RESUMEN

Envenomation by venomous fish, although not always fatal, is capable of causing damage to homeostasis by activating the inflammatory process, with the formation of edema, excruciating pain, necrosis that is difficult to heal, as well as hemodynamic and cardiorespiratory changes. Despite the wide variety of pharmacological treatments used to manage acute symptoms, none are effective in controlling envenomation. Knowing the essential role of neutralizing polyclonal antibodies in the treatment of envenoming for other species, such as snakes, this work aimed to produce a polyclonal antiserum in mice and test its ability to neutralize the main toxic effects induced by the venoms of the main venomous Brazilian fish. We found that the antiserum recognizes the main toxins present in the different venoms of Thalassophryne nattereri, Scorpaena plumieri, Potamotrygon gr. Orbignyi, and Cathorops spixii and was effective in pre-incubation trials. In an independent test, the antiserum applied immediately to the topical application of T. nattereri, P. gr orbygnyi, and C. spixii venoms completely abolished the toxic effects on the microcirculation, preventing alterations such as arteriolar contraction, slowing of blood flow in postcapillary venules, venular stasis, myofibrillar hypercontraction, and increased leukocyte rolling and adherence. The edematogenic and nociceptive activities induced by these venoms were also neutralized by the immediate application of the antiserum. Importantly, the antiserum prevented the acute inflammatory response in the lungs induced by the S. plumieri venom. The success of antiserum containing neutralizing polyclonal antibodies in controlling the toxic effects induced by different venoms offers a new strategy for the treatment of fish envenomation in Brazil.

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RESUMEN

Thalassophryne nattereri toadfish (niquim) envenomation, common in the hands and feet of bathers and fishermen in the north and northeast regions of Brazil, is characterized by local symptoms such as immediate edema and intense pain. These symptoms progress to necrosis that lasts for an extended period of time, with delayed healing. Wound healing is a complex process characterized by the interdependent role of keratinocytes, fibroblasts, and endothelial and innate cells such as neutrophils and macrophages. Macrophages and neutrophils are actively recruited to clear debris during the inflammatory phase of wound repair, promoting the production of pro-inflammatory mediators, and in the late stage, macrophages promote tissue repair. Our hypothesis is that injury caused by T. nattereri venom (VTn) leads to senescent wounds. In this study, we provide valuable information about the mechanism(s) behind the dysregulated inflammation in wound healing induced by VTn. We demonstrate in mouse paws injected with the venom the installation of γH2AX/p16Ink4a-dependent senescence with persistent neutrophilic inflammation in the proliferation and remodeling phases. VTn induced an imbalance of M1/M2 macrophages by maintaining a high number of TNF-α-producing M1 macrophages in the wound but without the ability to eliminate the persistent neutrophils. Chronic neutrophilic inflammation and senescence were mediated by cytokines such as IL-1α and IL-1ß in a caspase-1- and caspase-11-dependent manner. In addition, previous blocking with anti-IL-1α and anti-IL-ß neutralizing antibodies and caspase-1 (Ac YVAD-CMK) and caspase-11 (Wedelolactone) inhibitors was essential to control the pro-inflammatory activity of M1 macrophages induced by VTn injection, skewing towards an anti-inflammatory state, and was sufficient to block neutrophil recruitment and senescence.

Asunto(s)

RESUMEN

Envenomation by venomous fish, although not always fatal, is capable of causing damage to homeostasis by activating the inflammatory process, with the formation of edema, excruciating pain, necrosis that is difficult to heal, as well as hemodynamic and cardiorespiratory changes. Despite the wide variety of pharmacological treatments used to manage acute symptoms, none are effective in controlling envenomation. Knowing the essential role of neutralizing polyclonal antibodies in the treatment of envenoming for other species, such as snakes, this work aimed to produce a polyclonal antiserum in mice and test its ability to neutralize the main toxic effects induced by the venoms of the main venomous Brazilian fish. We found that the antiserum recognizes the main toxins present in the different venoms of Thalassophryne nattereri, Scorpaena plumieri, Potamotrygon gr. Orbignyi, and Cathorops spixii and was effective in pre-incubation trials. In an independent test, the antiserum applied immediately to the topical application of T. nattereri, P. gr orbygnyi, and C. spixii venoms completely abolished the toxic effects on the microcirculation, preventing alterations such as arteriolar contraction, slowing of blood flow in postcapillary venules, venular stasis, myofibrillar hypercontraction, and increased leukocyte rolling and adherence. The edematogenic and nociceptive activities induced by these venoms were also neutralized by the immediate application of the antiserum. Importantly, the antiserum prevented the acute inflammatory response in the lungs induced by the S. plumieri venom. The success of antiserum containing neutralizing polyclonal antibodies in controlling the toxic effects induced by different venoms offers a new strategy for the treatment of fish envenomation in Brazil.

RESUMEN

Thalassophryne nattereri toadfish (niquim) envenomation, common in the hands and feet of bathers and fishermen in the north and northeast regions of Brazil, is characterized by local symptoms such as immediate edema and intense pain. These symptoms progress to necrosis that lasts for an extended period of time, with delayed healing. Wound healing is a complex process characterized by the interdependent role of keratinocytes, fibroblasts, and endothelial and innate cells such as neutrophils and macrophages. Macrophages and neutrophils are actively recruited to clear debris during the inflammatory phase of wound repair, promoting the production of pro-inflammatory mediators, and in the late stage, macrophages promote tissue repair. Our hypothesis is that injury caused by T. nattereri venom (VTn) leads to senescent wounds. In this study, we provide valuable information about the mechanism(s) behind the dysregulated inflammation in wound healing induced by VTn. We demonstrate in mouse paws injected with the venom the installation of γH2AX/p16Ink4a-dependent senescence with persistent neutrophilic inflammation in the proliferation and remodeling phases. VTn induced an imbalance of M1/M2 macrophages by maintaining a high number of TNF-α-producing M1 macrophages in the wound but without the ability to eliminate the persistent neutrophils. Chronic neutrophilic inflammation and senescence were mediated by cytokines such as IL-1α and IL-1β in a caspase-1- and caspase-11-dependent manner. In addition, previous blocking with anti-IL-1α and anti-IL-β neutralizing antibodies and caspase-1 (Ac YVAD-CMK) and caspase-11 (Wedelolactone) inhibitors was essential to control the pro-inflammatory activity of M1 macrophages induced by VTn injection, skewing towards an anti-inflammatory state, and was sufficient to block neutrophil recruitment and senescence.

RESUMEN

Since the first record of the five founder members of the group of Natterin proteins in the venom of the medically significant fish Thalassophryne nattereri, new sequences have been identified in other species. In this work, we performed a detailed screening using available genome databases across a wide range of species to identify sequence members of the Natterin group, sequence similarities, conserved domains, and evolutionary relationships. The high-throughput tools have enabled us to dramatically expand the number of members within this group of proteins, which has a remote origin (around 400 million years ago) and is spread across Eukarya organisms, even in plants and primitive Agnathans jawless fish. Overall, the survey resulted in 331 species presenting Natterin-like proteins, mainly fish, and 859 putative genes. Besides fish, the groups with more species included in our analysis were insects and birds. The number and variety of annotations increased the knowledge of the obtained sequences in detail, such as the conserved motif AGIP in the pore-forming loop involved in the transmembrane barrel insertion, allowing us to classify them as important constituents of the innate immune defense system as effector molecules activating immune cells by interacting with conserved intracellular signaling mechanisms in the hosts.

Asunto(s)

RESUMEN

Since the first record of the five founder members of the group of Natterin proteins in the venom of the medically significant fish Thalassophryne nattereri, new sequences have been identified in other species. In this work, we performed a detailed screening using available genome databases across a wide range of species to identify sequence members of the Natterin group, sequence similarities, conserved domains, and evolutionary relationships. The high-throughput tools have enabled us to dramatically expand the number of members within this group of proteins, which has a remote origin (around 400 million years ago) and is spread across Eukarya organisms, even in plants and primitive Agnathans jawless fish. Overall, the survey resulted in 331 species presenting Natterin-like proteins, mainly fish, and 859 putative genes. Besides fish, the groups with more species included in our analysis were insects and birds. The number and variety of annotations increased the knowledge of the obtained sequences in detail, such as the conserved motif AGIP in the pore-forming loop involved in the transmembrane barrel insertion, allowing us to classify them as important constituents of the innate immune defense system as effector molecules activating immune cells by interacting with conserved intracellular signaling mechanisms in the hosts.

RESUMEN

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.

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The venom of marine animals is a rich source of compounds with remarkable functional specificity and diversity. Thalassophryne nattereri is a small venomous fish inhabiting the northern and northeastern coast of Brazil, and represents a relatively frequent cause of injuries. Its venom causes severe inflammatory response followed frequently by the necrosis of the affected area. This venom presents characterized components such as proteases (Natterins 1-4) and a lectin (Nattectin) with complex effects on the human organism. A specific inhibitor of tissue kallikrein (TKI) reduces the nociception and the edema caused by the venom in mice. Our study sought to investigate the proteolytic activities against vasopeptides Angiotensin I, Angiotensin II, Angiotensin 1-9 and Bradykinin. The venom indicated angiotensin conversion against angiotensin I, as well as kininase against bradykinin. Captopril conducted the total inhibition of the converting activity, featuring the first report of ACE activity in fish venoms.

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Thalassophryne nattereri (niquim) is a venomous fish found off North and Northeast coast of Brazil, where it is known by the severity of the accidents involving humans. This review article is divided into four topics. The first one provides a brief description of the animal biology and its distribution off Brazilian coastal waters, the venom apparatus, signs and symptoms observed in envenomated humans and also describes envenomation in mice. The second topic describes the use of modern genetic approach and mass spectrometry for identification of highly expressed genes in its venom glands and the sequence of major toxins. The third chapter offers a detailed study of tissue injury induced by the venom and reveals the role of toxins that impair inflammation reduction. Finally, the fourth section expands the understanding of many extrinsic and intrinsic essential factors in maintaining survival of memory B cell compartment. Our results demonstrate the wide possibilities for research in the area of toxinology, also the necessity of interconnection among biochemistry, pharmacology and immunology areas for the expansion of knowledge and for generation of innovation.

RESUMEN

Thalassophryne nattereri (niquim) is a venomous fish found off North and Northeast coast of Brazil, where it is known by the severity of the accidents involving humans. This review article is divided into four topics. The first one provides a brief description of the animal biology and its distribution off Brazilian coastal waters, the venom apparatus, signs and symptoms observed in envenomated humans and also describes envenomation in mice. The second topic describes the use of modern genetic approach and mass spectrometry for identification of highly expressed genes in its venom glands and the sequence of major toxins. The third chapter offers a detailed study of tissue injury induced by the venom and reveals the role of toxins that impair inflammation reduction. Finally, the fourth section expands the understanding of many extrinsic and intrinsic essential factors in maintaining survival of memory B cell compartment. Our results demonstrate the wide possibilities for research in the area of toxinology, also the necessity of interconnection among biochemistry, pharmacology and immunology areas for the expansion of knowledge and for generation of innovation.

Asunto(s)

RESUMEN

Thalassophryne nattereri (niquim) is a venomous fish found off North and Northeast coast of Brazil, where it is known by the severity of the accidents involving humans. This review article is divided into four topics. The first one provides a brief description of the animal biology and its distribution off Brazilian coastal waters, the venom apparatus, signs and symptoms observed in envenomated humans and also describes envenomation in mice. The second topic describes the use of modern genetic approach and mass spectrometry for identification of highly expressed genes in its venom glands and the sequence of major toxins. The third chapter offers a detailed study of tissue injury induced by the venom and reveals the role of toxins that impair inflammation reduction. Finally, the fourth section expands the understanding of many extrinsic and intrinsic essential factors in maintaining survival of memory B cell compartment. Our results demonstrate the wide possibilities for research in the area of toxinology, also the necessity of interconnection among biochemistry, pharmacology and immunology areas for the expansion of knowledge and for generation of innovation.

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Thalassophryne nattereri (niquim) is a venomous fish responsible for numerous accidents involving fishermen in northern and northeastern Brazil. The aim of the present investigation was to evaluate the action of antivenom on renal effects caused by Thalassophryne nattereri venom. Isolated kidneys of Wistar rats were perfused with a previously dialyzed Krebs-Henseleit solution containing 6 g% bovine serum albumin. The antivenom action was studied through perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). The niquim venom (1 miug/mL), the antivenom alone (1 miug/mL) or the venom incubated with antivenom were added to the system 30 minutes after the beginning of each perfusion. Previous works have shown venom induced-alterations of renal function parameters. In the isolated rat Kidney, T. nattereri venom (1 miug/mL) increased the perfusion pressure and renal vascular resistance at 60, 90 and 120 minutes. UF and GFR also increased at 60, 90 and 120 minutes when compared with the control group; however, no effects were observed on the percent of sodium (% TNa more control equal 81.1 more or less 0.86; % TNa more 60 equal 78.04 more or less 1.18; % TNa more 90 equal -5.16 more or less 3.34; %TNa more 120 equal 79.49 more or less 0.87) and potassium (%TKcontrol equal 72.29 more or less 1.12; %TK more 60 equal 75.41 more or less 0.65; % TK more 90 equal 71.23 more or less 2.55; % TK more 120 equal 76.62 more or less 1.04) tubular transporto. The administration of the antivenom (1 miug/mL) incubated with venom (1 miug/mL) reduced the changes in PP, RVR, UF and GFR provoked by Thalassophryne nattereri venom. The group perfused with venom alone showed a moderate deposit of a proteinaceous material in the tubules and urinary space.(...)

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Thalassophryne nattereri (niquim) is a venomous fish found on the northern and northeastern coasts of Brazil. Every year, hundreds of humans are affected by the poison, which causes excruciating local pain, edema, and necrosis, and can lead to permanent disabilities. In experimental models, T. nattereri venom induces edema and nociception, which are correlated to human symptoms and dependent on venom kininogenase activity; myotoxicity; impairment of blood flow; platelet lysis and cytotoxicity on endothelial cells. These effects were observed with minute amounts of venom. To characterize the primary structure of T. nattereri venom toxins, a list of transcripts within the venom gland was made using the expressed sequence tag (EST) strategy. Here we report the analysis of 775 ESTs that were obtained from a directional cDNA library of T. nattereri venom gland.

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A novel family of proteins with kininogenase activity and unique primary structure was characterized using combined pharmacological, proteomic and transcriptomic approaches of Thalassophryne nattereri fish venom. The major venom components were isolated and submitted to bioassays corresponding to its main effects: nociception and edema. These activities were mostly located in one fraction (MS3), which was further fractionated. The isolated protein, named natterin, was able to induce edema, nociception and cleave human kininogen and kininogen-derived synthetic peptides, releasing kallidin (Lys-bradykinin). The enzymatic digestion was inhibited by kallikrein inhibitors as Trasylol and TKI. Natterin N-terminal peptide showed no similarity with already known proteins present in databanks. Primary structure of natterin was obtained by a transcriptomic approach using a representative cDNA library constructed from T. nattereri venom glands. Several expressed sequence tags (ESTs) were obtained and processed by bioinformatics revealing a major group (18%) of related sequences unknown to gene or protein sequence databases. This group included sequences showing the N-terminus of isolated natterin and was named Natterin family. Analysis of this family allowed us to identify five related sequences, which we called natterin 1-4 and P. Natterin 1 and 2 sequences include the N-terminus of the isolated natterin. Furthermore, internal peptides of natterin 1-3 were found in major spots of whole venom submitted to mass spectrometry/2DGE. Similarly to the ESTs, the complete sequences of natterins did not show any significant similarity with already described tissue kallikreins, kininogenases or any proteinase, all being entirely new. These data present a new task for the knowledge of the action of kininogenases and may help in understanding the mechanisms of T. nattereri fish envenoming, which is an important medical problem in North and Northeast of Brazil.

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