Zebrafish embryo sensitivity test as in vivo platform to anti-Shiga toxin compound screening

Shiga toxin-producing Escherichia coli (STEC) pathotype secretes two types of AB5 cytotoxins (Stx1 and Stx2), responsible for complications such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in infected patients, which could lead to sequels and death. Currently, there is no effective treatment against the cytotoxic effect of these toxins. However, in order to approve any therapy molecule, an animal experiment is required in order to evaluate the efficacy and safety of therapeutic approaches. The use of alternative small host models is growing among human infectious disease studies, particularly the vertebrate zebrafish model, since relevant results have been described for pathogen-host interaction. In this sense, the present work aimed to analyze the toxic effect of Shiga toxins in zebrafish embryo model in order to standardize this method in the future to be used as a fast, simple, and efficient methodology for the screening of therapeutic molecules. Herein, we demonstrated that the embryos were sensitive in a dose-dependent manner to both Stx toxins, with LD50 of 22 µg/mL for Stx1 and 33 µg/mL for Stx2, and the use of anti-Stx polyclonal antibody abolished the toxic effect. Therefore, this methodology can be a rapid alternative method for selecting promising compounds against Stx toxins, such as recombinant antibodies.

A metal-free blue chromophore derived from plant pigments

Blue natural pigments are rare, especially among plants. However, flowering species that evolved to attract Hymenoptera pollinators are colored by blue anthocyanin-metal complexes. Plants lacking anthocyanins are pigmented by betalains but are unable to produce blue hues. By extending the p-system of betalains, we designed a photostable and metal-free blue dye named BeetBlue that did not show toxicity to human hepatic and retinal pigment epithelial cells and does not affect zebrafish embryonal development. This chiral dye can be conveniently synthesized from betalamic acid obtained from hydrolyzed red beetroot juice or by enzymatic oxidation of L-dopa. BeetBlue is blue in the solid form and in solution of acidified polar molecular solvents, including water. Its capacity to dye natural matrices makes BeetBlue the prototype of a new class of low-cost bioinspired chromophores suitable for a myriad of applications requiring a blue hue.

Design of bioactive peptides derived from CART sequence isolated from the toadfish Thalassophryne nattereri

The emergence of bacterial resistance due to the indiscriminate use of antibiotics warrants the need for developing new bioactive agents. In this context, antimicrobial peptides are highly useful for managing resistant microbial strains. In this study, we report the isolation and characterization of peptides obtained from the venom of the toadfish Thalassophryne nattereri. These peptides were active against Gram-positive and Gram-negative bacteria and fungi. The primary amino acid sequences showed similarity to Cocaine and Amphetamine Regulated Transcript peptides, and two peptide analogs—Tn CRT2 and Tn CRT3—were designed using the AMPA algorithm based on these sequences. The analogs were subjected to physicochemical analysis and antimicrobial screening and were biologically active at concentrations ranging from 2.1 to 13 µM. Zeta potential analysis showed that the peptide analogs increased the positive charge on the cell surface of Gram-positive and Gram-negative bacteria. The toxicity of Tn CRT2 and Tn CRT3 were analyzed in vitro using a hemolytic assay and tetrazolium salt reduction in fibroblasts and was found to be significant only at high concentrations (up to 40 µM). These results suggest that this methodological approach is appropriate to design novel antimicrobial peptides to fight bacterial infections and represents a new and promising discovery in fish venom.

Severe impacts of the Brumadinho dam failure (Minas Gerais, Brazil) on the water quality of the Paraopeba River

On 25 January 2019, Córrego do Feijão's tailing dam at Brumadinho city (Minas Gerais, Brazil) breached, leaving over 250 people dead. At least 12 million cubic meters of ore tailing were spread into Paraopeba River and the surrounding area. To evaluate the short-term impacts of the Brumadinho dam rupture on the environment, we performed biogeochemical, microbiological and ecotoxicological analyses across 464 km of the Paraopeba River in the week following the disaster (1 February 2019) and four months latter (27–29 May 2019). Immediately after the disaster, the water turbidity was 3000 NTU, 30 times greater than the standard recommended by the Brazilian Resolution for Water Quality (CONAMA 357). Up to a 60-fold increase in iron tolerant microbial colony forming unities was observed up to 115 km downstream of the dam failure in May 2019 (compared with February 2019), suggesting changes in microbial metabolic profiles. In the second sampling (May 2019), the ecotoxicological analyses indicate higher zebrafish embryo mortality (up to ~85% embryo mortality) rates in Retiro Baixo (304 km from dam failure location). However, increased zebrafish mortality in Retiro Baixo and Três Marias reservoirs may not be related exclusively to the dam failure. The causal nexus of mortality may be associated with other factors (e.g. local sewage pollution). Our study suggests that independent monitoring programs are needed to quantify the extent of potential impacts caused by the anthropogenic use of the river and to promote the recovery of the impacted area.

Anaphylaxis induced by Thalassophryne nattereri venom in mice is an IgE/IgG1-mediated, IL-4-dependent phenomenon

We hypothesized that beyond the Thalassophryne nattereri venoms ability to induce in mice a strong specific-Th2 response with high levels of specific IgE/IgG1, it would be able to trigger anaphylaxis in sensitized individuals. To investigate whether the venom is capable of inducing an allergic reaction in mice and characterize soluble and cellular mediators involved in this process, BALB/c female mice were sensitized intraperitoneally with decreasing-dose of venom at weekly intervals for 4 weeks and challenged by intraperitoneal, oral or epicutaneous routes with venom 2 weeks later. Our data show that sensitized-mice challenged by all routes showed intense symptoms of anaphylaxis, dependent on the anaphylactic IgG1 and IgE antibodies and mast cells. The late-phase reaction developed after initial symptoms was characterized by the influx of eosinophils, dependent on IL-5, IL-17A and eotaxin produced by Th2 cells in inflamed lungs and skin draining lymph-nodes. Using C57BL/6 deficient mice we demonstrated that IL-4 KO mice failed to develop anaphylactic symptoms or local Th2 inflammation, producing low levels of IgG1 and increased levels of IgG2a. Together our results demonstrated that the venom of T. nattereri has allergenic proteins that can trigger an allergic process, a phenomenon IgE-IgG1 dependent, IL-4-mediated and negatively regulated by IFN-gama.

In vivo toxicity and antimicrobial activity of AuPt bimetallic nanoparticles

Despite the potential antimicrobial activity of metallic nanoparticles, the increasing concerns about nanosafety have been holding back the use of these materials in therapeutics and biomedical devices. In the last years, several studies called attention to metallic nanoparticles toxicity. In the most part of in vitro studies performed with mammalian cells, metallic NPs reduced cell viability and induced genotoxicity and inflammatory responses. Bimetallic NPs have attracted great attention because they present distinct and even more advanced characteristics when compared to nanoparticles formed by a single metal. Recently, bimetallic NPs have emerged as an alternative to improve the antimicrobial activity of metallic nanoparticles, aiming at the broadening of the action spectrum and the reduction of the toxicity. However, the biocompatibility of bimetallic nanoparticles has been demonstrated only by in vitro studies. In the present work, the toxicity of AuPt nanoparticles was addressed both in vitro and in vivo. In addition, the antimicrobial activity of AuPt bimetallic nanoparticles has been evaluated in comparison with Au and Ag nanoparticles. The nanoparticles were characterized by ultraviolet-visible spectroscopy, dynamic light scattering, transmission electron microscopy, inductively coupled plasma optical emission spectroscopy, and X-ray diffraction. The antimicrobial activity was studied against Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus. The toxicity of nanoparticles was evaluated in vitro by analyzing their toxicity against human fibroblast cells (HS68 cell line) and in vivo by embryonic toxicity test in zebrafish (Danio rerio). The results confirmed the intrinsic antimicrobial activity of the three types of nanoparticles but different toxicity. Bimetallic nanoparticles showed enhanced antimicrobial activity in comparison with Au nanoparticles but lower antimicrobial activity compared with Ag nanoparticles. However, AuPt nanoparticles showed great advantage over Ag nanoparticles due to the absence of cytotoxicity and lower toxicity in vivo.

Stingray (Potamotrygon rex) maturity is associated with inflammatory capacity of the venom

Several studies have been carried out with venom from sting and mucus of stingrays of marine or fluvial environments to compare the toxicity of their venom. However, studies demonstrating the existence of the influence of both sex and the maturation stage of stingrays on the variability of the toxic effects of venom are still scarce. Here, we investigated whether the sex and/or the stage maturation of the Potamotrygon rex stingray influence the toxic capacity of the venom to develop acute inflammation in mice. We carried out the main toxic activities in mice using venom from female or male of young and adult stingrays. Our results described here show that the nociception is mainly induced by venom from young female stingrays. In contrast, we observed the action of venom from both sex of adult stingrays in the induction of exudative phase of inflammatory process, including vascular leakage and neutrophil infiltration. Our data illustrate that the composition of the venom of P. rex is influenced by the stage of maturity of the stingray, modulating the production of peptides and proteins capable of acting on leukocytes-endothelial interactions and favoring neutrophil infiltration to the damage tissue.

Acute-phase proteins during inflammatory reaction by bacterial infection: fish-model

Acute-phase protein (APPs) serum levels have been studied in many human diseases, and their components contribute to host defense during the evolution of infectious diseases by acting as part of the innate immune system. Based on the importance of establishing new experimental models, the present investigation evaluated the modulation of APPs following inflammatory stimulus by the inoculation of Aeromonas hydrophila in tilapias. Fish were sampled 6 and 24 hours post-infection. Tilapias presented increase of positive APPs such as ceruloplasmin, haptoglobin, alpha-2-macroglobulin and complement C3, as well as decrease of negative APPs such as albumin and transferrin. The protein response of tilapias during the course of bacterial infection showed correlation with the kinetics of cellular accumulation in the inflamed focus with significant increase of granulocytes, thrombocytes, lymphocytes and macrophages. However, granulocytes were the predominant cells, associated with increment in the reactive oxygen species (ROS) production. Showing responses similar to those observed in humans, the modulation of APPs and the kinetics of cellular accumulation in the exudate demonstrate the feasibility of this alternative experimental model for advances and studies to understand changes in pathophysiological mechanisms of acute inflammatory reaction due to bacterial infection.

Heparin-binding protein release is strongly induced by Leptospira species and is a candidate for an early diagnostic marker of human Leptospirosis

Background Leptospirosis, caused by spirochetes of the genus Leptospira, is one of the most widespread zoonoses worldwide. Efficient diagnostic methods for early diagnosis of leptospirosis are still lacking, and acute disease presents with nonspecific symptomatology and is often misdiagnosed. The leptospires pathogenic processes and virulence mechanisms remain virtually unknown. In severe infections, hemostatic impairment is frequently observed, and pathophysiological complications often develop when the host response is modulated by the pathogen. The neutrophil heparin-binding protein (HBP) is an inflammatory mediator and potent inducer of vascular leakage. Results In this study, we found that leptospires and their secreted products induce the release of HBP from stimulated neutrophils through a controlled degranulation mechanism. We acknowledged 2 leptospiral proteins as able to induce HBP degranulation. These findings have clinical implications, as high levels of HBP were detected in serum from patients with leptospirosis, especially at the early phase of the disease. Conclusion In conclusion, we describe a new mechanism by which the leptospirosis pathophysiological complications may arise, such as vascular leakage and edema formation. We also propose HBP as a new early screening biomarker for human leptospirosis.

Multiple functional therapeutic effects of TnP: a small stable synthetic peptide derived from fish venom in a mouse model of multiple sclerosis

The pathological condition of multiple sclerosis (MS) relies on innate and adaptive immunity. New types of agents that beneficially modify the course of MS, stopping the progression and repairing the damage appear promising. Here, we studied TnP, a small stable synthetic peptide derived from fish venom in the control of inflammation and demyelination in experimental autoimmune encephalomyelitis as prophylactic treatment. TnP decreased the number of the perivascular infiltrates in spinal cord, and the activity of MMP-9 by F4/80+ macrophages were decreased after different regimen treatments. TnP reduces in the central nervous system the infiltration of IFN-gamma-producing Th1 and IL-17A-producing Th17 cells. Also, treatment with therapeutic TnP promotes the emergence of functional Treg in the central nervous system entirely dependent on IL-10. Therapeutic TnP treatment accelerates the remyelination process in a cuprizone model of demyelination. These findings support the beneficial effects of TnP and provides a new therapeutic opportunity for the treatment of MS.

Neutrophils releasing IL-17A into NETs are essential to plasma cell differentiation in inflamed tissue dependent on IL-1R

Interleukin (IL) 17A in chronic inflammation is also produced by innate immune cells as neutrophils. Mice with chronic humoral response induced by venom of Thalassophryne nattereri (VTn) proved to be a good tool for evaluating the impact of IL-17A on the development of long-lived plasma cells in the inflamed peritoneal cavity. Here, we report that VTn induces IL-17A production by neutrophils accumulating in the peritoneal cavity and triggers the extrusion of IL-17A along with neutrophil extracellular traps (NETs). Neutrophil depletion reduced the number of IL17A-producing cells in VTn-immunized mice and blocked the differentiation of long-lived plasma cells. Specific antibody production and survival of long-lived plasma cells was ablated in VTn-immunized mice deficient in CD4, while CD28 signaling had the opposite effect on differentiation of long-lived plasma cells. Further, maturation of long-lived plasma cells in inflamed peritoneal cavity was IL-1R1 and COX-2 dependent. Finally, when both the Raf-MEK-ERK pathway and the IL-17A or IL-1R1 activities were blocked, neutrophils were unable to promote the differentiation of memory B cells into long-lived plasma cells, confirming the essential role of neutrophils and IL-17A along with NETs in an IL-1/IL-1R-dependent manner as the novel helping partner for plasma cell differentiation in chronically inflamed tissues.

Stingray venom activates IL-33 producing cardiomyocytes, but not mast cell, to promote acute neutrophil-mediated injury

One of the hallmarks of acute inflammation is neutrophil infiltration of tissues. We investigated molecular mechanisms implicated in acute neutrophilic inflammation induced by the venom of a freshwater stingray (Potamotrygon cf. henlei) in mice. Ray venom induced early mobilization of neutrophil in the microvasculature of cremaster mice and infiltration of the peritoneal cavity 2 hours after injury, in a dose-response manner. IL-1 beta, IL-6, TNF-alpha, and KC were produced. The neutrophilic infiltration did not occur in mice with ST2 receptor and MyD88 adapters neutralized, or in those with PI3K and p38 MAPK signaling blocked. Drastic reduction of neutrophil infiltration to peritoneal cavities was observed in ST2(-/-), TLR2/TLR4(-/-), MyD88(-/-), TRIF-/- and IL-17A(-/-) mice, and a partial reduction was observed in IL-18R(-/-) mice. Mast cell Kit W(sh)/W(sh)-, AHR-, NLRP3-, ICE-, IL-1 beta-, P2RX7-, CD39-, IL-17RA-, and TBX21 KO mice retain the ability to induce neutrophilia in peritoneal cavity after ray venom injection. IL- 6 and TNF-alpha alone were insufficient for promote neutrophilia in the absence of ST2 signaling. Finally, abundant production of IL-33 by cardiomyocytes was observed. These results refine our understanding of the importance of the IL-33/ST2 axis and IL-33-producing cardiomyocytes in the early acute neutrophilia induced by freshwater stingray venoms.

The Anti-Inflammatory Effects of the Methanolic Extract and Fractions from Davilla elliptica St. Hil. (Dilleniaceae) on Bothrops jararaca Envenomation.

Inflammation and haemorrhage are the main characteristics of tissue injury in botropic envenomation. Although some studies have shown that anti-venom prevents systemic reactions, it is not efficient in preventing tissue injury at the site of the bite. Therefore, this work was undertaken to investigate the anti-inflammatory effects of the methanolic extract and fractions from D. elliptica and to evaluate the role of matrix metalloproteinases (MMPs) in this process. Effects of the extract and fractions from D. elliptica were evaluated using a carrageenan-induced paw oedema model in rats, and leukocyte rolling was visualized by intravital. The quantification of MMPs activities (MMP-2 and MMP-9) extracted from the dermis of mice treated with extract and fractions alone or incubated with venom was determined by zymographic analyses. Our results show that intraperitoneal (i.p.) injection of fractions significantly reduced paw oedema after the carrageenan challenge. Treatment with the tannins fraction also resulted in considerable inhibition of the rolling of leukocytes and this fraction was able to decrease the activation of MMP-9. These results confirmed the anti-inflammatory activity of the methanolic extract and tannins fraction of D. elliptica and showed that the dermonecrosis properties of B. jararaca venom might be mediated through the inhibition of MMP-9 activity.