Dietary sequestration of defensive steroids in nuchal glands of the Asian snake Rhabdophis tigrinus

The Asian snake Rhabdophis tigrinus possesses specialized defensive glands on its neck that contain steroidal toxins known as bufadienolides. We hypothesized that R. tigrinus does not synthesize these defensive steroids but instead sequesters the toxins from toads it consumes as prey. To test this hypothesis, we conducted chemical analyses on the glandular fluid from snakes collected in toad-free and toad-present localities. We also performed feeding experiments in which hatchling R. tigrinus were reared on controlled diets that either included or lacked toads. We demonstrate that the cardiotonic steroids in the nuchal glands of R. tigrinus are obtained from dietary toads. We further show that mothers containing high levels of bufadienolides can provision their offspring with toxins. Hatchlings had bufadienolides in their nuchal glands only if they were fed toads or were born to a dam with high concentrations of these compounds. Because geographic patterns in the availability of toxic prey are reflected in the chemical composition of the glandular fluid, snakes in toad-free regions are left undefended by steroidal toxins. Our findings confirm that the sequestration of dietary toxins underlies geographic variation in antipredatory behavior in this species and provide a unique example of sequestered defensive compounds in a specialized vertebrate structure.     

Clinical trial of an F(ab')2 polyvalent equine antivenom for African snake bites in Benin

We report the results of a trial designed to measure the safety and efficacy of African Antivipmyn, a new freeze-dried polyvalent equine F(ab')(2)-based antivenom. We tested 289 envenomations. After treatment, 19% of treated patients had undesirable events, all benign. A possible adverse effect was attributed to this antivenom in 11% of the patients. Bleeding was observed in 48% of the patients; it stopped within 2 hours after treatment with antivenom in 60% of the patients. Blood incoagulability was observed in 80% of the patients. Restoration of coagulation was attained within 4 hours in 60% of the patients. Nine patients died; 6 arrived at the hospital in the final stage of complications and 5 arrived at the hospital more than 60 hours after the bite. The value of blood coagulation tests in diagnosis of envenomation and bleeding as an indicator of renewal of treatment are emphasized.     

Biological properties of the venom of the red-necked keel-back snake (Rhabdophis subminiatus)

Studies in mice on the biological activity of the venom of the red-necked keel-back snake, Rhabdophis subminiatus, showed that the venom contained a potent Factor X activator and had intense defibrinogenating activity; the overall proteolytic activity of the venom was low, and this correlated well with its negligible fibrinogenolytic and fibrinolytic activities. Only one antivenom tested was shown to have weak neutralizing activity against the venom in mice. This species of snake has recently been added to the schedule of the Dangerous Wild Animals Act, 1976.     

Severe coagulopathy after a bite from a 'harmless' snake (Rhabdophis subminiatus)

European physicians may occasionally be confronted with cases of snake venom poisoning. The increasing interest in snakes as pets has unfortunately resulted in the importation of a number of venomous snakes. We here report the third known case in the medical literature of a patient who was exposed to severe coagulopathy after being bitten by a 'harmless' ringsnake (Rhabdophis subminiatus). The prolonged bleeding diathesis and the complete depletion of fibrinogen observed in our patient are consistent with the presence of a factor X activator in the venom, as has been described in a murine model. In agreement with the two previous case reports, we found no evidence of severe organ damage, despite active intravascular coagulation.     

Red-back spider (Latrodectus hasselti) antivenom prevents the toxicity of widow spider venoms

STUDY OBJECTIVES: Widow spiders of the genus Latrodectus are found worldwide and produce similar clinical envenomation syndromes. In Australia, red-back spider antivenom (RBS-AV) is effective therapy for Latrodectus hasselti envenomation and it has been reported to reverse envenomation by other widow spiders. This study assessed the efficacy of RBS-AV in preventing in vitro and in vivo toxicity of widow spider venoms of North America and Europe. METHODS: The binding of RBS-AV to alpha-latrotoxin and Latrodectus venoms (Latrodectus spp mactans, hesperus, lugubris, tredecimguttatus, hasselti) was assayed using Western blotting. Prevention of in vitro toxicity to alpha-latrotoxin and the same venoms was tested by pretreating an isolated chick biventer cervicis nerve-muscle preparation with RBS-AV. Prevention of in vivo toxicity was determined by a lethality study in male Balb/c mice (2.5 to 5x median lethal dose [LD50]) or alpha-latrotoxin (10x LD50) preincubated with antivenom or without RBS-AV (control). RESULTS: In Western blots, RBS-AV bound to alpha-latrotoxin and similar widow spider proteins in all venoms tested, indicating antigenic similarity with proteins found in RBS venom. Antivenom prevented the typical in vitro muscle contracture and loss of twitch tension seen with alpha-latrotoxin and the venoms tested. Control mice rapidly developed signs of envenomation, but mice treated with RBS-AV remained free of signs of envenomation. CONCLUSION: RBS-AV prevented both in vitro and in vivo toxicity from Latrodectus venoms and alpha-latrotoxin in mice. These data suggest that RBS-AV may be clinically effective in the treatment of envenomation resulting from the bite of other widow spiders.     

Immediate and delayed allergic reactions to Crotalidae polyvalent immune Fab (ovine) antivenom

Allergic reactions are the most commonly reported adverse events after administration of antivenoms. Conventional horse serum-based crotalid antivenom used in the United States (Antivenin [Crotalidae] polyvalent) can lead to both immediate and delayed hypersensitivity reactions. Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV) has recently been approved for use in the United States. Experience from premarketing trials of this product and in the administration of other types of Fab, such as in digoxin poisoning, has demonstrated these fragments to be safe and effective, with a low incidence of sequella; however, allergic reactions can occur when any animal-protein derivatives are administered to human subjects. We report in detail the nature and course of allergic reactions that occurred in 4 patients treated with FabAV. Cases of anaphylaxis, acute urticaria, angioedema, and delayed serum sickness are described. All reactions were easily treated with some combination of antihistamines, epinephrine, and steroids, with prompt resolution of signs and symptoms enabling further dosing of antivenom as required. Several of these cases may have resulted from batches of antivenom contaminated with Fc fragments. The overall incidence of immediate and delayed allergic reactions to this product appears so far to be lower than that reported with conventional whole-immunoglobulin G (IgG) antivenom, but postmarketing surveillance is warranted.     

Crotalidae polyvalent immune Fab (ovine) antivenom is efficacious for envenomations by Southern Pacific rattlesnakes (Crotalus helleri)

STUDY OBJECTIVE: Southern Pacific rattlesnake (Crotalus helleri ) venom is not 1 of the 4 venoms used to produce Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV). There is currently no published clinical experience regarding the efficacy of this new antivenom for confirmed C helleri envenomation, and animal data suggest greatly diminished efficacy. We assessed the efficacy of FabAV for patients with confirmed C helleri envenomation. METHODS: We conducted a prospective observational study of 23 consecutive rattlesnake envenomations that were treated with FabAV at our center. Patients were excluded if the species of snake could not be confirmed, if FabAV antivenom was not given, or if Antivenin (Crotalidae) polyvalent (equine) was given. We collected serial physical examination and laboratory data over a 24-hour period to serially evaluate the severity score and performed follow-up to evaluate delayed reactions. RESULTS: There were 15 patients who received FabAV and had the species of rattlesnake confirmed (9 C helleri, 4 C scutulatus scutulatus, 1 C mitchellii pyrrhus, 1 C ruber ruber ). C helleri envenomations demonstrated similar improvement in serial snakebite severity scores to those of other species. Three patients treated with scheduled dosing had recurrence of progressive swelling (2 C helleri and 1 C mitchellii pyrrhus ) during the 24-hour study period. CONCLUSION: We observed similar improvement in FabAV-treated patients with C helleri envenomation compared with those of other species and conclude that this treatment in standard doses appears efficacious for bites by this species. Progressive swelling may recur despite scheduled dosing.     

西方马脑炎疫苗研究进展

西方马脑炎是由西方马脑炎病毒感染引起的,经蚊虫传播的急性人兽共患传染病,可引起严重的脑损伤。此外,西方马脑炎病毒也被视为潜在的生物战剂(生物恐怖剂)。目前西方马脑炎尚无获批疫苗和有效药物,研发有效疫苗用于相关人群的免疫接种很有必要。传统灭活病毒疫苗具有良好的免疫保护效能,但为了避免其制备过程中病毒培养对昂贵生物安全3级实验室的依赖以及操作者潜在感染的风险,研究者采用新的生物学技术研发西方马脑炎亚单位疫苗、DNA疫苗、嵌合体疫苗和重组活载体疫苗等新型疫苗。本文对疫苗的研究进展进行综述。     

Low incidence of early reactions to horse-derived F(ab')(2) antivenom for snakebites in Thailand

Although antivenom is effective for systemic effects of snake envenomation, the possibility of fatal hypersensitivity reactions must be considered. The risks vary greatly among different products and cannot be excluded by negative skin test. However, clinical outcomes of patients with positive skin test remain to be determined. The medical records of 254 cases receiving antivenoms during 1997-2006 were reviewed. Most were for green pit vipers (84%) and cobras (13%). Early reactions occurred in 9 (3.5%) including 3 (1.2%) with hypotension. The incidence was more common after cobra than viper antivenoms (12.5% vs. 2.3%, respectively, p=0.016). Skin test was negative in 7/7 tested cases. All reactions resolved. Antivenoms were re-administered in five without recurrent reactions. Overall, skin test was positive in 10/211 (4.7%). Five of them underwent desensitization. Antivenom can be given in all 10 without reactions. In conclusion, the incidence of early reactions to antivenoms was low in Thailand and skin test is not helpful at all in predicting this adverse reaction.     

Surveillance of the clinical use of mamushi (Gloydius blomhoffii) antivenom in tertiary care centers in Japan

We report the results of the first large-scale questionnaire surveillance on the clinical use of pit viper antivenom in tertiary care centers in Japan. The questionnaire surveillance was conducted over a period of 3 years (April 2006 to March 2009). Completed questionnaires were received from the tertiary care centers of 108 (49.3%) medical institutions. In that period, 574 cases of pit viper bites, including 2 severe cases, were reported. Antivenom was administered in 44% of the cases of pit viper bites, and of these cases, 2.4% had adverse reactions but no severe symptoms. Approximately half of the clinicians indicated that antivenom was effective. Antivenom was recognized to be safe; however, the remarkable finding was that although the severity of treated cases was unclear, some clinicians reported using cepharanthine as the first choice of treatment for pit viper bites.     

Standardization of regional reference for mamushi (Gloydius blomhoffii) antivenom in Japan, Korea, and China

The mamushi (Gloydius blomhoffii) snakes that inhabit Japan, Korea, and China produce venoms with similar serological characters to each other. Individual domestic standard mamushi antivenoms have been used for national quality control (potency testing) of mamushi antivenom products in these countries, because of the lack of an international standard material authorized by the World Health Organization. This precludes comparison of the results of product potency testing among countries. We established a regional reference antivenom for these three Asian countries. This collaborative study indicated that the regional reference mamushi antivenom has an anti-lethal titer of 33,000 U/vial and anti-hemorrhagic titer of 36,000 U/vial. This reference can be used routinely for quality control, including national control of mamushi antivenom products.     

Pseudotyped viruses permit rapid detection of neutralizing antibodies in human and equine serum against Venezuelan equine encephalitis virus.

Virus envelope proteins are the primary targets of neutralizing antibody responses. The epitopes recognized differ sufficiently between virus subtypes and species to distinguish viruses and provide an important basis for disease diagnosis. Venezuelan equine encephalitis virus (VEEV) causes acute febrile illness in humans and has high mortality in equines. The most specific detection methods for serum antibodies use live virus in neutralization assays or in blocking enzyme linked immunosorbent assays. However, work with Venezuelan equine encephalitis virus requires biosafety level 3 containment and select agent security in the United States. We report two new assays for detection of Venezuelan equine encephalitis virus neutralizing antibody responses, based on virus pseudotypes. The first provides detection by marker gene expression after 20 hours and is particularly suited for high-throughput screening; the second uses a new, rapid virus entry assay to give readouts within 1 hour. Both assays are safe, sensitive, and in general recapitulate neutralizing antibody titers obtained by conventional plaque reduction assays. Each is suitable as a rapid primary screen for detection of neutralizing antibodies against Venezuelan equine encephalitis virus.     

Cross neutralization of common Southeast Asian viperid venoms by a Thai polyvalent snake antivenom (Hemato Polyvalent Snake Antivenom)

Snake envenomation is a serious public health threat in many rural areas of Asia and Africa. Antivenom has hitherto been the definite treatment for snake envenomation. Owing to a lack of local production of specific antivenom, most countries in these regions fully depend on foreign supplies of antivenoms. Often, the effectiveness of the imported antivenoms against local medically important species has not been validated. This study aimed to assess cross-neutralizing capacity of a recently developed polyvalent antivenom, Hemato Polyvalent Snake Antivenom (HPAV), against venoms of a common viper and some pit vipers from Southeast Asia. Neutralisation assays showed that HPAV was able to effectively neutralize lethality of the common Southeast Asian viperid venoms examined (Calloselasma, Crytelytrops, Popeia, and Daboia sp.) except for Tropidolaemus wagleri venom. HPAV also effectively neutralized the procoagulant and hemorrhagic activities of all the venoms examined, corroboratively supporting the capability of HPAV in neutralizing viperid venoms which are principally hematoxic. The study also indicated that HPAV fully prevented the occurrence of hematuria and proteinuria in mice envenomed with Thai Daboia siamensis venom but was only partially effective against venoms of Myanmar D. siamensis. Thus, HPAV appears to be useful against its homologous venoms and venoms from Southeast Asian viperids including several medically important pit vipers belonging to the Trimeresurus complex. Nevertheless, the effectiveness of HPAV as a paraspecific antivenom for treatment of viperid envenomation in Southeast Asian region requires further assessment from future clinical trials     

Truncated equine LH beta and asparagine(56)-deglycosylated equine LH alpha combine to produce a potent FSH antagonist

Hybrid hormone preparations were prepared by combining intact and Asn(56)-deglycosylated (N(56)dg) equine (e) LH or FSH alpha subunit preparations with truncated, des(121-149)eLH beta (eLH beta t), immunopurified, intact eLH beta or equine chorionic gonadotropin beta (eCG beta) preparations, and eFSH beta. The LH receptor-binding potencies of N(56)dg-eLH alpha:eLH beta t and N(56)dg-eFSH alpha:eLH beta t hybrids were equivalent to that of eLH; however, both N(56)dg-alpha preparations were only 3-4% as active as eLH in the rat testis Leydig cell bioassay. In the granulosa cell FSH bioassay, eLH alpha:eLH beta t stimulated progesterone synthesis and induced aromatase activity, while N(56)dg-eLH alpha:eLH beta t was completely inactive at doses up to 5 microg. N(56)dg-eLH alpha:eLH beta t inhibited progesterone production and aromatase induction elicited by 0.3 ng eFSH or 2 ng human (h) FSH. The inhibitory activities of N(56)dg-eLH alpha:eLH beta and N(56)dg-eCG alpha:eLH beta t were only 10% that of N(56)dg-eLH alpha:eLH beta t. N(56)dg-eLH alpha:eCG beta did not inhibit progesterone synthesis stimulated by eFSH at all and appeared to further stimulate aromatase induction at the highest dose tested. Preincubation of N(56)dg-eLH alpha:eLH beta and N(56)dg-eLH alpha:eLH beta t for 72 h at 37 C resulted in no loss of FSH receptor-binding activity. Preincubation resulted in 50% loss of receptor-binding activity by the eFSH preparation due to subunit dissociation, while 88% of N(56)dg-eLH alpha:eFSH beta activity was lost following 72 h, 37 C preincubation. While alpha Asn(56) oligosaccharide had no effect on eLH beta hybrid stability, it did contribute to the stability of the eFSH heterodimer.     

Polycarbonate-polyether (PC-PE) flat sheet membrane: manufacture, structure and performance

Polycarbonate-polyether (PC-PE) polymer, prepared by solution or interfacial polymerization, was used to produce a dry PC-PE diffusion membrane by means of the phase inversion technique. The mechanical stabilities and diffusive permeabilities of chloride, phosphate and vitamin B12 were determined for this membrane. Additionally, close control over manufacturing parameters allowed the study of the effects of varied membrane thickness (at constant ultrafiltration rate) and varied ultrafiltration rate (at constant membrane thickness) on solute diffusive permeabilities. Both increased ultrafiltration rate and decreased thickness resulted in an increase in solute diffusive permeabilities.