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Background: The venom of Bothrops lanceolatus, a viperid species endemic to the Lesser Antillean Island of Martinique, induces thrombosis in a number of patients. Previous clinical observations indicate that thrombotic events are more common in patients bitten by juvenile specimens. There is a need to develop an experimental model of this effect in order to study the mechanisms involved. Methodology/Principal findings: The venoms of juvenile and adult specimens of B. lanceolatus were compared by (a) describing their proteome, (b) assessing their ability to induce thrombosis in a mouse model, and (c) evaluating their in vitro procoagulant activity and in vivo hemostasis alterations. Venom proteomes of juvenile and adult specimens were highly similar, albeit showing some differences. When injected by the intraperitoneal (i.p.) route, the venom of juvenile specimens induced the formation of abundant thrombi in the pulmonary vasculature, whereas this effect was less frequent in the case of adult venom. Thrombosis was not abrogated by the metalloproteinase inhibitor Batimastat. Both venoms showed a weak in vitro procoagulant effect on citrated mouse plasma and bovine fibrinogen. When administered intravenously (i.v.) venoms did not affect classical clotting tests (prothrombin time and activated partial thromboplastin time) but caused a partial drop in fibrinogen concentration. The venom of juvenile specimens induced partial alterations in some rotational thromboelastometry parameters after i.v. injection. When venoms were administered i.p., only minor alterations in classical clotting tests were observed with juvenile venom, and no changes occurred for either venom in rotational thromboelastometry parameters. Both juvenile and adult venoms induced a marked thrombocytopenia after i.p. injection. Conclusions/Significance: An experimental model of the thrombotic effect induced by B. lanceolatus venom was developed. This effect is more pronounced in the case of venom of juvenile specimens, despite the observation that juvenile and adult venom proteomes are similar. Adult and juvenile venoms do not induce a consumption coagulopathy characteristic of other Bothrops sp venoms. Both venoms induce a conspicuous thrombocytopenia. This experimental model reproduces the main clinical findings described in these envenomings and should be useful to understand the mechanisms of the thrombotic effect. Author summary: Envenomings by the viperid species Bothrops lanceolatus, endemic of the Caribbean Island of Martinique, are characterized by a thrombotic effect responsible for infarcts in various organs. Until now, no experimental in vivo models of this effect have been described. In this study, we developed a mouse model of thrombosis by using the intraperitoneal route of venom injection. The venom of juvenile specimens of B. lanceolatus induced the formation of abundant thrombi in the lungs, whereas the effect was much less pronounced with the venom of adult specimens. This difference in the ability of juvenile and adult venoms occurs despite both venoms having highly similar proteomic profiles. Both adult and juvenile venoms showed a weak in vitro procoagulant effect on plasma and fibrinogen, underscoring a thrombin-like (pseudo-procoagulant) activity. In vivo, the venoms did not affect the classical clotting tests (prothrombin time and activated partial thromboplastin time) but induced a partial drop in fibrinogen concentration and limited alterations in rotational thromboelastometry parameters when injected by the i.v. route. In contrast, few alterations of these parameters were observed after i.p. injection of venoms, in conditions in which thrombosis occurred, hence evidencing the lack of a consumption coagulopathy. After i.p. injection both venoms induced a pronounced thrombocytopenia. This experimental model reproduces some of the main clinical manifestations of envenoming by this species. This model can be used to identify the toxins responsible for the thrombotic effect, to study the mechanism(s) of thrombosis and to assess the preclinical efficacy of antivenoms and novel therapies. [ABSTRACT FROM AUTHOR]

The variable domain of New Antigen Receptors (vNAR) from sharks, present special characteristics in comparison to the conventional antibody molecules such as: small size (12–15 kDa), thermal and chemical stability and great tissue penetration, that makes them a good alternative source as therapeutic or diagnostic agents. Therefore, it is essential to improve techniques used for the development and selection of vNAR antibodies that recognize distinct antigens. The development of synthetic antibody libraries offers a fast option for the generation of antibodies with the desired characteristics. In this work three synthetic antibody libraries were constructed; without cysteines (Cys), with one Cys and with two Cys residues within its CDR3, with the objective of determining whether the presence or absence of Cys in the CDR3 favors the isolation of vNAR clones from a synthetic library. The libraries were validated selecting against six mammalian proteins. At least one vNAR was found for each of the antigens, and a clone coming from the library without Cys in the CDR3 was selected with all the antigens. In vitro angiogenesis assay with the isolated anti-VEGF antibodies, suggest that these vNARs are capable of inhibiting in vitro angiogenesis. In silico analysis of anti-VEGF antibodies showed that vNARs from synthetic libraries could rival antibodies with affinity maturation by in silico modeling. [ABSTRACT FROM AUTHOR]

The precise mechanisms by which Snake Venom Metalloproteinases (SVMPs) disrupt the microvasculature and cause haemorrhage have not been completely elucidated, and novel in vivo models are needed. In the present study, we compared the effects induced by BaP1, a PI SVMP isolated from Bothrops asper venom, and CsH1, a PIII SVMP from Crotalus simus venom, on cremaster muscle microvasculature by topical application of the toxins on isolated tissue (i.e., ex vivo model), and by intra-scrotal administration of the toxins (i.e., in vivo model). The whole tissue was fixed and immunostained to visualize the three components of blood vessels by confocal microscopy. In the ex vivo model, BaP1 was able to degrade type IV collagen and laminin from the BM of microvessels. Moreover, both SVMPs degraded type IV collagen from the BM in capillaries to a higher extent than in PCV and arterioles. CsH1 had a stronger effect on type IV collagen than BaP1. In the in vivo model, the effect of BaP1 on type IV collagen was widespread to the BM of arterioles and PCV. On the other hand, BaP1 was able to disrupt the endothelial barrier in PCV and to increase vascular permeability. Moreover, this toxin increased the size of gaps between pericytes in PCV and created new gaps between smooth muscle cells in arterioles in ex vivo conditions. These effects were not observed in the case of CsH1. In conclusion, our findings demonstrate that both SVMPs degrade type IV collagen from the BM in capillaries in vivo. Moreover, while the action of CsH1 is more directed to the BM of microvessels, the effects of BaP1 are widespread to other microvascular components. This study provides new insights in the mechanism of haemorrhage and other pathological effects induced by these toxins. [ABSTRACT FROM AUTHOR]

The time-course of the pathological effects induced by the venom of the snake Bothrops asper in muscle tissue was investigated by a combination of histology, proteomic analysis of exudates collected in the vicinity of damaged muscle, and immunodetection of extracellular matrix proteins in exudates. Proteomic assay of exudates has become an excellent new methodological tool to detect key biomarkers of tissue alterations for a more integrative perspective of snake venom-induced pathology. The time-course analysis of the intracellular proteins showed an early presence of cytosolic and mitochondrial proteins in exudates, while cytoskeletal proteins increased later on. This underscores the rapid cytotoxic effect of venom, especially in muscle fibers, due to the action of myotoxic phospholipases A2, followed by the action of proteinases in the cytoskeleton of damaged muscle fibers. Similarly, the early presence of basement membrane (BM) and other extracellular matrix (ECM) proteins in exudates reflects the rapid microvascular damage and hemorrhage induced by snake venom metalloproteinases. The presence of fragments of type IV collagen and perlecan one hour after envenoming suggests that hydrolysis of these mechanically/structurally-relevant BM components plays a key role in the genesis of hemorrhage. On the other hand, the increment of some ECM proteins in the exudate at later time intervals is likely a consequence of the action of endogenous matrix metalloproteinases (MMPs) or of de novo synthesis of ECM proteins during tissue remodeling as part of the inflammatory reaction. Our results offer relevant insights for a more integrative and systematic understanding of the time-course dynamics of muscle tissue damage induced by B. asper venom and possibly other viperid venoms. [ABSTRACT FROM AUTHOR]

Hemorrhage is a clinically important manifestation of viperid snakebite envenomings, and is induced by snake venom metalloproteinases (SVMPs). Hemorrhagic and non-hemorrhagic SVMPs hydrolyze some basement membrane (BM) and associated extracellular matrix (ECM) proteins. Nevertheless, only hemorrhagic SVMPs are able to disrupt microvessels; the mechanisms behind this functional difference remain largely unknown. We compared the proteolytic activity of the hemorrhagic P-I SVMP BaP1, from the venom of Bothrops asper, and the non-hemorrhagic P-I SVMP leucurolysin-a (leuc-a), from the venom of Bothrops leucurus, on several substrates in vitro and in vivo, focusing on BM proteins. When incubated with Matrigel, a soluble extract of BM, both enzymes hydrolyzed laminin, nidogen and perlecan, albeit BaP1 did it at a faster rate. Type IV collagen was readily digested by BaP1 while leuc-a only induced a slight hydrolysis. Degradation of BM proteins in vivo was studied in mouse gastrocnemius muscle. Western blot analysis of muscle tissue homogenates showed a similar degradation of laminin chains by both enzymes, whereas nidogen was cleaved to a higher extent by BaP1, and perlecan and type IV collagen were readily digested by BaP1 but not by leuc-a. Immunohistochemistry of muscle tissue samples showed a decrease in the immunostaining of type IV collagen after injection of BaP1, but not by leuc-a. Proteomic analysis by LC/MS/MS of exudates collected from injected muscle revealed higher amounts of perlecan, and types VI and XV collagens, in exudates from BaP1-injected tissue. The differences in the hemorrhagic activity of these SVMPs could be explained by their variable ability to degrade key BM and associated ECM substrates in vivo, particularly perlecan and several non-fibrillar collagens, which play a mechanical stabilizing role in microvessel structure. These results underscore the key role played by these ECM components in the mechanical stability of microvessels. [ABSTRACT FROM AUTHOR]

Background: Viperid snakebite envenoming is characterized by prominent local tissue damage, including muscle necrosis. A frequent outcome of such local pathology is deficient skeletal muscle regeneration, which causes muscle dysfunction, muscle loss and fibrosis, thus provoking permanent sequelae that greatly affect the quality of life of patients. The causes of such poor regenerative outcome of skeletal muscle after viperid snakebites are not fully understood. Methodology/Principal Findings: A murine model of muscle necrosis and regeneration was adapted to study the effects of the venom and isolated toxins of Bothrops asper, the medically most important snake in Central America. Gastrocnemius muscle was injected with either B. asper venom, a myotoxic phospholipase A2 (Mtx), a hemorrhagic metalloproteinase (SVMP), or saline solution. At various time intervals, during one month, tissue samples were collected and analyzed by histology, and by immunocytochemical and immunohistochemical techniques aimed at detecting muscle fibers, collagen, endothelial cells, myoblasts, myotubes, macrophages, TUNEL-positive nuclei, and axons. A successful regenerative response was observed in muscle injected with Mtx, which induces myonecrosis but does not affect the microvasculature. In contrast, poor regeneration, with fibrosis and atrophic fibers, occurred when muscle was injected with venom or SVMP, both of which provoke necrosis, microvascular damage leading to hemorrhage, and poor axonal regeneration. Conclusions/Significance: The deficient skeletal muscle regeneration after injection of B. asper venom is likely to depend on the widespread damage to the microvasculature, which affects the removal of necrotic debris by phagocytes, and the provision of nutrients and oxygen required for regeneration. In addition, deficient axonal regeneration is likely to contribute to the poor regenerative outcome in this model. [ABSTRACT FROM AUTHOR]

Snake venom hemorrhagic metalloproteinases (SVMPs) of the PI, PII and PIII classes were compared in terms of tissue localization and their ability to hydrolyze basement membrane components in vivo, as well as by a proteomics analysis of exudates collected in tissue injected with these enzymes. Immunohistochemical analyses of co-localization of these SVMPs with type IV collagen revealed that PII and PIII enzymes co-localized with type IV collagen in capillaries, arterioles and post-capillary venules to a higher extent than PI SVMP, which showed a more widespread distribution in the tissue. The patterns of hydrolysis by these three SVMPs of laminin, type VI collagen and nidogen in vivo greatly differ, whereas the three enzymes showed a similar pattern of degradation of type IV collagen, supporting the concept that hydrolysis of this component is critical for the destabilization of microvessel structure leading to hemorrhage. Proteomic analysis of wound exudate revealed similarities and differences between the action of the three SVMPs. Higher extent of proteolysis was observed for the PI enzyme regarding several extracellular matrix components and fibrinogen, whereas exudates from mice injected with PII and PIII SVMPs had higher amounts of some intracellular proteins. Our results provide novel clues for understanding the mechanisms by which SVMPs induce damage to the microvasculature and generate hemorrhage.

Background: To unravel the strategy by which Brucella abortus establishes chronic infections, we explored its early interaction with innate immunity., Methodology/principal Findings: Brucella did not induce proinflammatory responses as demonstrated by the absence of leukocyte recruitment, humoral or cellular blood changes in mice. Brucella hampered neutrophil (PMN) function and PMN depletion did not influence the course of infection. Brucella barely induced proinflammatory cytokines and consumed complement, and was strongly resistant to bactericidal peptides, PMN extracts and serum. Brucella LPS (BrLPS), NH-polysaccharides, cyclic glucans, outer membrane fragments or disrupted bacterial cells displayed low biological activity in mice and cells. The lack of proinflammatory responses was not due to conspicuous inhibitory mechanisms mediated by the invading Brucella or its products. When activated 24 h post-infection macrophages did not kill Brucella, indicating that the replication niche was not fusiogenic with lysosomes. Brucella intracellular replication did not interrupt the cell cycle or caused cytotoxicity in WT, TLR4 and TLR2 knockout cells. TNF-alpha-induction was TLR4- and TLR2-dependent for live but not for killed B. abortus. However, intracellular replication in TLR4, TLR2 and TLR4/2 knockout cells was not altered and the infection course and anti-Brucella immunity development upon BrLPS injection was unaffected in TLR4 mutant mice., Conclusion/significance: We propose that Brucella has developed a stealth strategy through PAMPs reduction, modification and hiding, ensuring by this manner low stimulatory activity and toxicity for cells. This strategy allows Brucella to reach its replication niche before activation of antimicrobial mechanisms by adaptive immunity. This model is consistent with clinical profiles observed in humans and natural hosts at the onset of infection and could be valid for those intracellular pathogens phylogenetically related to Brucella that also cause long lasting infections.