Corrigendum to "CpG-ODN formulated with a nanostructure as adjuvant for anticrotalic serum production. Studies in mice" [Toxicon 215 (2022) 28-36].

Antivenom is the only safe and effective treatment to neutralize snake venom. Specific anti-venom used to treat snake bite is usually obtained from horses after hyperimmunization with crude snake venom in combination with Freund's Adjuvant. Freund's complete and incomplete adjuvant can cause severe local and systemic acute and chronic inflammation, its potentially severe inflammatory effects have led many researchers to seek alternative immunological adjuvants. CpG-ODN formulated in a 6-O-ascorbyl palmitate nanostructure (Coa-ASC16) was more efficient as adjuvant than CpG-ODN alone using ovalbumin (OVA) as an antigen model. Particularly, immunization of mice with OVA/CpG-ODN/Coa-ASC16 resulted in high OVA specific antibody titers and IFN-γ and IL-17 secretion compared to immunization with OVA/CpG-ODN. First of all, we estimated the effect of Coa-ASC16 nanostructure preparation on venom activity. Additionally, in order to evaluate the immune response induced by this adjuvant strategy using Crotalus durissus terrificus (C. d. terrificus) venom (CdtV), we determined the titer of antibodies (IgG, IgG1 and IgG2) and their specificity. BALB/c mice were subcutaneously immunizated on days 0, 15 and 30 with CdtV/CpG-ODN/Coa-ASC16 or CdtV/Freund's Adyuvant (complete first and incomplete-booster) (dose/mice: CdtV: 6-10 µg, CpG-ODN: 30 µg). On day 45 mice were sacrificed. The neutralizing ability of serum from animals immunized with CdtV/CpG-ODN/Coa-ASC16 or CdtV/Freund's adjuvant was tested against PLA2 activity and lethality. In both immunized group mice, the antibody titers in plasma were high (1 × 105), with a similar IgG1/IgG2a ratio. The antibodies recognized phospholipase A2 and thrombin-like proteins, the main toxins from C. d. terrificus venom. Macroscopic and microscopic analysis at the site of injection of mice injected with Freund's adjuvant showed local damage (with non-infectious abscesses) and hypertrophy of inguinal lymph nodes, whereas mice injected with CpG-ODN/Coa/ASC16 did not. Our results show that CpG-ODN/Coa-ASC16 produces a humoral response as strong and specific as Freund's adjuvant, with minor or null local deleterious effect, demonstrating the potentiality and advisability of an alternative formulation as a new adjuvant option for future immunizations to produce C. d. terrificus antivenom.

Traces of Bothrops snake venoms in necrotic muscle preclude myotube formation in vitro.

Deficient skeletal muscle regeneration, which often leads to permanent sequelae, is a common clinical finding in envenomations caused by snakes of the family Viperidae, such as those of Bothrops alternatus and B. diporus in South America. The causes of such poor muscle regenerative outcome are still incompletely understood. Using a murine experimental model of envenomation by the venoms of these two species, we assessed whether traces of venom components that remain in muscle tissue days after envenomation affect myoblasts and myotube formation in culture. The kinetics of drop in venom concentration in the tissue was assessed by ELISA and Western blot, and by the quantification of venom phospholipase A2 activity. A rapid drop of venom components was observed in muscle, although a band of 58-63 kDa remained even 168 h after venom injection, and venom phospholipase A2 activity was detected in muscle tissue days after envenomation. Muscle homogenates from envenomated animals were cytotoxic to myoblasts in culture and inhibited the formation of myotubes even in conditions where homogenates were devoid of cytotoxicity. These deleterious effects were abrogated when homogenates were incubated with antivenom. Our findings agree with previous observations with the venom of Bothrops asper and provide further evidence that one of the causes of the poor skeletal muscle regeneration after Bothrops sp venom-induced myonecrosis is the deleterious action on myogenic cells of traces of venom components remaining in the tissue.

Fast venomic analysis of Crotalus durissus terrificus from northeastern Argentina.

The complete knowledge of the toxins that make up venoms is the base for the treatment of snake accidents victims and the selection of specimens for the preparation of venom pools for antivenom production. In this work, we used a fast and direct venomics approach to identify the toxin families in the C.d. terrificus venom, a Southern American Neotropical rattlesnake. The RP-HPLC separation profile of pooled venom from adult specimens followed by mass spectrometry analysis revealed that C.d. terrificus' venom proteome is composed of 12 protein families, which are unevenly distributed in the venom, e.g., there are few major proteins in the venom's composition phospholipase A2, serine proteinase, crotamine and L-amino acid oxidase. At the same time, the proteome analysis revealed a small set of proteins with low quantity (less than 1.5%), both enzymes (metaloprotease, phospholipase B and 5'-nucleotidase) and proteins (Bradykinin potentiating and C-type natriuretic peptides, C-type lectin convulxin and nerve growth factor). To sum up, this research is the first venomic report of C.d.terrificus venom from Argentina. This proved to be crotamine positive venom that has a lower metalloprotease content than C.d. terrificus venoms from other regions. This information could be used in the discovery of future pharmacological agents or targets in antivenom therapy.

Biochemical characterization and cytotoxic effect of the skin secretion from the red-spotted Argentina frog Argenteohyla siemersi (Anura: Hylidae).

BACKGROUND: Argenteohyla siemersi (red-spotted Argentina frog) is a casque-headed tree frog species belonging to the Hylidae family. This species has a complex combination of anti-predator defense mechanisms that include a highly lethal skin secretion. However, biochemical composition and biological effects of this secretion have not yet been studied. METHODS: The A. siemersi skin secretion samples were analyzed by mass spectrometry and chromatographic analysis (MALDI-TOF/MS, RP-HPLC and GC-MS). Proteins were also studied by SDS-PAGE. Among the biological activities evaluated, several enzymatic activities (hemolytic, phospholipase A2, clotting, proteolytic and amidolytic) were assessed. Furthermore, the cytotoxic activity (cytolysis and fluorescence staining) was evaluated on myoblasts of the C2C12 cell line. RESULTS: The MALDI-TOF/MS analysis identified polypeptides and proteins in the aqueous solution of A. siemersi skin secretion. SDS-PAGE revealed the presence of proteins with molecular masses from 15 to 55 kDa. Steroids, but no alkaloids or peptides (less than 5 KDa), were detected using mass spectrometry. Skin secretion revealed the presence of lipids in methanolic extract, as analyzed by CG-MS. This secretion showed hemolytic and phospholipase A2 activities, but was devoid of amidolytic, proteolytic or clotting activities. Moreover, dose-dependent cytotoxicity in cultured C2C12 myoblasts of the skin secretion was demonstrated. Morphological analysis, quantification of lactate dehydrogenase release and fluorescence staining indicated that the cell death triggered by this secretion involved necrosis. CONCLUSIONS: Results presented herein evidence the biochemical composition and biological effects of A. siemersi skin secretion and contribute to the knowledge on the defense mechanisms of casque-headed frogs.

Biochemical characterization and cytotoxic effect of the skin secretion from the red-spotted Argentina frog Argenteohyla siemersi (Anura: Hylidae)

Argenteohyla siemersi (red-spotted Argentina frog) is a casque-headed tree frog species belonging to the Hylidae family. This species has a complex combination of anti-predator defense mechanisms that include a highly lethal skin secretion. However, biochemical composition and biological effects of this secretion have not yet been studied. Methods: The A. siemersi skin secretion samples were analyzed by mass spectrometry and chromatographic analysis (MALDI-TOF/MS, RP-HPLC and GC-MS). Proteins were also studied by SDS-PAGE. Among the biological activities evaluated, several enzymatic activities (hemolytic, phospholipase A2, clotting, proteolytic and amidolytic) were assessed. Furthermore, the cytotoxic activity (cytolysis and fluorescence staining) was evaluated on myoblasts of the C2C12 cell line. Results: The MALDI-TOF/MS analysis identified polypeptides and proteins in the aqueous solution of A. siemersi skin secretion. SDS-PAGE revealed the presence of proteins with molecular masses from 15 to 55 kDa. Steroids, but no alkaloids or peptides (less than 5 KDa), were detected using mass spectrometry. Skin secretion revealed the presence of lipids in methanolic extract, as analyzed by CG-MS. This secretion showed hemolytic and phospholipase A2 activities, but was devoid of amidolytic, proteolytic or clotting activities. Moreover, dose-dependent cytotoxicity in cultured C2C12 myoblasts of the skin secretion was demonstrated. Morphological analysis, quantification of lactate dehydrogenase release and fluorescence staining indicated that the cell death triggered by this secretion involved necrosis. Conclusions: Results presented herein evidence the biochemical composition and biological effects of A. siemersi skin secretion and contribute to the knowledge on the defense mechanisms of casque-headed frogs.(AU)

Fast venomic analysis of Crotalus durissus terrificus from northeastern Argentina

The complete knowledge of the toxins that make up venoms is the base for the treatment of snake accidents victims and the selection of specimens for the preparation of venom pools for antivenom production. In this work, we used a fast and direct venomics approach to identify the toxin families in the C.d. terrificus venom, a Southern American Neotropical rattlesnake. The RP-HPLC separation profile of pooled venom from adult specimens followed by mass spectrometry analysis revealed that C.d. terrificus’ venom proteome is composed of 12 protein families, which are unevenly distributed in the venom, e.g., there are few major proteins in the venom's composition phospholipase A2, serine proteinase, crotamine and L-amino acid oxidase. At the same time, the proteome analysis revealed a small set of proteins with low quantity (less than 1.5%), both enzymes (metaloprotease, phospholipase B and 5′-nucleotidase) and proteins (Bradykinin potentiating and C-type natriuretic peptides, C-type lectin convulxin and nerve growth factor). To sum up, this research is the first venomic report of C.d.terrificus venom from Argentina. This proved to be crotamine positive venom that has a lower metalloprotease content than C.d. terrificus venoms from other regions. This information could be used in the discovery of future pharmacological agents or targets in antivenom therapy.

Biochemical characterization and cytotoxic effect of the skin secretion from the red-spotted Argentina frog Argenteohyla siemersi (Anura: Hylidae)

Background: Argenteohyla siemersi (red-spotted Argentina frog) is a casque-headed tree frog species belonging to the Hylidae family. This species has a complex combination of anti-predator defense mechanisms that include a highly lethal skin secretion. However, biochemical composition and biological effects of this secretion have not yet been studied. Methods: The A. siemersi skin secretion samples were analyzed by mass spectrometry and chromatographic analysis (MALDI-TOF/MS, RP-HPLC and GC-MS). Proteins were also studied by SDS-PAGE. Among the biological activities evaluated, several enzymatic activities (hemolytic, phospholipase A2, clotting, proteolytic and amidolytic) were assessed. Furthermore, the cytotoxic activity (cytolysis and fluorescence staining) was evaluated on myoblasts of the C2C12 cell line. Results: The MALDI-TOF/MS analysis identified polypeptides and proteins in the aqueous solution of A. siemersi skin secretion. SDS-PAGE revealed the presence of proteins with molecular masses from 15 to 55 kDa. Steroids, but no alkaloids or peptides (less than 5 KDa), were detected using mass spectrometry. Skin secretion revealed the presence of lipids in methanolic extract, as analyzed by CG-MS. This secretion showed hemolytic and phospholipase A2 activities, but was devoid of amidolytic, proteolytic or clotting activities. Moreover, dose-dependent cytotoxicity in cultured C2C12 myoblasts of the skin secretion was demonstrated. Morphological analysis, quantification of lactate dehydrogenase release and fluorescence staining indicated that the cell death triggered by this secretion involved necrosis. Conclusions: Results presented herein evidence the biochemical composition and biological effects of A. siemersi skin secretion and contribute to the knowledge on the defense mechanisms of casque-headed frogs.

Biochemical characterization and cytotoxic effect of the skin secretion from the red-spotted Argentina frog Argenteohyla siemersi (Anura: Hylidae)

Background: Argenteohyla siemersi (red-spotted Argentina frog) is a casque-headed tree frog species belonging to the Hylidae family. This species has a complex combination of anti-predator defense mechanisms that include a highly lethal skin secretion. However, biochemical composition and biological effects of this secretion have not yet been studied. Methods: The A. siemersi skin secretion samples were analyzed by mass spectrometry and chromatographic analysis (MALDI-TOF/MS, RP-HPLC and GC-MS). Proteins were also studied by SDS-PAGE. Among the biological activities evaluated, several enzymatic activities (hemolytic, phospholipase A2, clotting, proteolytic and amidolytic) were assessed. Furthermore, the cytotoxic activity (cytolysis and fluorescence staining) was evaluated on myoblasts of the C2C12 cell line. Results: The MALDI-TOF/MS analysis identified polypeptides and proteins in the aqueous solution of A. siemersi skin secretion. SDS-PAGE revealed the presence of proteins with molecular masses from 15 to 55 kDa. Steroids, but no alkaloids or peptides (less than 5 KDa), were detected using mass spectrometry. Skin secretion revealed the presence of lipids in methanolic extract, as analyzed by CG-MS. This secretion showed hemolytic and phospholipase A2 activities, but was devoid of amidolytic, proteolytic or clotting activities. Moreover, dose-dependent cytotoxicity in cultured C2C12 myoblasts of the skin secretion was demonstrated. Morphological analysis, quantification of lactate dehydrogenase release and fluorescence staining indicated that the cell death triggered by this secretion involved necrosis. Conclusions: Results presented herein evidence the biochemical composition and biological effects of A. siemersi skin secretion and contribute to the knowledge on the defense mechanisms of casque-headed frogs.

P9a(Cdt-PLA2) from Crotalus durissus terrificus as good immunogen to be employed in the production of crotalic anti-PLA2 IgG.

Four proteins with phospholipase A2 (PLA2) activity, designated P9a(Cdt-PLA2), P9b(Cdt-PLA2), P10a(Cdt-PLA2) and P10b(Cdt-PLA2) were purified from the venom of Crotalus durissus terrificus by two chromatographic steps: a gel filtration and reversed phase HPLC. The profile obtained clearly shows that three of them have a similar abundance. The molecular mass, 14193.8340Da for P9a(Cdt-PLA2), 14134.9102Da for P9b(Cdt-PLA2), 14242.6289Da for P10a(Cdt-PLA2) and 14183.8730Da for P10b(Cdt-PLA2), were initially evaluated by SDS-PAGE and confirmed by ESI-Q-TOF spectrometry, and all of them displayed a monomeric conformation. Also, partial amino acid sequence of each protein was obtained and their alignments with other crotalic PLA2 revealed a high degree of identity among them. Additionally, we studied some pharmacological activities like neurotoxicity, myotoxicity and lethality, which prompted us to pick two of them, P9a(Cdt-PLA2) and P10a(Cdt-PLA2) that resulted to be less toxic that the others, and further characterize them to be used as immunogen. We next injected these last proteins in mice to produce antitoxins against them and ELISA and dot blots reveled that both toxins do not show immunogenic differences, unlike those other pharmacologic activities tested. Furthermore, the antibodies produced cross-reacted with all the isoforms purified demonstrating the feasibility of using only one of them and ensuring the cross-reaction of all. The results obtained show that P9a(Cdt-PLA2) isoform has the lowest toxicity and also a good purification performance; thus this protein may be a promising candidate to be employed in the production of crotalic antitoxins.