Refinement and Neutralization Evaluation of the F(ab')2 Type of Antivenom against the Deadly Jellyfish Nemopilema nomurai Toxins.

Jellyfish stings threaten people's health and even life in coastal areas worldwide. Nemopilema nomurai is one of the most dangerous jellyfish in the East Asian Marginal Seas, which not only stings hundreds of thousands of people every year but also is assumed to be responsible for most deaths by jellyfish stings in China. However, there is no effective first-aid drug, such as antivenoms, for the treatment of severe stings by N. nomurai to date. In this study, we prepared a N. nomurai antiserum from rabbits using inactivated N. nomurai toxins (NnTXs) and isolated the IgG type of antivenom (IgG-AntiNnTXs) from the antiserum. Subsequently, IgG-AntiNnTXs were refined with multiple optimizations to remove Fc fragments. Finally, the F(ab')2 type of antivenom (F(ab')2-AntiNnTXs) was purified using Superdex 200 and protein A columns. The neutralization efficacy of both types of antivenom was analyzed in vitro and in vivo, and the results showed that both IgG and F(ab')2 types of antivenom have some neutralization effect on the metalloproteinase activity of NnTXs in vitro and could also decrease the mortality of mice in the first 4 h after injection. This study provides some useful information for the development of an effective antivenom for N. nomurai stings in the future.

Preparation and Neutralization Efficacy of Novel Jellyfish Antivenoms against Cyanea nozakii Toxins.

Jellyfish stings are a common issue globally, particularly in coastal areas in the summer. Victims can suffer pain, itching, swelling, shock, and even death. Usually, hot water, vinegar, or alumen is used to treat the normal symptoms of a jellyfish sting. However, a specific antivenom may be an effective treatment to deal with severe jellyfish stings. Cyanea nozakii often reach a diameter of 60 cm and are responsible for hundreds of thousands of stings per year in coastal Chinese waters. However, there has been no specific C. nozakii antivenom until now, and so the development of this antivenom is very important. Herein, we collected C. nozakii antisera from tentacle extract venom immunized rabbits and purified the immunoglobulin (IgG) fraction antivenom (AntiCnTXs). Subsequently, two complete procedures to produce a refined F(ab')2 type of antivenom (F(ab')2-AntiCnTXs) and Fab type of antivenom (Fab-AntiCnTXs) by multiple optimizations and purification were established. The neutralization efficacy of these three types of antivenoms was compared and analyzed in vitro and in vivo, and the results showed that all types of antibodies displayed some neutralization effect on the lethality of C. nozakii venom toxins, with the neutralization efficacy as follows: F(ab')2-AntiCnTXs ≥ AntiCnTXs > Fab-AntiCnTXs. This study describes the preparation of novel C. nozakii jellyfish antivenom preparations towards the goal of developing a new, effective treatment for jellyfish stings.

Functional Elucidation of Nemopilema nomurai and Cyanea nozakii Nematocyst Venoms' Lytic Activity Using Mass Spectrometry and Zymography.

BACKGROUND: Medusozoans utilize explosively discharging penetrant nematocysts to inject venom into prey. These venoms are composed of highly complex proteins and peptides with extensive bioactivities, as observed in vitro. Diverse enzymatic toxins have been putatively identified in the venom of jellyfish, Nemopilema nomurai and Cyanea nozakii, through examination of their proteomes and transcriptomes. However, functional examination of putative enzymatic components identified in proteomic approaches to elucidate potential bioactivities is critically needed. METHODS: In this study, enzymatic toxins were functionally identified using a combined approach consisting of in gel zymography and liquid chromatography tandem mass spectrometry (LC-MS/MS). The potential roles of metalloproteinases and lipases in hemolytic activity were explored using specific inhibitors. RESULTS: Zymography indicated that nematocyst venom possessed protease-, lipase- and hyaluronidase-class activities. Further, proteomic approaches using LC-MS/MS indicated sequence homology of proteolytic bands observed in zymography to extant zinc metalloproteinase-disintegrins and astacin metalloproteinases. Moreover, pre-incubation of the metalloproteinase inhibitor batimastat with N. nomurai nematocyst venom resulted in an approximate 62% reduction of hemolysis compared to venom exposed sheep erythrocytes, suggesting that metalloproteinases contribute to hemolytic activity. Additionally, species within the molecular mass range of 14-18 kDa exhibited both egg yolk and erythrocyte lytic activities in gel overlay assays. CONCLUSION: For the first time, our findings demonstrate the contribution of jellyfish venom metalloproteinase and suggest the involvement of lipase species to hemolytic activity. Investigations of this relationship will facilitate a better understanding of the constituents and toxicity of jellyfish venom.

Prophylactic treatment of jellyfish stings--a randomised trial.

BACKGROUND: Contact with jellyfish can cause skin irritation and manifestations. We wanted to investigate the prophylactic effect of a sun cream containing an inhibitor against jellyfish stings. MATERIAL AND METHOD: We recruited 38 persons who were randomised such that each received two of three possible treatments, one on each underarm. Prophylactic treatment with sun cream containing jellyfish sting inhibitor, ordinary sun cream, and no cream. Their underarms were exposed to wet jellyfish tentacles in a watchglass. The following were recorded: time before pain, skin changes after four minutes, and pain intensity after 10 minutes, registered on a VAS scale. RESULTS: Thirteen of 25 subjects who had the sun cream with jellyfish sting inhibitor did not register any pain after 4 minutes' exposure, compared with two of 25 and two of 26 who had received pre-treatment with ordinary sun cream (p = 0.32) and no pre-treatment (p < 0.001), respectively. On average, subjects who had received prophylactic treatment with sun cream containing jellyfish sting inhibitor recorded a lower VAS score for pain/discomfort after 10 minutes. The difference was 10.6 mm (95 % CI 3.1-17.9) compared with ordinary sun cream and 14.2 mm (95 % CI 6.9-21.5) compared with no pre-treatment. A smaller number of subjects were found to have underarms with inflamed skin when prophylactic cream containing jellyfish sting inhibitor was used (6 of 25) than when ordinary sun cream was used (11 of 25) or no pre-treatment (12 of 26). There were no statistically significant differences between ordinary sun cream and no pre-treatment for any of the three outcomes. INTERPRETATION: Prophylactic treatment with jellyfish sting inhibitor reduces the risk of subjects developing symptoms after exposure to jellyfish tentacles.

Evidence-based treatment of jellyfish stings in North America and Hawaii.

We performed a systematic review of the evidence supporting various treatments for envenomation by jellyfish (cnidarian) and related organisms in North America and Hawaii. Our review produced 19 pertinent primary articles. Current research demonstrates variable response to treatment, often with conflicting results according to species studied, which contributes to considerable confusion about what treatment is warranted and efficacious. Our review suggests that vinegar causes pain exacerbation or nematocyst discharge in the majority of species. Hot water and topical lidocaine appear more widely beneficial in improving pain symptoms and are preferentially recommended. Unfortunately, they may be difficult to obtain at the site of envenomation, such as the beach or diving sites. In these instances, removing the nematocysts and washing the area with saltwater may be considered. If the envenomation is thought to be due to the bluebottle (Physalia), vinegar may be beneficial.

Evaluation of the effects of various chemicals on discharge of and pain caused by jellyfish nematocysts.

Jellyfish tentacles in contact with human skin can produce pain swelling and redness. The pain is due to discharge of jellyfish nematocysts and associated toxins and discharge can be caused by a variety of mechanical and chemical stimuli. A series of tests were carried out with chemicals traditionally used to treat jellyfish stings e.g. acetic acid ammonia meat tenderizer baking soda and urea to determine if these chemicals stimulated or inhibited nematocyst discharge and if they brought relief to testers who were exposed to jellyfish tentacles. Chrysaora quinquecirrha (sea nettle) Chiropsalmus quadrumanus (sea wasp) and Physalia physalis (Portuguese man-of-war) were used in the study. It was found that many of the chemicals traditionally used to treat jellyfish stings stimulated nematocyst discharge and did not relieve the pain. However there was immediate relief when a common anesthetic lidocaine was sprayed on the skin of testers in contact with jellyfish tentacles. Initial exposure of tentacle suspensions to lidocaine prevented the nematocyst discharge by subsequent exposure to acetic acid ethanol ammonia or bromelain. Thus lidocaine in addition to acting as an anesthetic on skin in contact with jellyfish tentacles inhibited nematocyst discharge possibly by blocking sodium and/or calcium channels of the nematocytes.

Irukandji syndrome case series from Australia's Tropical Northern Territory.

STUDY OBJECTIVE: We describe Irukandji syndrome (a painful hypercatecholaminergic condition caused by jellyfish envenoming) in Australia's Northern Territory. METHODS: We collected prospectively a standardized data set on patients presenting to health facilities in the Northern Territory. Additional cases were identified retrospectively. Data collected included demographic, geographic, seasonal, and environmental features, as well as sting details, clinical manifestations, investigations, management, and outcomes. RESULTS: From 1990 to 2007, Irukandji syndrome affected 87 people. Age ranged from 1 to 51 years (64% male victims; 41% children [63% indigenous]). Activities associated with stings included snorkeling or scuba diving (35%) and swimming (29%). Stings commonly occurred in water greater than 2 m deep (63%), with fine weather (73%) and still or light breeze (70%). Seasonal variation was bimodal; peaks in May and October corresponded to prevailing offshore winds in the Darwin and Gove areas, respectively. Pain was severe (65%), with rapid onset (<30 minutes in 79%). Sting lesions (visible in 63%) were mild, and nematocysts (detected in 7 cases) had variable morphology. Systemic features were common, including hypertension and ECG abnormalities. Severe complications included troponin-level increases (2 cases) and cardiomyopathy with ventricular tachycardia (1 case), but no fatalities. Management included vinegar as first aid (66%), parenteral opioids (70%) (range 2 to 82.5 mg morphine equivalents in adults), and magnesium sulfate (3 cases). Hospital admission (49%) and aeromedical retrieval (16%) were commonplace. CONCLUSION: Irukandji syndrome in the Northern Territory was clinically consistent with previous studies but had distinct seasonal, geographic, and environmental features. Indigenous children in remote coastal communities are at risk, and there is room for improvement in prevention and management.

An examination of the cardiovascular effects of an 'Irukandji' jellyfish, Alatina nr mordens.

Irukandji syndrome is usually characterized by delayed severe abdominal, back and chest pain associated with autonomic effects including diaphoresis, hypertension and, in severe cases, myocardial injury and pulmonary oedema. It is most often associated with envenoming by the jellyfish Carukia barnesi, but a number of other jellyfish, including Alatina mordens, are now known to produce Irukandji syndrome. In the present study, nematocyst-derived venom from A. nr mordens (150-250 microg/kg, i.v.) produced a long-lasting pressor effect in anaesthetised rats. This pressor response (250 microg/kg, i.v.) was significantly inhibited by prior administration of the alpha-adrenoceptor antagonist prazosin (200 microg/kg, i.v.) but not by CSL box jellyfish antivenom (300 U/kg, i.v.). A. nr mordens venom 250 microg/kg (i.v.) caused marked increases in plasma adrenaline and noradrenaline concentrations following administration in anaesthetised rats. The venom did not contain appreciable amounts of either adrenaline or noradrenaline. A. nr mordens venom (25 microg/ml) produced a contractile response in rat electrically stimulated vas deferens which was markedly reduced in tissues pre-treated with reserpine (0.1mM) or guanethidine (0.1mM). Sodium dodecyl sulphate (SDS)-PAGE analysis showed that A. nr mordens venom is comprised of multiple protein bands ranging from 10 to 200 kDa. Western blot analysis using CSL box jellyfish antivenom indicated several antigenic proteins in A. nr mordens venom, however, it did not detect all proteins present in the venom. This study characterizes the in vitro and in vivo effects of A. nr mordens venom and indicates that the cardiovascular effects are at least partially mediated by endogenous catecholamine release.

A randomized, controlled field trial for the prevention of jellyfish stings with a topical sting inhibitor.

BACKGROUND: Jellyfish stings are a common occurrence among ocean goers worldwide with an estimated 150 million envenomations annually. Fatalities and hospitalizations occur annually, particularly in the Indo-Pacific regions. A new topical jellyfish sting inhibitor based on the mucous coating of the clown fish prevents 85% of jellyfish stings in laboratory settings. The field effectiveness is unknown. The objective is to evaluate the field efficacy of the jellyfish sting inhibitor, Safe Sea. METHODS: A double-blind, randomized, placebo-controlled trial occurred at the Dry Tortugas National Park, FL, USA and Sapodilla Cayes, Belize. Participants were healthy volunteers planning to snorkel for 30 to 45 minutes. Ten minutes prior to swimming, each participant was directly observed applying a blinded sample of Safe Sea (Nidaria Technology Ltd, Jordan Valley, Israel) to one side of their body and a blinded sample of Coppertone (Schering-Plough, Kenilworth, NJ, USA) to the contralateral side as placebo control. Masked 26 g samples of both Safe Sea SPF15 and Coppertone SPF15 were provided in identical containers to achieve 2 mg/cm(2) coverage. Sides were randomly chosen by participants. The incidence of jellyfish stings was the main outcome measure. This was assessed by participant interview and examination as subjects exited the water. RESULTS: A total of 82 observed water exposures occurred. Thirteen jellyfish stings occurred during the study period for a 16% incidence. Eleven jellyfish stings occurred with placebo, two with the sting inhibitor, resulting in a relative risk reduction of 82% (95% confidence interval: 21%-96%; p= 0.02). No seabather's eruption or side effects occurred. CONCLUSIONS: Safe Sea is a topical barrier cream effective at preventing >80% jellyfish stings under real-world conditions.

A comparison of the toxinological characteristics of two Cassiopea and Aurelia species.

A comparison of the toxinological properties of nematocyst venoms from Old and New World Cassiopea and Aurelia species was undertaken. The cnidom of venomous Cassiopea andromeda (Ca) and Aurelia (Aa(RS)) from the Red Sea was identical to that of nonvenomous Bahamian Cassiopea xamancha (Cx) and Chesapeake Bay Aurelia aurita (Aa(CB)), respectively. A clean nematocyst preparation of Ca and both Aurelias could be obtained but algal particles could not be separated completely from the Cx nematocysts. Further purification of all four nematocyst preparations showed significant differences in the action of their protein. Only the Cassiopea had coexisting dermonecrotic and vasopermeability producing properties and Ca's hemolytic activity was associated with mouse lethality. The protein, hemolysin and phospholipase gel filtration eluant curves of Ca venom were similar. Venomous Aa(RS) actively stung lips and contained more potent mouse lethal, demonecrotic, vasopermeability plus hemolytic factors than Aa(CB). Cross reactivity of convalescent human serum obtained from patients stung by Ca and venomous Cx collected in Central America occurred. This was also observed between sera of bathers stung by Aa(RS) and stinging Aurelia which appeared in Florida during the recent El Niño year. IgG was stimulated by several nematocyst proteins since many venom subfractions tested positive at high titers against convalescent sera. T-cell proliferation of mice primed with either Aurelia venom was positive against the homologous preparation with cross reactivity to the heterologous venom. Crude venoms of both Red Sea jellyfish metabolically stimulated cultured human hepatocytes more than their New World counterparts. This data shows that considerable similarities and differences exist in the venoms of these Old and New World Cassiopea and Aurelia medusae with the Eastern species being more potent.

Toxicity of sea nettle toxin to human hepatocytes and the protective effects of phosphorylating and alkylating agents.

The sea nettle jellyfish toxin (SNTX), which contains several polypeptides, was highly toxic to human hepatocytes. The Cytosensor microphysiometer was used continuously to monitor cell media acidification rate as an index of cellular metabolic activity. Cells exposed to > 1 microg SNTX protein/ml media exhibited a transient increase in metabolic activity, followed by a sharp decrease and cell death within minutes. The kinetics of the transient increase and subsequent decline increased with higher concentrations of SNTX. The biphasic and time-dependent response of hepatocytes to SNTX suggests that more than one mechanism may be involved in the toxicity of its different polypeptides. SNTX-induced cytotoxicity of hepatocytes was reduced by the presence of high titer antibodies against a heterologous jellyfish. Phenobarbital-induced cells became more vulnerable to SNTX, suggesting that some toxin component(s) require(s) bioactivation. Short-term exposure (1-2 h) to 10 microg/ml of the calcium ionophore calcimycin, or the non-selective monovalent cation ionophore gramicidin, had no effect on metabolic activity. However, 165 microg/ml gramicidin or 53 microg/ml calcimycin produced slight transient activation followed by steady decline in metabolic activity, while 20 h exposure to either ionophore produced total cell death. Exposure to even a 10-fold lower concentration of either ionophore killed 88% and 75%, respectively. This contrasts with the toxicity of SNTX which is detectable in minutes with as little as 3 microg/ml. Since pre-exposure to the organophosphate anticholinesterases VX and paraoxon, or the chemotherapeutic alkylating agents cyclophosphamide and mechlorethamine reduced the cytotoxic effects of SNTX, it suggests that phosphorylation or alkylation of cell protein(s) interferes with SNTX toxicity.

The effects of antivenom and verapamil on the haemodynamic actions of Chironex fleckeri (box jellyfish) venom.

The efficacy of antivenom and verapamil against Chironex fleckeri (box jellyfish) venom was investigated in monitored mechanically ventilated piglets. Chironex fleckeri tentacle extract alone, a mixture of tentacle extract with antivenom, and verapamil before tentacle extract were administered intravenously to groups of animals. Tentacle extract caused severe systemic hypotension, cardiac dysrrhythmias, pulmonary hypertension, haemolysis and hyperkalaemia. These effects were prevented by pre-incubation of tentacle extract with antivenom. Verapamil did not prevent any effect of venom, exacerbated cardiovascular collapse and increased mortality. We conclude that antivenom neutralizes the cardiovascular, haemolytic and hyperkalaemic effects of box jellyfish venom. Verapamil does not prevent any of these effects and is contra-indicated for treatment of envenomation.

Antivenom use in Australia. Premedication, adverse reactions and the use of venom detection kits.

OBJECTIVES: To analyse reports of antivenom use and sequelae in Australia from July 1 1989 to June 30 1990. The value of snake venom detection kits (VDKs) was also analysed. METHODS: Information was obtained from antivenom usage reports returned to the Commonwealth Serum Laboratories and from personal letters sent to those reporting doctors. Information on VDKs was obtained from antivenom usage reports or from questionnaires packaged with VDKs. RESULTS: Reported antivenoms used were: red-back spider, 258 cases; funnel-web spider, 3 cases; stonefish, 26 cases; box jellyfish, 6 cases; snake, 91 cases. Immediate reactions followed administration of red-back spider antivenom in only two patients and snake antivenoms in four patients. Delayed reactions (serum sickness) followed use of red-back spider antivenom in three patients, stonefish antivenom in two, and snake antivenoms in three. No reaction was life-threatening. Premedication was used in the majority of red-back spider bites and in 66 of 86 snake bites. Oral corticosteroids were given prophylactically after some uses of snake antivenom to prevent serum sickness. VDKs were used in 181 cases of snake bite and were reported as being useful in selecting appropriate snake antivenom in 31% of cases of antivenom use. (Only 10 of these 181 were also reported on the antivenom usage report.) Appropriate first aid was given in 61% of cases. There were 50% fewer snake bites reported than 10 years ago. CONCLUSIONS: Antivenoms in Australia are well tolerated with few immediate or delayed reactions. The use of premedication and prophylactic oral corticosteroids for four to five days after antivenom administration may be responsible for this low reaction rate. VDK results help select the appropriate antivenom; however, in some cases positive results were obtained from urine samples from patients with no symptoms of envenomation.

Neutralization of toxic effects of different crude jellyfish venoms by an extract of Ipomoea pes-caprae (L.) R. Br.

An extract (IPA) of the plant Ipomoea pes-caprae (L.) R. Br., previously shown to be clinically effective toward dermatitis caused by venomous jellyfishes, was studied as to its ability to neutralize toxic activities of jellyfish venoms. Different venoms exhibited different degrees of activity. When IPA was incubated with active venoms, it inhibited the actions of all jellyfish venoms tested, with IC50 values in the range of 0.3-0.8 mgIPA/mg venom for proteolytic action, and with about 10 times lower IC50 values for the neutralization of haemolytic action. These activities of IPA support the previously reported effectiveness in the treatment of dermatitis caused by jellyfish sting.

Pathophysiology of sea nettle (Chrysaora quinquecirrha), envenomation in a rat model and the effects of hyperbaric oxygen and verapamil treatment.

The effect of lethal sea nettle envenomation on the morphology and blood flow in various rat organs was characterized and the influence of two antidotes (hyperbaric oxygen and verapamil) was compared. Either antidote slightly prolonged survival, but the protective effects were not statistically significant. The venom caused no histologic alterations in brain, heart, or lungs but induced hepatic and renal necrosis. Hepatocytes in mid-zonal regions and renal tubular epithelium were the cell types predominantly affected. Hyperbaric oxygen and verapamil did not decrease the hepatic injury. The venom did not influence central hemodynamics until preterminally and it diminished blood flow to brain, but not to liver or kidney. Hyperbaric oxygenation protected against venom-induced decreases in blood flow to the brain. These results add toxic hepatic and renal necrosis and cerebral ischemia to the pathophysiology of envenomation in this model.

Pharmacological effects of various venoms on cutaneous capillary leakage.

Studies to counteract the cutaneous vasopermeability actions of a wasp (Vespa orientalis), an anemone (Bolocera tuediae) and three jellyfish (Chironex fleckeri, Chrysaora quinquecirrha and Physalia physalis) venoms were conducted by using various pharmacological antagonists. Piripost (a leukotriene inhibitor) reduced vasopermeability if administered 5 min prior to challenge with the jellyfish venoms. Methysergide counteracted the vasopermeability of three of four coelenterate venoms, whereas indomethacin was effective against capillary leakage induced by Chironex venom. These studies indicate that anti-dermonecrotic therapy against various venoms will have to be species-specific.

Partial purification of Chironex fleckeri (sea wasp) venom by immunochromatography with antivenom.

Chironex fleckeri crude venom was partially purified using immobilized commercially available ovoid antivenom. The antibody preparation reacted with lethal, hemolytic, dermonecrotic and mouse writhing (pain) factors in the crude venom. The lethal activity was purified five fold, while the specific eluate contained lower quantities of hemolytic, dermonecrotic and mouse writhing activities than did the crude venom.

Platelet aggregation caused by a partially purified jellyfish toxin from Carybdea rastonii.

A partially purified toxin (pCrTX) was obtained from the tentacles of the jellyfish, Carybdea rastonii. When pCrTX (3 X 10(-8) - 3 X 10(-7) g/ml) was added to citrated platelet-rich plasma, aggregation was produced in a concentration-dependent manner. Scanning electron microscopic examination revealed that both pCrTX and collagen produced aggregates of platelets possessing many pseudopods. The concentration which produced 50% aggregation for pCrTX was 1.8 X 10(-7) g/ml, as compared to 2.3 X 10(-6) g/ml for collagen. The pCrTX-induced aggregation was only slightly inhibited by indomethacin and quinacrine in concentrations sufficient to inhibit arachidonic acid- and collagen-induced aggregation. pCrTX was less active in washed platelets suspended in Ca2+ free medium, whereas the pCrTX-induced aggregation was significantly augmented in the presence of Ca2+. The augmentation of aggregation by Ca2+ was only slightly attenuated by pretreatment with 100 microM verapamil. pCrTX significantly increased the concentration of cytoplasmic free Ca2+ ([Ca2+]i) and depolarized the platelet membrane in concentrations that produced aggregation. The increase in [Ca2+]i caused by pCrTX was little affected by verapamil. The depolarization by pCrTX was unchanged in the presence or absence of Ca2+, or by sodium or potassium transport inhibitors. The movement of 22Na+ into platelets was significantly increased by pCrTX. This increase in the movement of 22N+ into platelets was unaffected by tetrodotoxin. On the other hand, pCrTX-induced aggregation, depolarization and the increase in [Ca2+]i were all significantly attenuated in low Na+ medium. These results suggest that pCrTX causes a massive depolarization by increasing cation permeability indiscriminately and this generalized depolarization permits an inward movement of calcium down an electrochemical gradient which, in turn triggers platelet aggregation.

Response of the box-jellyfish (Chironex fleckeri) cardiotoxin to intravenous administration of verapamil.

Verapamil, a calcium antagonist, has been shown to be effective in delaying death in mice after intravenous challenge with box-jellyfish (Chironex fleckeri) venom. Death from a challenge of up to three mouse-lethal doses of venom was significantly delayed after the prior intravenous administration of verapamil (P = 0.003). A smaller dose of verapamil could "rescue" mice previously envenomed with 1.25 mouse-lethal doses (P = 0.0001). These data suggest that verapamil injections should be added to the first-aid procedures performed on the beach for victims of box-jellyfish stings.