the metropolitan region of the state of São Paulo. To date, there is no study in the literature about the toxins present in its venom. Thus, in this work, a transcriptomic characterization of the Cryptops iheringi venom gland, as well as a proteomic analysis of its venom, were performed to obtain a toxin profile of this species. These methods indicated that 57.9% of the sequences showed to be putative toxins unknown in public databases; among them, we pointed out a novel putative toxin named Cryptoxin-1. The recombinant form of this new toxin was able to promote edema in mice footpads with massive neutrophils infiltration, linking this toxin to envenomation symptoms observed in accidents with humans. Our findings may elucidate the role of this toxin in the venom, as well as the possibility to explore other proteins found in this work.

Modulation of adhesion molecules expression by different metalloproteases isolated from Bothrops snakes

Snake venom metalloproteinases (SVMP) are involved in local inflammatory reactions observed after snakebites. Based on domain composition, they are classified as PI (pro-domain + proteolytic domain), PII (PI + disintegrin-like domains), or PIII (PII + cysteine-rich domains). Here, we studied the role of different SVMPs domains in inducing the expression of adhesion molecules at the microcirculation of the cremaster muscle of mice. We used Jararhagin (Jar)—a PIII SVMP with intense hemorrhagic activity, and Jar-C—a Jar devoid of the catalytic domain, with no hemorrhagic activity, both isolated from B. jararaca venom and BnP-1—a weakly hemorrhagic P1 SVMP from B. neuwiedi venom. Toxins (0.5 µg) or PBS (100 µL) were injected into the scrotum of mice, and 2, 4, or 24 h later, the protein and gene expression of CD54 and CD31 in the endothelium, and integrins (CD11a and CD11b), expressed in leukocytes were evaluated. Toxins induced significant increases in CD54, CD11a, and CD11b at the initial time and a time-related increase in CD31 expression. In conclusion, our results suggest that, despite differences in hemorrhagic activities and domain composition of the SVMPs used in this study, they behave similarly to the induction of expression of adhesion molecules that promote leukocyte recruitment.

Cationic and biocompatible polymer/lipid nanoparticles as immunoadjuvants

Nanostructures have been of paramount importance for developing immunoadjuvants. They must be cationic and non-cytotoxic, easily assembling with usually oppositely charged antigens such as proteins, haptens or nucleic acids for use in vaccines. We obtained optimal hybrid nanoparticles (NPs) from the biocompatible polymer poly(methyl methacrylate) (PMMA) and the cationic lipid dioctadecyl dimethyl ammonium bromide (DODAB) by emulsion polymerization of methyl methacrylate (MMA) in the presence of DODAB. NPs adsorbed ovalbumin (OVA) as a model antigen and we determined their adjuvant properties. Interestingly, they elicited high double immune responses of the cellular and humoral types overcoming the poor biocompatibility of DODAB-based adjuvants of the bilayer type. The results suggested that the novel adjuvant would be possibly of use in a variety of vaccines.

Robust immune response induced by Schistosoma mansoni TSP-2 antigen coupled to bacterial outer membrane vesicles

Purpose: The use of adjuvants can significantly strengthen a vaccine’s efficacy. We sought to explore the immunization efficacy of bacterial outer membrane vesicles (OMVs) displaying the Schistosoma mansoni antigen, SmTSP-2, through a biotin-rhizavidin coupling approach. The rationale is to exploit the nanoparticulate structure and the adjuvant properties of OMVs to induce a robust antigen-specific immune response, in light of developing new vaccines against S. mansoni. Materials and Methods: OMVs were obtained from Neisseria lactamica and conjugated with biotin. The recombinant SmTSP-2 in fusion with the biotin-binding protein rhizavidin (rRzvSmTSP-2) was produced in E. coli and coupled to biotinylated OMVs to generate an OMV complex displaying SmTSP-2 on the membrane surface (OMV:rSmTSP-2). Transmission electron microscopy (TEM) and dynamic light scattering analysis were used to determine particle charge and size. The immunogenicity of the vaccine complex was evaluated in C57BL/6 mice. Results: The rRzvSmTSP-2 protein was successfully coupled to biotinylated OMVs and purified by size-exclusion chromatography. The OMV:rSmTSP-2 nanoparticles showed an average size of 200 nm, with zeta potential around – 28 mV. Mouse Bone Marrow Dendritic Cells were activated by the nanoparticles as determined by increased expression of the co-stimulatory molecules CD40 and CD86, and the proinflammatory cytokines (TNF-α, IL-6 and IL-12) or IL-10. Splenocytes of mice immunized with OMV:rSmTSP-2 nanoparticles reacted to an in vitro challenge with SmTSP-2 with an increased production of IL-6, IL-10 and IL-17 and displayed a higher number of CD4+ and CD8+ T lymphocytes expressing IFN-γ, IL-4 and IL-2, compared to mice immunized with the antigen alone. Immunization of mice with OMV:rSmTSP-2 induced a 100-fold increase in specific anti-SmTSP-2 IgG antibody titers, as compared to the group receiving the recombinant rSmTSP-2 protein alone or even co-administered with unconjugated OMV. Conclusion: Our results demonstrate that the SmTSP-2 antigen coupled with OMVs is highly immunogenic in mice, supporting the potential effectiveness of this platform for improved antigen delivery in novel vaccine strategies.

The modulatory effect of crotoxin and its phospholipase A2 subunit from Crotalus durissus terrificus venom on dendritic cells interferes with the generation of effector CD4+ T lymphocytes

Dendritic Cells (DCs) direct either cellular immune response or tolerance. The crotoxin (CTX) and its CB subunit (phospholipase A2) isolated from Crotalus durissus terrificus rattlesnake venom modulate the DCs maturation induced by a TLR4 agonist. Here, we analyzed the potential effect of CTX and CB subunit on the functional ability of DCs to induce anti-ovalbumin (OVA) immune response. Thus, CTX and CB inhibited the maturation of OVA/LPS-stimulated BM-DCs from BALB/c mice, which means inhibition of costimulatory and MHC-II molecules expression and proinflammatory cytokines secretion, accompanied by high expression of ICOSL, PD-L1/2, IL-10 and TGF-β mRNA expression. The addition of CTX and CB in cultures of BM-DCs incubated with ConA or OVA/LPS inhibited the proliferation of CD3+ or CD4+T cells from OVA-immunized mice. In in vitro experiment of co-cultures of purified CD4+T cells of DO11.10 mice with OVA/LPS-stimulated BM-DCs, the CTX or CB induced lowest percentage of Th1 and Th2 and CTX induced increase of Treg cells. In in vivo, CTX and CB induced lower percentage of CD4+IFNγ+ and CD4+IL-4+ cells, as well as promoted CD4+CD25+IL-10+ population in OVA/LPS-immunized mice. CTX in vivo also inhibited the maturation of DCs. Our findings demonstrate that the modulatory action of CTX and CB on DCs interferes with the generation of adaptive immunity and, therefore contribute for the understanding of the mechanisms involved in the generation of cellular immunity, which can be useful for new therapeutic approaches for immune disorders.

Inflammatory effect of Bothropstoxin-I from the Bothrops jararacussu venom mediated by NLRP3 inflammasome involves ATP and P2X7 receptor

Muscle tissue damage is one of the local effects described in bothropic envenomations. Bothropstoxin-I (BthTX-I), from B. jararacussu venom, is a K49-phospholipase A2 that induces a massive muscle tissue injury, and, consequently, local inflammatory reaction. The NLRP3 inflammasome is a sensor that triggers inflammation by activating caspase 1 and releasing IL-1b and/or inducing pyroptotic cell death in response to tissue damage. We, therefore, aimed to address activation of NLRP3 inflammasome by BthTX-I-associated injury and the mechanism involved in this process. Intramuscular injection of BthTX-I results in infiltration of neutrophils and macrophages in gastrocnemius muscle, which is reduced in NLRP3- and Caspase-1-deficient mice. The in vitro IL-1β production induced by BthTX-I- inperitoneal macrophages requires caspase 1/11, ASC and NLRP3 and is dependent of ATP-induced K+ efflux and P2X7R. BthTX-I induces a dramatic release of ATP from C2C12 myotubes, therefore representing the major mechanism for P2X7R-dependent inflammasome activation in macrophages. A similar result was obtained when human monocyte-derived macrophages were treated with BthTX-I. These findings demonstrated the inflammatory effect of BthTX-I on muscle tissue, pointing out a role for the ATP released by damaged cells for the NLRP3 activation on macrophages, contributing to the understanding of the microenvironment of the tissue damage of the Bothrops envenomation.

Biocompatible lipid polymer cationic nanoparticles for antigen presentation

Biocompatible lipid polymer nanoparticles (NPs) previously used as antimicrobial agents are explored here as immuno-adjuvants. Poly (methyl methacrylate) (PMMA)/dioctadecyldimethylammonium bromide (DODAB)/poly (diallyldimethylammonium chloride) (PDDA) nanoparticles (NPs) were prepared by emulsion polymerization of methyl methacrylate (MMA) in the presence of DODAB and PDDA, with azobisisobutyronitrile (AIBN) as the initiator. NPs characterization after dialysis by dynamic light-scattering yielded 225 ± 2 nm hydrodynamic diameter (Dz), 73 ± 1 mV zeta-potential (ζ), and 0.10 ± 0.01 polydispersity (P). Ovalbumin (OVA) adsorption reduced ζ to 45 ± 2 mV. Balb/c mice immunized with NPs/OVA produced enhanced OVA-specific IgG1 and IgG2a, exhibited moderate delayed type hypersensitivity reaction, and enhanced cytokines production (IL-4, IL-10, IL-2, IFN-γ) by cultured spleen cells. There was no cytotoxicity against cultured macrophages and fibroblasts. Advantages of the PMMA/DODAB/PDDA NPs were high biocompatibility, zeta-potential, colloidal stability, and antigen adsorption. Both humoral and cellular antigen-specific immune responses were obtained.

Extracellular vesicles isolated from mesenchymal stromal cells modulate CD4+ T lymphocytes toward a regulatory profile

Mesenchymal stromal cells (MSCs) can generate immunological tolerance due to their regulatory activity in many immune cells. Extracellular vesicles (EVs) release is a pivotal mechanism by which MSCs exert their actions. In this study, we evaluate whether mesenchymal stromal cell extracellular vesicles (MSC-EVs) can modulate T cell response. MSCs were expanded and EVs were obtained by differential ultracentrifugation of the supernatant. The incorporation of MSC-EVs by T cells was detected by confocal microscopy. Expression of surface markers was detected by flow cytometry or CytoFLEX and cytokines were detected by RT-PCR, FACS and confocal microscopy and a miRNA PCR array was performed. We demonstrated that MSC-EVs were incorporated by lymphocytes in vitro and decreased T cell proliferation and Th1 differentiation. Interestingly, in Th1 polarization, MSC-EVs increased Foxp3 expression and generated a subpopulation of IFN-gama+/Foxp3+T cells with suppressive capacity. A differential expression profile of miRNAs in MSC-EVs-treated Th1 cells was seen, and also a modulation of one of their target genes, TGFbR2. MSC-EVs altered the metabolism of Th1-differentiated T cells, suggesting the involvement of the TGF-ß pathway in this metabolic modulation. The addition of MSC-EVs in vivo, in an OVA immunization model, generated cells Foxp3+. Thus, our findings suggest that MSC-EVs are able to specifically modulate activated T cells at an alternative regulatory profile by miRNAs and metabolism shifting

Simple Nanoparticles from the Assembly of Cationic Polymer and Antigen as Immunoadjuvants

Since antigens are negatively charged, they combine well with positively charged adjuvants. Here, ovalbumin (OVA) (0.1 mg·mL-1) and poly (diallyldimethylammonium chloride) (PDDA) (0.01 mg·mL-1) yielded PDDA/OVA assemblies characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM) as spherical nanoparticles (NPs) of 170 ± 4 nm hydrodynamic diameter, 30 ± 2 mV of zeta-potential and 0.11 ± 0.01 of polydispersity. Mice immunization with the NPs elicited high OVA-specific IgG1 and low OVA-specific IgG2a production, indicating a Th-2 response. Delayed-type hypersensitivity reaction (DTH) was low and comparable to the one elicited by Al(OH)3/OVA, suggesting again a Th-2 response. PDDA advantages as an adjuvant were simplicity (a single-component adjuvant), low concentration needed (0.01 mg·mL-1 PDDA) combined with antigen yielding neglectable cytotoxicity, and high stability of PDDA/OVA dispersions. The NPs elicited much higher OVA-specific antibodies production than Al(OH)3/OVA. In vivo, the nano-metric size possibly assured antigen presentation by antigen-presenting cells (APC) at the lymph nodes, in contrast to the location of Al(OH)3/OVA microparticles at the site of injection for longer periods with stimulation of local dendritic cells. In the future, it will be interesting to evaluate combinations of the antigen with NPs carrying both PDDA and elicitors of the Th-1 response

Inflammatory reaction induced by two metalloproteinases isolated from bothrops atrox venom and by fragments generated from the hydrolysis of basement membrane components

Snake venom metalloproteinases (SVMPs) play an important role in local tissue damage of snakebite patients, mostly by hydrolysis of basement membrane (BM) components. We evaluated the proinflammatory activity of SVMPs Atroxlysin-Ia (ATXL) and Batroxrhagin (BATXH) from Bothrops atrox venom and their hydrolysis products of Matrigel. BALB/c mice were injected with SVMPs (2 µg), for assessment of paw edema and peritoneal leukocyte accumulation. Both SVMPs induced edema, representing an increase of ~70% of the paw size. Leukocyte infiltrates reached levels of 6 × 106 with ATXL and 5 × 106 with BATXH. TNF-a was identified in the supernatant of BATXH—or venom-stimulated MPAC cells. Incubation of Matrigel with the SVMPs generated fragments, including peptides from Laminin, identified by LC–MS/MS. The Matrigel hydrolysis peptides caused edema that increased 30% the paw size and promoted leukocyte accumulation (4–5 × 106) to the peritoneal cavity, significantly higher than Matrigel control peptides 1 and 4 h after injection. Our findings suggest that ATXL and BATXH are involved in the inflammatory reaction observed in B. atrox envenomings by direct action on inflammatory cells or by releasing proinflammatory peptides from BM proteins that may amplify the direct action of SVMPs through activation of endogenous signaling pathways

Correction: CD28 deficiency leads to accumulation of germinal-center independent IgM+ experienced B cells and to production of protective IgM during experimental malaria.

[This corrects the article DOI: 10.1371/journal.pone.0202522.].

Differential macrophage subsets in muscle damage induced by a K49-PLA2 from Bothrops jararacussu venom modulate the time course of the regeneration process

Bothrops snakes cause around 80% of snakebites in Brazil, with muscle tissue damage as an important consequence, which may cause dysfunction on the affected limb. Bothropstoxin-I (BthTX-I) from Bothrops jararacussu is a K49-phospholipase A2, involved in the injury and envenomation’s inflammatory response. Immune system components act in the resolution of tissue damage and regeneration. Thus, macrophages exert a crucial role in the elimination of dead tissue and muscle repair. Here, we studied the cellular influx and presence of classical and alternative macrophages (M1 and M2) during muscle injury induced by BthTX-I and the regeneration process. BthTX-I elicited intense inflammatory response characterized by neutrophil migration, then increased influx of M1 macrophages followed by M2 population that declined, resulting in tissue regeneration. The high expressions of TNF-a and IL6 were changed by increased TGF-ß expression after BthTX-I injection, coinciding with the iNOs and arginase expression and the peaks of M1 and M2 macrophages in muscle tissue. A coordinated sequence of PAX7, MyoD, and myogenin expression involved in muscle regenerative process appeared after BthTX-I injection. Together, these results demonstrate a direct correlation between the macrophage subsets, cytokine microenvironment, and the myogenesis process. This information may be useful for new envenomation and muscular dysfunction therapies

When spider and snake get along: fusion of a snake disintegrin with a spider phospholipase D to explore their synergistic effects on a tumor cell

Venoms of spiders and snakes contain toxins extremely active and, thus, provide a natural source for the development of new biotechnological tools. Among the diversity of toxins present in the venom of spiders from genus Loxosceles, the phospholipases D (PLDs) show high hydrolytic activity upon lysophosphatidylcholine (LPC) and sphingomyelin (SM), generating bioactive phospholipids such as cyclic phosphatidic acid (cPA). Since this mediator has been shown to play a major role in complex signaling pathways, including inhibition of tumor cells, the PLDs may hold the key to learn how toxins could be used for therapeutic purposes. However, the strong platelet aggregation of PLDs and their lack of selectivity impose a major limitation. On the other hand, disintegrins present in the venoms of Viperidae snakes are a potent inhibitor of platelet aggregation and possess high affinity and specificity to molecules called integrins that are highly expressed in some tumor cells, such as murine melanoma B16F10. Therefore, disintegrins might be suitable molecules to carry the PLDs to the malignant cells, so both toxins may work synergistically to eliminate these cells. Thus, in this work, a recombinant PLD from Loxosceles gaucho spider was recombinantly fused to a disintegrin from Echis carinatus snake to form a hybrid toxin called Rechistatin. This recombinant toxin was successfully expressed in bacteria, showed binding activity in B16F10 murine melanoma cells and exerted a synergistic cytotoxicity effect on these cells. Therefore, the approach presented in this work may represent a new strategy to explore new potential applications for spider PLDs.

Design and production of a recombinant hybrid toxin to raise protective antibodies against Loxosceles spider venom

Human accidents with spiders of the genus Loxosceles are an important health problem affecting thousands of people worldwide. Patients evolve to severe local injuries and, in many cases, to systemic disturbances as acute renal failure, in which cases antivenoms are considered to be the most effective treatment. However, for antivenom production, the extraction of the venom used in the immunization process is laborious and the yield is very low. Thus, many groups have been exploring the use of recombinant Loxosceles toxins, particularly phospholipases D (PLDs), to produce the antivenom. Nonetheless, some important venom activities are not neutralized by anti-PLD antibodies. Astacin-like metalloproteases (ALMPs) are the second most expressed toxin acting on the extracellular matrix, indicating the importance of its inclusion in the antigen’s formulation to provide a better antivenom. Here we show the construction of a hybrid recombinant immunogen, called LgRec1ALP1, composed of hydrophilic regions of the PLD and the ALMP toxins from Loxosceles gaucho. Although the LgRec1ALP1 was expressed as inclusion bodies, it resulted in good yields and it was effective to produce neutralizing antibodies in mice. The antiserum neutralized fibrinogenolytic, platelet aggregation and dermonecrotic activities elicited by L. gaucho, L. laeta, and L. intermedia venoms, indicating that the hybrid recombinant antigen may be a valuable source for the production of protective antibodies against Loxosceles ssp. venoms. In addition, the hybrid recombinant toxin approach may enrich and expand the alternative antigens for antisera production for other venoms.

CD28 deficiency leads to accumulation of germinal-center independent IgM+ experienced B cells and to production of protective IgM during experimental malaria

Protective immunity to blood-stage malaria is attributed to Plasmodium-specific IgG and effector-memory T helper 1 (Th1) cells. However, mice lacking the costimulatory receptor CD28 (CD28KO) maintain chronic parasitemia at low levels and do not succumb to infection, suggesting that other immune responses contribute to parasite control. We report here that CD28KO mice develop long-lasting non-sterile immunity and survive lethal parasite challenge. This protection correlated with a progressive increase of anti-parasite IgM serum levels during chronic infection. Serum IgM from chronically infected CD28KO mice recognize erythrocytes infected with mature parasites, and effectively control Plasmodium infection by promoting parasite lysis and uptake. These antibodies also recognize autoantigens and antigens from other pathogens. Chronically infected CD28KO mice have high numbers of IgM+ plasmocytes and experienced B cells, exhibiting a germinal-center independent Fas+GL7-CD38+CD73- phenotype. These cells are also present in chronically infected C57BL/6 mice although in lower numbers. Finally, IgM+ experienced B cells from cured C57BL/6 and CD28KO mice proliferate and produce anti-parasite IgM in response to infected erythrocytes. This study demonstrates that CD28 deficiency results in the generation of germinal-center independent IgM+ experienced B cells and the production of protective IgM during experimental malaria, providing evidence for an additional mechanism by which the immune system controls Plasmodium infection

Crotoxin isolated from Crotalus durissus terrificus venom modulates the functional activity of dendritic cells via formyl peptide receptors

The Crotalus durissus terrificus rattlesnake venom, its main toxin, crotoxin (CTX), and its crotapotin (CA) and phospholipase A(2) (CB) subunits modulate the immune system. Formyl peptide receptors (FPRs) and lipoxin A(4) (LXA(4)) are involved in CTX's effect on macrophages and neutrophils. Dendritic cells (DCs) are plasticity cells involved in the induction of adaptive immunity and tolerance maintenance. Therefore, we evaluated the effect of CTX, CA or CB on the maturation of DCs derived from murine bone marrow (BM). According to data, CTX and CB-but not CA-induced an increase of MHC-II, but not costimulatory molecules on DCs. Furthermore, CTX and CB inhibited the expression of costimulatory and MHC-II molecules, secretion of proinflammatory cytokines and NF-kappa Bp65 and p38/ERK1/2-MAPK signaling pathways by LPS-incubated DCs. Differently, CTX and CB induced IL-10, PGE(2) and LXA(4) secretion in LPS-incubated DCs. Lower proliferation and IL-2 secretion were verified in coculture of CD3(+) cells and DCs incubated with LPS plus CTX or CB compared with LPS-incubated DCs. The effect of CTX and CB on DCs was abolished in cultures incubated with a FPRs antagonist. Hence, CTX and CB exert a modulation on functional activity of DCs; we also checked the involvement the FPR family on cell activities.

Experimental immunization with Thalassophryne nattereri fish venom: striking IL-5 production and impaired of B220+ cells.

Murine experimental model have been useful to understanding the toxic as well as the pharmacological properties of the Thalassophryne nattereri venom. However, the specific immune response to T. nattereri venom in mice is yet unclear. Our results showed that the venom elicited in BALB/c mice high levels of specific IgG1 and total IgE isotype with high affinity, accompanied by a striking IL-5 production, what point out to a Th2-like response. Meanwhile, the production of IFN-gamma by lymphocytes pool expanded upon mitogen stimulus, suggests that the venom was also able to activate Th1 clones. Elevated number of antigen-presenting cells expressing CD11c or CD11b from day 4 to 6 supported ongoing antigen presentation process in the primary response and efficient T-cell expansion (increase of CD4(+) cells). In contrast, decreased B220 expression was observed, suggesting that the formation of memory long lived cell compartment. In conclusion, T. natterri venom stimulates an association of cytokine of both Th1 and Th2 profile, with a notable IL-5 production and specific IgG1 and total IgE isotypes secretion. Furthermore, our finding showed that T. natterri venom can affect the B cell fate and induce a memory antibody response through the secretion of protective IgG subclasses. Further studies with the venom protein toxins may provide clues to molecular mechanism regulating proliferation and differentiation of antibody-secreting cells in our model. A better understating of how T. natterri venom can modulate immune response could be useful in therapeutic strategies.