Moderate-to-severe Vipera berus envenoming requiring ViperaTAb antivenom therapy in the UK.

BACKGROUND: Bites by the European adder (Vipera berus) in the UK are uncommon but potentially life threatening, and can be associated with marked limb swelling and disability. Following an interruption in Zagreb Imunoloski zavod antivenom supply around 2012, the UK changed its national choice of antivenom for Vipera berus to ViperaTAb, an ovine Fab monospecific antivenom. In the absence of randomised controlled trials, we established an audit to review its use in clinical practice. METHODS: A prospective audit of ViperaTAb use was conducted from March 2016 until November 2020 by the UK National Poison Information Service (NPIS). Users of the NPIS online toxicology database, TOXBASE, considering the use of antivenom for V. berus envenoming were invited to discuss the case with the on-call clinical toxicology consultant. Information was collected prospectively on indications, administration, adverse reactions and outcome of patients administered ViperaTAb antivenom. RESULTS: One hundred and seventy patients were administered ViperaTAb antivenom over five years. One hundred and thirty-two were adults and 38 children (median age and range: 38, 2-87 years). Bites occurred across the UK, but most commonly in coastal regions of Wales and of South-West and East England. Median time to presentation was 2.1 (IQR 1.5-4.0) h and to antivenom administration from presentation was 2.0 (IQR 0.9-3.6) h. A minority of patients presented to hospital more than 12 h after being bitten (n = 19, 11.2%) or received antivenom more than 12 h after presenting to hospital (n = 17, 10.0%). Features of systemic envenoming were present in 64/170 (37.6%) patients, including 23 (13.5%) with anaphylaxis and 26 (15.3%) with hypotension (nine with both). Clinician assessment considered the initial antivenom to have been effective in 122/169 (72.2%) patients. Repeated dosing was common, occurring in 55/169 (32.5%), predominantly due to persisting or worsening local effects (46/51, 90.2%). There were three cases of probable early adverse reaction. No deaths occurred during the study. Complications of envenoming were rare but included four patients that underwent surgery, three patients each with acute kidney injury, mild coagulopathy, or thrombocytopenia (one severe). The median duration of hospital stay was 43.7 (IQR 22.5-66.5) h, longer for children than adults (52.5 vs 41.3 h). CONCLUSION: ViperaTAb antivenom appears to be effective and safe and should be administered as soon as possible for patients meeting clinical criteria. Patients require close observation following antivenom to detect adverse reactions and progression or recurrence of envenoming. Close collaboration with expert NPIS consultant advice can help optimise antivenom timing, ensure repeated dosing is given appropriately, and avoid unnecessary surgical intervention. All hospitals, particularly those located in areas of relatively high incidence, should stock sufficient antivenom available at short notice, 24 h a day.

Extensive Variation in the Activities of Pseudocerastes and Eristicophis Viper Venoms Suggests Divergent Envenoming Strategies Are Used for Prey Capture.

Snakes of the genera Pseudocerastes and Eristicophis (Viperidae: Viperinae) are known as the desert vipers due to their association with the arid environments of the Middle East. These species have received limited research attention and little is known about their venom or ecology. In this study, a comprehensive analysis of desert viper venoms was conducted by visualising the venom proteomes via gel electrophoresis and assessing the crude venoms for their cytotoxic, haemotoxic, and neurotoxic properties. Plasmas sourced from human, toad, and chicken were used as models to assess possible prey-linked venom activity. The venoms demonstrated substantial divergence in composition and bioactivity across all experiments. Pseudocerastes urarachnoides venom activated human coagulation factors X and prothrombin and demonstrated potent procoagulant activity in human, toad, and chicken plasmas, in stark contrast to the potent neurotoxic venom of P. fieldi. The venom of E. macmahonii also induced coagulation, though this did not appear to be via the activation of factor X or prothrombin. The coagulant properties of P. fieldi and P. persicus venoms varied among plasmas, demonstrating strong anticoagulant activity in the amphibian and human plasmas but no significant effect in that of bird. This is conjectured to reflect prey-specific toxin activity, though further ecological studies are required to confirm any dietary associations. This study reinforces the notion that phylogenetic relatedness of snakes cannot readily predict venom protein composition or function. The significant venom variation between these species raises serious concerns regarding antivenom paraspecificity. Future assessment of antivenom is crucial.

Effect of light emitting diode photobiomodulation on murine macrophage function after Bothrops envenomation.

Several reports have suggested that photobiomodulation, owing to its analgesic, anti-inflammatory, and healing effects, may be an effective therapeutic option for local effects of snakebites when the availability and accessibility of conventional serum therapy are inefficient and far from medical care centers. Although there have been studies that demonstrate the application of photobiomodulation in the treatment of local adverse events due to snakebites from snakes of the genus Bothrops, its role in the activation of leukocytes, particularly macrophages, has not been evaluated. Here, we assessed the effect of light-emitting diode (LED) treatment on macrophage activation induced by B. jararacussu venom (BjV). LED treatment caused an increase in the viability of macrophages incubated with BjV. This treatment reduced reactive oxygen species (ROS) and nitric oxide (NO) production by macrophages after incubation with BjV. However, LED treatment did not interfere with IL-1ß and IL-10 production by macrophages after incubation with BjV. In conclusion, this study showed that LED treatment has the potential to be used in combination with conventional serum therapy to prevent or minimize the progression of local to severe symptoms after Bothrops envenomation.

Mesenchymal Stromal Cell-Based Therapies as Promising Treatments for Muscle Regeneration After Snakebite Envenoming.

Snakebite envenoming is a global neglected disease with an incidence of up to 2.7 million new cases every year. Although antivenoms are so-far the most effective treatment to reverse the acute systemic effects induced by snakebite envenoming, they have a limited therapeutic potential, being unable to completely neutralize the local venom effects. Local damage, such as dermonecrosis and myonecrosis, can lead to permanent sequelae with physical, social, and psychological implications. The strong inflammatory process induced by snake venoms is associated with poor tissue regeneration, in particular the lack of or reduced skeletal muscle regeneration. Mesenchymal stromal cells (MSCs)-based therapies have shown both anti-inflammatory and pro-regenerative properties. We postulate that using allogeneic MSCs or their cell-free products can induce skeletal muscle regeneration in snakebite victims, improving all the three steps of the skeletal muscle regeneration process, mainly by anti-inflammatory activity, paracrine effects, neovascularization induction, and inhibition of tissue damage, instrumental for microenvironment remodeling and regeneration. Since snakebite envenoming occurs mainly in areas with poor healthcare, we enlist the principles and potential of MSCs-based therapies and discuss regulatory issues, good manufacturing practices, transportation, storage, and related-procedures that could allow the administration of these therapies, looking forward to a safe and cost-effective treatment for a so far unsolved and neglected health problem.

Predictors of hypopituitarism due to vasculotoxic snake bite with acute kidney injury.

PURPOSE: Hypopituitarism frequently develops following vasculotoxic snake bite complicated by acute kidney injury (AKI). Well defined prospective studies of prevalence of hypopituitarism and its predictors in vasculotoxic snake bites complicated by AKI are unavailable. METHODS: Fifty-one consecutive patients of AKI following vasculotoxic snake bite were evaluated for various clinical/biochemical parameters (including Free T4, TSH, Cortisol, ACTH, total testosterone, FSH, LH, prolactin, and IGF-1). Diabetes insipidus was evaluated in relevant cases. Twenty minutes whole blood clotting time (WBCT) at presentation was measured in all. MRI of hypothalamo-pituitary region was done at 3 months in subjects with hypopituitarism to rule out structural lesion. RESULTS: 21.6% (11/51) patients developed hypopituitarism at baseline (within 7 days), 39.3% (13 /33) at 3 months developed hypopituitarism. Cortisol deficiency was the commonest abnormality. Subjects who developed hypopituitarism at baseline were younger compared to those without hypopituitarism (35.67 years vs. 46.59 years, p = 0.032) and required more sessions of hemodialysis (8 vs. 3, p = 0.041). Binary logistic regression confirmed that development of hypopituitarism could be predicted by increased number of sessions of hemodialysis (OR 1.51, p = 0.008) and 20 min WBCT (OR 1.2, p = 0.038). CONCLUSION: Hypopituitarism is common following vasculotoxic snake bite in subjects who develop AKI requiring hemodialysis. Hypopituitarism can develop as early as 7 days following snake bite and should be evaluated for particularly in younger subjects, especially those requiring increasing number of sessions of hemodialysis and in subjects with abnormal 20 min WBCT at presentation.

Phylovenomics of Daboia russelii across the Indian subcontinent. Bioactivities and comparative in vivo neutralization and in vitro third-generation antivenomics of antivenoms against venoms from India, Bangladesh and Sri Lanka.

Russell's viper (Daboia russelii) is, together with Naja naja, Bungarus caeruleus and Echis carinatus, a member of the medically important 'Big Four' species responsible for causing a large number of morbidity and mortality cases across the Indian subcontinent. Despite the wide distribution of Russell's viper and the well-documented ubiquity of the phenomenon of geographic variability of intraspecific snake venom composition, Indian polyvalent antivenoms against the "Big Four" venoms are raised against venoms sourced mainly from Chennai in the southeastern Indian state of Tamil Nadu. Biochemical and venomics investigations have consistently revealed notable compositional, functional, and immunological differences among geographic variants of Russell's viper venoms across the Indian subcontinent. However, these studies, carried out by different laboratories using different protocols and involving venoms from a single geographical region, make the comparison of the different venoms difficult. To bridge this gap, we have conducted bioactivities and proteomic analyses of D. russelii venoms from the three corners of the Indian subcontinent, Pakistan, Bangladesh, and Tamil Nandu (India) and Sri Lanka, along with comparative in vivo neutralization and in vitro third-generation antivenomics of antivenoms used in India, Bangladesh and Sri Lanka. These analyses let us to propose two alternative routes of radiation for Russell's viper in the Indian subcontinent. Both radiations, towards the northeast of India and Bangladesh and towards south India and Sri Lanka, have a common origin in Pakistan, and provide a phylovenomics ground for rationalizing the geographic variability in venom composition and their distinct immunoreactivity against available antivenoms. BIOLOGICAL SIGNIFICANCE: Russell's viper (Daboia russelii), the Indian cobra (Naja naja), the common krait (Bungarus caeruleus), and the saw-scaled viper (Echis carinatus) constitute the 'Big Four' snake species responsible for most snakebite envenomings and deaths in the Indian subcontinent. Despite the medical relevance of Daboia russelii, and the well documented variations in the clinical manifestations of envenomings by this wide distributed species, which are doubtless functionally related to differences in venom composition of its geographic variants, antivenoms for the clinical treatment of envenomings by D. russelii across the Indian subcontinent are invariably raised using venom sourced mainly from the southeastern Indian state of Tamil Nadu. We have applied a phylovenomics approach to compare the venom proteomes of Russell's vipers from the three corners of the Indian subcontinent, Pakistan, Bangladesh, and South India/Sri Lanka, and have assessed the in vitro (third-generation antivenomics) and in vivo preclinical efficacy of a panel of homologous antivenoms. The identification of two dispersal routes of ancestral D. russelii into the Indian subcontinent provides the ground for rationalizing the variability in composition and immunoreactivity of the venoms of extant geographic variants of Russell's viper. Such knowledge is relevant for envisioning strategies to improve the clinical coverage of anti- D. russelii antivenoms.

Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release.

Pain-producing animal venoms contain evolutionarily honed toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. From a functional screen, we have identified a secreted phospholipase A2 (sPLA2)-like protein, BomoTx, from the Brazilian lancehead pit viper (Bothrops moojeni). BomoTx is closely related to a group of Lys49 myotoxins that have been shown to promote ATP release from myotubes through an unknown mechanism. Here we show that BomoTx excites a cohort of sensory neurons via ATP release and consequent activation of P2X2 and/or P2X3 purinergic receptors. We provide pharmacological and electrophysiological evidence to support pannexin hemichannels as downstream mediators of toxin-evoked ATP release. At the behavioral level, BomoTx elicits nonneurogenic inflammatory pain, thermal hyperalgesia, and mechanical allodynia, of which the latter is completely dependent on purinergic signaling. Thus, we reveal a role of regulated endogenous nucleotide release in nociception and provide a detailed mechanism of a pain-inducing Lys49 myotoxin from Bothrops species, which are responsible for the majority of snake-related deaths and injuries in Latin America.

Analgesic Effect of Photobiomodulation on Bothrops Moojeni Venom-Induced Hyperalgesia: A Mechanism Dependent on Neuronal Inhibition, Cytokines and Kinin Receptors Modulation.

BACKGROUND: Envenoming induced by Bothrops snakebites is characterized by drastic local tissue damage that involves an intense inflammatory reaction and local hyperalgesia which are not neutralized by conventional antivenom treatment. Herein, the effectiveness of photobiomodulation to reduce inflammatory hyperalgesia induced by Bothrops moojeni venom (Bmv), as well as the mechanisms involved was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Bmv (1 µg) was injected through the intraplantar route in the right hind paw of mice. Mechanical hyperalgesia and allodynia were evaluated by von Frey filaments at different time points after venom injection. Low level laser therapy (LLLT) was applied at the site of Bmv injection at wavelength of red 685 nm with energy density of 2.2 J/cm2 at 30 min and 3 h after venom inoculation. Neuronal activation in the dorsal horn spinal cord was determined by immunohistochemistry of Fos protein and the mRNA expression of IL-6, TNF-α, IL-10, B1 and B2 kinin receptors were evaluated by Real time-PCR 6 h after venom injection. Photobiomodulation reversed Bmv-induced mechanical hyperalgesia and allodynia and decreased Fos expression, induced by Bmv as well as the mRNA levels of IL-6, TNF-α and B1 and B2 kinin receptors. Finally, an increase on IL-10, was observed following LLLT. CONCLUSION/SIGNIFICANCE: These data demonstrate that LLLT interferes with mechanisms involved in nociception and hyperalgesia and modulates Bmv-induced nociceptive signal. The use of photobiomodulation in reducing local pain induced by Bothropic venoms should be considered as a novel therapeutic tool for the treatment of local symptoms induced after bothropic snakebites.

Snake venomics of monocled cobra (Naja kaouthia) and investigation of human IgG response against venom toxins.

The venom proteome of the monocled cobra, Naja kaouthia, from Thailand, was characterized by RP-HPLC, SDS-PAGE, and MALDI-TOF-TOF analyses, yielding 38 different proteins that were either identified or assigned to families. Estimation of relative protein abundances revealed that venom is dominated by three-finger toxins (77.5%; including 24.3% cytotoxins and 53.2% neurotoxins) and phospholipases A2 (13.5%). It also contains lower proportions of components belonging to nerve growth factor, ohanin/vespryn, cysteine-rich secretory protein, C-type lectin/lectin-like, nucleotidase, phosphodiesterase, metalloproteinase, l-amino acid oxidase, cobra venom factor, and cytidyltransferase protein families. Small amounts of three nucleosides were also evidenced: adenosine, guanosine, and inosine. The most relevant lethal components, categorized by means of a 'toxicity score', were α-neurotoxins, followed by cytotoxins/cardiotoxins. IgGs isolated from a person who had repeatedly self-immunized with a variety of snake venoms were immunoprofiled by ELISA against all venom fractions. Stronger responses against larger toxins, but lower against the most critical α-neurotoxins were obtained. As expected, no neutralization potential against N. kaouthia venom was therefore detected. Combined, our results display a high level of venom complexity, unveil the most relevant toxins to be neutralized, and provide prospects of discovering human IgGs with toxin neutralizing abilities through use of phage display screening.

The snake venom rhodocytin from Calloselasma rhodostoma- a clinically important toxin and a useful experimental tool for studies of C-type lectin-like receptor 2 (CLEC-2).

The snake venom, rhodocytin, from the Malayan viper, Calloselasma rhodostoma, and the endogenous podoplanin are identified as ligands for the C-type lectin-like receptor 2 (CLEC-2). The snakebites caused by Calloselasma rhodostoma cause a local reaction with swelling, bleeding and eventually necrosis, together with a systemic effect on blood coagulation with distant bleedings that can occur in many different organs. This clinical picture suggests that toxins in the venom have effects on endothelial cells and vessel permeability, extravasation and, possibly, activation of immunocompetent cells, as well as effects on platelets and the coagulation cascade. Based on the available biological studies, it seems likely that ligation of CLEC-2 contributes to local extravasation, inflammation and, possibly, local necrosis, due to microthrombi and ischemia, whereas other toxins may be more important for the distant hemorrhagic complications. However, the venom contains several toxins and both local, as well as distant, symptoms are probably complex reactions that cannot be explained by the effects of rhodocytin and CLEC-2 alone. The in vivo reactions to rhodocytin are thus examples of toxin-induced crosstalk between coagulation (platelets), endothelium and inflammation (immunocompetent cells). Very few studies have addressed this crosstalk as a part of the pathogenesis behind local and systemic reactions to Calloselasma rhodostoma bites. The author suggests that detailed biological studies based on an up-to-date methodology of local and systemic reactions to Calloselasma rhodostoma bites should be used as a hypothesis-generating basis for future functional studies of the CLEC-2 receptor. It will not be possible to study the effects of purified toxins in humans, but the development of animal models (e.g., cutaneous injections of rhodocytin to mimic snakebites) would supplement studies in humans.

Hyponatraemia, rhabdomyolysis, alterations in blood pressure and persistent mydriasis in patients envenomed by Malayan kraits (Bungarus candidus) in southern Viet Nam.

Between 1998 and 2007, 42 patients admitted to Choray hospital, Ho Chi Minh City, and to two hospitals in adjacent regions in southern Viet Nam brought the Malayan kraits (Bungarus candidus) that had been responsible for biting them. Half of the patients had been bitten while they were asleep. Fang marks and numbness were the only local features of the bites. Common signs of neurotoxic envenoming included bilateral ptosis, persistently dilated pupils, limb weakness, breathlessness, hypersalivation, dysphonia and dysphagia. Thirty patients (71.4%) required endotracheal intubation of whom all but one were mechanically ventilated. Fourteen patients (33.3%) developed hypertension, 13 (31.0%) shock, 31 (73.8%) hyponatraemia (plasma sodium concentration < 130 mEq/l) and 30 (71.4%) showed evidence of mild rhabdomyolysis (peak plasma creatine kinase concentration 1375 +/- 140 micro/l). None developed acute kidney injury. All the patients were treated with a new monospecific B. candidus antivenom. There were no fatalities. Hyponatraemia has been reported previously in victims of Chinese kraits (Bungarus multicinctus) in northern Viet Nam and rhabdomyolysis in patients envenomed by B. niger in Bangladesh. These features of envenoming pose new problems for the management of krait bite cases in South east Asia and should stimulate a search for the causative venom toxins.

Processing of pro-tumor necrosis factor-alpha by venom metalloproteinases: a hypothesis explaining local tissue damage following snake bite.

Venom-induced necrosis is a common local debilitating sequela of bites by many vipers, frequently resulting in severe permanent scarring and deformity. Antivenoms are not effective under these circumstances unless administered within a few minutes of the bite; this is unlikely to occur in the rural tropics where most victims take a long time to reach medical care. We have shown that two venom zinc metalloproteinases (jararhagin from Bothrops jararaca venom and a metalloproteinase from Echis pyramidum leakeyi venom) successfully cleaved the recombinant glutathione-S-transferase-tumor necrosis factor-alpha fusion protein (GST-TNF-alpha) substrate to form biologically active TNF-alpha which was shown to be neutralized by ovine TNF-alpha Fab antibodies. This resulted in a reduction of venom-induced necrosis in mice when injected intravenously or intradermally both before and after intradermal injections of E.p.leakeyi venom. A peptidomimetic (POL 647) was also found to inhibit the Echis metalloproteinase, thus preventing the processing of the TNF precursor; this was shown using a TNF-alpha-sensitive cell culture assay and electrophoresis. These observations demonstrate the possible importance of TNF-alpha in the development of the resulting necrotic lesion and leads to the hypothesis that increased levels of venom metalloproteinases following snake bite release active TNF-alpha. This cytokine may contribute to the local necrosis and also induce the production of endogenous matrix metalloproteinases, which in turn generate a positive feedback mechanism resulting in continued cleavage of pro-TNF-alpha. The results indicate that inhibition or neutralization of endogenous TNF-alpha appears to result in a significant reduction in venom-induced necrosis. This could help to explain the clinical observations that treatment of local necrosis following snake bite by antivenom is only minimally successful.