ABSTRACT
The measurement of free venom with enzyme immunoassay in serum of patients with snake envenoming is used to confirm snake identification and to determine if sufficient antivenom has been given. Recent studies with Russell's viper (RV; Daboia russelii) envenoming have detected free venom post-antivenom despite recovery of coagulopathy. This raises the question as to whether this assay also measures venom-antivenom (VAV) complexes. In this study we developed an assay to measure VAV complexes and investigate the binding of venom and antivenom in vitro. The assay consisted of rabbit anti-snake venom IgG attached to a microplate which binds the venom component of VAV and anti-horse IgG antibodies conjugated to horseradish peroxidase to detect the antivenom portion of VAV. A known amount of venom or toxin was incubated with increasing antivenom concentrations and VAV was detected as absorbance at 450 nm and plotted against AV concentration. Pseudonaja textilis (brown snake), Notechis scutatus (tiger snake), Oxyuranus scutellatus (taipan), Tropidechis carinatus (rough-scaled snake), Pseudechis porphyriacus (red-bellied black snake) and D. russelii mixtures with appropriate antivenoms were assayed. Measured VAV initially increased with increasing antivenom concentration until it reached a maximum after which the VAV concentration decreased with further increasing antivenom concentrations. The VAV curves for two Australian snake venom-antivenom mixtures, Hoplocephalus stephensii and Ancanthophis antarcticus, had broad VAV peaks with two maxima. Two fractions isolated from N. scutatus venom and Russell's viper factor X activator toxin produced similar VAV curves to their whole venoms. The antivenom concentration for which the maximum VAV occurred was linearly related to the venom concentration, and this slope or ratio was consistent with that used to define the neutralisation units for Australian antivenoms. The maximal VAV point appears to represent the antivenom concentration where every venom molecule (toxin) is attached to at least one antivenom molecule (antibody) on average and may be a useful measure of antivenom efficacy. In vivo this would mean that for a defined antivenom concentration, venom components will be eliminated and are trapped in the central compartment.
Subject(s)
Antigen-Antibody Complex/immunology , Antivenins/immunology , Snake Venoms/immunology , Snakes/metabolism , Animals , Australia , Chromatography, High Pressure Liquid , Horses , Immunoenzyme Techniques , Immunoglobulin G/immunology , Linear Models , Models, Biological , Rabbits , Species SpecificityABSTRACT
CONTEXT: Mulga snakes (Pseudechis australis) are venomous snakes with a wide distribution in Australia. Objective. The objective of this study was to describe mulga snake envenoming and the response of envenoming to antivenom therapy. MATERIALS AND METHODS: Definite mulga bites, based on expert identification or venom-specific enzyme immunoassay, were recruited from the Australian Snakebite Project. Demographics, information about the bite, clinical effects, laboratory investigations and antivenom treatment are recorded for all patients. Blood samples are collected to measure the serum venom concentrations pre- and post-antivenom therapy using enzyme immunoassay. RESULTS: There were 17 patients with definite mulga snake bites. The median age was 37 years (6-70 years); 16 were male and six were snake handlers. Thirteen patients had systemic envenoming with non-specific systemic symptoms (11), anticoagulant coagulopathy (10), myotoxicity (7) and haemolysis (6). Antivenom was given to ten patients; the median dose was one vial (range, one-three vials). Three patients had systemic hypersensitivity reactions post-antivenom. Antivenom reversed the coagulopathy in all cases. Antivenom appeared to prevent myotoxicity in three patients with high venom concentrations, given antivenom within 2 h of the bite. Median peak venom concentration in 12 envenomed patients with samples was 29 ng/mL (range, 0.6-624 ng/mL). There was a good correlation between venom concentrations and the area under the curve of the creatine kinase for patients receiving antivenom after 2 h. Higher venom concentrations were also associated with coagulopathy and haemolysis. Venom was not detected after antivenom administration except in one patient who had a venom concentration of 8.3 ng/ml after one vial of antivenom, but immediate reversal of the coagulopathy. DISCUSSION: Mulga snake envenoming is characterised by myotoxicity, anticoagulant coagulopathy and haemolysis, and has a spectrum of toxicity that is venom dose dependant. This study supports a dose of one vial of antivenom, given as soon as a systemic envenoming is identified, rather than waiting for the development of myotoxicity.
Subject(s)
Antivenins/therapeutic use , Blood Coagulation Disorders/chemically induced , Elapid Venoms/poisoning , Muscle, Skeletal/drug effects , Muscular Diseases/chemically induced , Neurotoxins/poisoning , Snake Bites/pathology , Adolescent , Adult , Aged , Australia/epidemiology , Blood Coagulation Disorders/pathology , Child , Female , Humans , Male , Middle Aged , Muscle, Skeletal/pathology , Muscular Diseases/pathology , Prospective Studies , Snake Bites/drug therapy , Snake Bites/epidemiology , Treatment Outcome , Young AdultABSTRACT
INTRODUCTION: Immunoturbidimetry studies the phenomenon of immunoprecipitation of antigens and antibodies in solution, where there is the formation of large, polymeric insoluble immunocomplexes that increase the turbidity of the solution. We used immunoturbidimetry to investigate the interaction between commercial snake antivenoms and snake venoms, as well as cross-reactivity between different snake venoms. METHODS: Serial dilutions of commercial snake antivenoms (100µl) in water were placed in the wells of a microtitre plate and 100µl of a venom solution (50µg/ml in water) was added. Absorbance readings were taken at 340nm every minute on a BioTek ELx808 plate reader at 37°C. Limits imposed were a 30minute cut-off and 0.004 as the lowest significant maximum increase. Reactions with rabbit antibodies were carried out similarly, except that antibody dilutions were in PBS. RESULTS: Mixing venom and antivenom/antibodies resulted in an immediate increase in turbidity, which either reached a maximum or continued to increase until a 30minute cut-off. There was a peak in absorbance readings for most Australian snake venoms mixed with the corresponding commercial antivenom, except for Pseudonaja textilis venom and brown snake antivenom. There was cross-reactivity between Naja naja venom from Sri Lanka and tiger snake antivenom indicated by turbidity when they were mixed. Mixing rabbit anti-snake antibodies with snake venoms resulted in increasing turbidity, but there was not a peak suggesting the antibodies were not sufficiently concentrated. The absorbance reading at pre-determined concentrations of rabbit antibodies mixed with different venoms was able to quantify the cross-reactivity between venoms. Indian antivenoms from two manufacturers were tested against four Sri Lankan snake venoms (Daboia russelli, N. naja, Echis carinatus and Bungarus caeruleus) and showed limited formation of immunocomplexes with antivenom from one manufacturer. DISCUSSION: The turbidity test provides an easy and rapid way to compare and characterise interactions between antivenoms and snake venoms.
Subject(s)
Antivenins/chemistry , Antivenins/metabolism , Snake Venoms/chemistry , Snake Venoms/metabolism , Animals , Antibodies/immunology , Antivenins/immunology , Australia , Cross Reactions/immunology , Elapidae , Rabbits , Snake Venoms/immunology , Sri LankaABSTRACT
We provide the first detailed description of the inner ear of the oldest artiodactyl, Diacodexis, based on a three-dimensional reconstruction extracted from computed tomography imagery of a skull of Diacodexis ilicis of earliest Wasatchian age (ca. 55 Ma). This description provides new anatomical data for the earliest artiodactyls, and reveals that the bony labyrinth of Diacodexis differs greatly from that of modern artiodactyls described so far. The bony labyrinth of Diacodexis presents a weakly coiled cochlea (720 °), a secondary common crus, a dorsal extension of the anterior semicircular canal more pronounced than that of the posterior one, and a small angle between the basal turn of the bony cochlear canal and the lateral semicircular canal. This suite of characters also occurs in basal eutherian mammals. Diacodexis strongly resembles small living tragulid ruminants in its overall body shape and hindlimb proportions. Comparison of the bony labyrinth of Diacodexis to that of the tragulid Moschiola meminna (Indian mouse deer) reveals great morphological difference in cochlear shape and semicircular canal disposition. The shape of the cochlea suggests that Diacodexis was a high-frequency hearing specialist, with a high low-frequency hearing limit (543 Hz at 60 dB). By comparison, the estimated low-frequency limit of Moschiola meminna is much lower (186.0 Hz at 60 dB). We also assess the locomotor agility of Diacodexis based on measurements of the semicircular canals. Locomotor agility estimates for Diacodexis range between 3.62 and 3.93, and suggest a degree of agility compatible with a nimble, fast running to jumping animal. These results are congruent with the postcranial functional analysis for this extinct taxon.
Subject(s)
Artiodactyla/anatomy & histology , Ear, Inner/anatomy & histology , Fossils , Animals , Biological Evolution , Imaging, Three-Dimensional , Motor Activity/physiology , Tomography, X-Ray ComputedABSTRACT
There is limited information on envenoming by snakes of the genus Hoplocephalus from Eastern Australia. We investigated the clinical and laboratory features of patients with definite Hoplocephalus spp. bites including antivenom treatment, recruited to the Australian Snakebite Project. There were 15 definite Hoplocephalus spp. bites based on expert identification including eight by Hoplocephalus stephensi (Stephen's banded snakes), four by Hoplocephalus bungaroides (broad-headed snake) and three by H. bitorquatus (pale-headed snake). Envenoming occurred in 13 patients and was similar for the three species with venom induced consumption coagulopathy (VICC) in all envenomings. Seven patients had an INR >12 and partial VICC, with only incomplete fibrinogen consumption, occurred in three patients. Systemic symptoms occurred in eight patients. Myotoxicity and neurotoxicity did not occur. H. stephensi venom was detected in all three H. stephensi envenomings (1.1, 44 and 81 ng/mL) for whom pre-antivenom blood samples were available, and not detected in one without envenoming. In two cases with post-antivenom blood samples, venom was not detected after tiger snake antivenom (TSAV) was given. In vitro binding studies demonstrated that TSAV concentrations of 50mU/mL are sufficient to bind the majority of free H. stephensi venom components at concentrations above those detected in envenomed patients (100 ng/mL). Eleven patients received antivenom, median dose 2 vials (Range: 1 to 5 vials), which was TSAV in all but one case, where polyvalent antivenom was used. Immediate hypersensitivity reactions occurred in six cases including one case of anaphylaxis. Envenoming by Hoplocephalus spp. causes VICC and systemic symptoms, making it clinically similar to brown snake (Pseudonaja spp.) envenoming. Based on in vitro studies reported here, patients may be treated with one vial of TSAV, although one vial of brown snake antivenom may also be sufficient.
Subject(s)
Antivenins/pharmacology , Blood Coagulation/drug effects , Elapid Venoms/toxicity , Elapidae , Immunologic Factors/pharmacology , Neurotoxins/toxicity , Snake Bites/drug therapy , Adolescent , Adult , Animals , Antivenins/adverse effects , Antivenins/metabolism , Australia , Child , Cohort Studies , Elapid Venoms/metabolism , Humans , Hypersensitivity/etiology , Immunologic Factors/adverse effects , Immunologic Factors/metabolism , Male , Middle Aged , Neurotoxins/metabolism , Prospective Studies , Protein Binding , Young AdultABSTRACT
The coagulant effects of Australasian black snakes (Pseudechis spp.) are poorly understood and differ to the procoagulant venoms of most dangerous snakes in Australia. This study aimed to investigate in vitro coagulant effects of Pseudechis venoms and the efficacy of commercial black snake antivenom (BlSAV), tiger snake antivenom (TSAV) and specific rabbit anti-snake IgG to neutralise these effects. Using a turbidimetric assay, all six Pseudechis venoms had anticoagulant activity, as well as phospholipase A(2) (PLA(2)) activity. Inhibition of PLA(2) activity removed anticoagulant effects of the venoms. Pseudechis porphyriacus was unique and had procoagulant activity independent of PLA2 activity. Both BlSAV and TSAV completely inhibited the coagulant and PLA2 activity of all Pseudechis venoms. PLA2 activity was also inhibited completely by p-Bromophenacyl bromide (pBPB) and partially by specific anti-N. scutatus IgG antibodies. Anti-N. scutatus IgG also completely inhibited anticoagulant activity of Pseudechis venom. All Pseudechis venoms showed immunological cross reactivity with specific anti-snake IgG antibodies to P. porphyriacus, Pseudechis australis and Notechis scutatus. Pseudechis venoms have in vitro anticoagulant activity that appears to be attributable to PLA(2) activity. Both antivenoms inhibited anticoagulant and PLA(2) activity at concentrations below those occurring in patients treated with one vial of antivenom. There was cross-neutralisation of Pseudechis venoms and N. scutatus antibodies that might be attributable to immunological similarities between the venoms.
Subject(s)
Antivenins/pharmacology , Blood Coagulation/drug effects , Elapid Venoms/toxicity , Acetophenones/pharmacology , Animals , Cross Reactions , Elapid Venoms/immunology , Humans , Phospholipases A2/metabolism , RabbitsABSTRACT
BACKGROUND: Limited information exists on the dynamics of hemostasis in patients with venom-induced consumption coagulopathy (VICC) from snake envenomation. OBJECTIVE: The aim of the present study was to investigate specific factor deficiencies and their time course in Australasian elapid envenomation. METHODS: We measured coagulation parameters and factor concentrations in patients recruited to the Australian Snakebite Project, an observational cohort study. There were 112 patients with complete VICC, defined as an international normalized ratio (INR) > 3, and 18 with partial VICC. Serial citrated plasma samples were collected from 0.5 to 60 h post-bite. INR, activated partial thromboplastin time (aPTT), coagulation factors (F)I, II, V, VII, VIII, IX, X, von Willebrand factor antigen (VWF:Ag) and D-dimer concentrations were measured. RESULTS: Complete VICC was characterized by near/total depletion of fibrinogen, FV and FVIII, with an INR and aPTT that exceeded the upper limits of detection, within 2 h of snakebite. Prothrombin levels never fell below 60% of normal, suggesting that the toxins were rapidly eliminated or inactivated and re-synthesis of clotting factors occurred irrespective of antivenom. Partial VICC caused limited depletion of fibrinogen and FV, and almost complete consumption of FVIII. Onset of VICC was more rapid with brown snake (Pseudonaja spp.) venom, which contains a group C prothrombin activator toxin, compared with the tiger snake group, which contains a group D prothrombin activator toxin and requires human FVa formation. Resolution of VICC occurred within 24-36 h irrespective of snake type. CONCLUSIONS: These results suggest that Australasian elapid prothrombin activators have a potent but short duration of action. Antivenom is unlikely to be administered in time to prevent VICC.
Subject(s)
Disseminated Intravascular Coagulation/blood , Snake Bites/blood , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Antivenins/pharmacology , Australia , Blood Coagulation/drug effects , Blood Coagulation Factors/metabolism , Child , Child, Preschool , Cohort Studies , Disseminated Intravascular Coagulation/etiology , Female , Humans , International Normalized Ratio , Male , Middle Aged , Partial Thromboplastin Time , Time Factors , VenomsABSTRACT
The detection and measurement of snake venom in blood is important for confirming snake identification, determining when sufficient antivenom has been given, detecting recurrence of envenoming, and in forensic investigation. Venom enzyme immunoassays (EIA) have had persistent problems with poor sensitivity and high background absorbance leading to false positive results. This is particularly problematic with Australasian snakes where small amounts of highly potent venom are injected, resulting in low concentrations being associated with severe clinical effects. We aimed to develop a venom EIA with a limit of detection (LoD) sufficient to accurately distinguish mild envenoming from background absorbance at picogram concentrations of venom in blood. Serum samples were obtained from patients with taipan bites (Oxyuranus spp.) before and after antivenom, and from rats given known venom doses. A sandwich EIA was developed using biotinylated rabbit anti-snake venom antibodies for detection. For low venom concentrations (i.e. <1 ng/mL) the assay was done before and after addition of antivenom to the sample (antivenom difference method). The LoD was 0.15 ng/mL for the standard assay and 0.1 ng/mL for the antivenom difference method. In 11 pre-antivenom samples the median venom concentration was 10 ng/mL (Range: 0.3-3212 ng/mL). In four patients with incomplete venom-induced consumption coagulopathy the median venom concentration was 2.4 ng/mL compared to 30 ng/mL in seven patients with complete venom-induced consumption coagulopathy. No venom was detected in any post-antivenom sample and the median antivenom dose prior to this first post-antivenom sample was 1.5 vials (1-3 vials), including 7 patients administered only 1 vial. In rats the assay distinguished a 3-fold difference in venom dose administered and there was small inter-individual variability. There was small but measurable cross-reactivity with black snake (Pseudechis), tiger snake (Notechis) and rough-scale snake (Tropidechis carinatus) venoms with the assay for low venom concentrations (<1 ng/mL). The use of biotinylation and the antivenom difference method in venom EIA produces a highly sensitive assay that will be useful for determining antivenom dose, forensic and clinical diagnosis.
Subject(s)
Elapid Venoms/blood , Elapidae/physiology , Immunoenzyme Techniques , Snake Bites/diagnosis , Adult , Aged , Animals , Antivenins/therapeutic use , Child , Child, Preschool , Cross Reactions , Elapid Venoms/immunology , Elapid Venoms/poisoning , Female , Humans , Limit of Detection , Male , Middle Aged , Predictive Value of Tests , Rabbits , Rats , Snake Bites/blood , Snake Bites/therapy , Young AdultABSTRACT
Knowledge of the phylogenetic position of the order Cetacea (whales, dolphins, and porpoises) within Mammalia is of central importance to evolutionary biologists studying the transformations of biological form and function that accompanied the shift from fully terrestrial to fully aquatic life in this clade. Phylogenies based on molecular data and those based on morphological data both place cetaceans among ungulates but are incongruent in other respects. Morphologists argue that cetaceans are most closely related to mesonychians, an extinct group of terrestrial ungulates. They have disagreed, however, as to whether Perissodactyla (odd-toed ungulates) or Artiodactyla (even-toed ungulates) is the extant clade most closely related to Cetacea, and have long maintained that each of these orders is monophyletic. The great majority of molecule-based phylogenies show, by contrast, not only that artiodactyls are the closest extant relatives of Cetacea, but also that Artiodactyla is paraphyletic unless cetaceans are nested within it, often as the sister group of hippopotamids. We tested morphological evidence for several hypotheses concerning the sister taxon relationships of Cetacea in a maximum parsimony analysis of 123 morphological characters from 10 extant and 30 extinct taxa. We advocate treating certain multistate characters as ordered because such a procedure incorporates information about hierarchical morphological transformation. In all most-parsimonious trees, whether multistate characters are ordered or unordered, Artiodactyla is the extant sister taxon of Cetacea. With certain multistate characters ordered, the extinct clade Mesonychia (Mesonychidae + Hapalodectidae) is the sister taxon of Cetacea, and Artiodactyla is monophyletic. When all fossils are removed from the analysis, Artiodactyla is paraphyletic with Cetacea nested inside, indicating that inclusion of mesonychians and other extinct stem taxa in a phylogenetic analysis of the ungulate clade is integral to the recovery of artiodactyl monophyly. Phylogenies derived from molecular data alone may risk recovering inconsistent branches because of an inability to sample extinct clades, which by a conservative estimate, amount to 89% of the ingroup. Addition of data from recently described astragali attributed to cetaceans does not overturn artiodactyl monophyly.
Subject(s)
Cetacea/classification , Phylogeny , Animals , Cetacea/geneticsABSTRACT
Growth factors and hormones activate protein translation by phosphorylation and inactivation of the translational repressors, the eIF4E-binding proteins (4E-BPs), through a wortmannin- and rapamycin-sensitive signaling pathway. The mechanism by which signals emanating from extracellular signals lead to phosphorylation of 4E-BPs is not well understood. Here we demonstrate that the activity of the serine/threonine kinase Akt/PKB is required in a signaling cascade that leads to phosphorylation and inactivation of 4E-BP1. PI 3-kinase elicits the phosphorylation of 4E-BP1 in a wortmannin- and rapamycin-sensitive manner, whereas activated Akt-mediated phosphorylation of 4E-BP1 is wortmannin resistant but rapamycin sensitive. A dominant negative mutant of Akt blocks insulin-mediated phosphorylation of 4E-BP1, indicating that Akt is required for the in vivo phosphorylation of 4E-BP1. Importantly, an activated Akt induces phosphorylation of 4E-BP1 on the same sites that are phosphorylated upon serum stimulation. Similar to what has been observed with serum and growth factors, phosphorylation of 4E-BP1 by Akt inhibits the interaction between 4E-BP1 and eIF-4E. Furthermore, phosphorylation of 4E-BP1 by Akt requires the activity of FRAP/mTOR. FRAP/mTOR may lie downstream of Akt in this signaling cascade. These results demonstrate that the PI 3-kinase-Akt signaling pathway, in concert with FRAP/mTOR, induces the phosphorylation of 4E-BP1.
Subject(s)
Eukaryotic Initiation Factors , Phosphoproteins/metabolism , Protein Biosynthesis , Protein Kinases , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolism , Adaptor Proteins, Signal Transducing , Androstadienes/pharmacology , Carrier Proteins/metabolism , Cell Cycle Proteins , Drug Resistance , Eukaryotic Initiation Factor-4E , Humans , Models, Genetic , Mutation , Peptide Initiation Factors/metabolism , Peptide Mapping , Phosphatidylinositol 3-Kinases/metabolism , Phosphopeptides/isolation & purification , Phosphorylation/drug effects , Phosphotransferases (Alcohol Group Acceptor)/genetics , Polyenes/pharmacology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-akt , Signal Transduction , Sirolimus , TOR Serine-Threonine Kinases , WortmanninABSTRACT
Diffusing photons provide information about the optical properties of turbid media. In biological tissues these optical properties may be correlated to physiological parameters, enabling one to probe effectively the physiological states of tissue for abnormalities such as tumors and hemorrhages. We show that positional uncertainty in the source and detector lead to significant random errors that degrade the optical information available from diffusing photons. We investigate the limits for the detection, localization, and characterization of optical inhomogeneities by using diffusing photons as a probe. Although detection is sufficient for tumor screening, full characterization of the optical properties is desirable for specification of the tumor. Our findings in model breast systems with realistic signal-to-noise ratios indicate that tumors as small as 0.3 cm in diameter can be unambiguously detected; however, simultaneous determination of tumor size and optical properties is possible only if its diameter is of the order of 1.0 cm or larger. On the other hand, if a priori information about the size (optical properties) is available, then the optical properties (size) of tumors as small as 0.3 cm in diameter can be determined.
ABSTRACT
The lifetime of a f luorophore generally varies in different environments, making the molecule a sensitive indicator of tissue oxygenation, pH, and glucose. However, lifetime measurements are complicated when the f luorophore is embedded in an optically thick, highly scattering medium such as human tissue. We formulate the inverse problem for f luorescence lifetime tomography using diffuse photon density waves, and we demonstrate the technique by deriving spatial images of heterogeneous f luorophore distribution and lifetime, using simulated measurements in heterogeneous turbid media.
ABSTRACT
We present analytic solutions for fluorescent diffuse photon density waves originating from fluorophores distributed in thick turbid media. Solutions are derived for a homogeneous turbid medium containing a uniform distribution of fluorophores and for a system that is homogeneous except for the presence of a single spherical inhomogeneity Generally the inhomogeneity has fluorophore concentration, and lifetime and optical properties that differ from those of the background. The analytic solutions are verified by numerical calculations and are used to determine the fluorophore lifetime and concentration changes required for the accurate detection of inhomogeneities in biologically relevant systems. The relative sensitivities of absorption and fluorescence methods are compared.
ABSTRACT
We present images of heterogeneous turbid media derived from measurements of diffuse photon-density waves traveling through highly scattering tissue phantoms. To our knowledge, the images are the first experimental reconstruction based on data collected in the frequency domain. We demonstrate images of both absorbing and scattering heterogeneities and show that this method is sensitive to the optical properties of the heterogeneity. The algorithm employs a differential measurement scheme that reduces the effect of errors resulting from incorrect estimation of the background optical properties. The relative advantages of sources with low and high modulation frequency are discussed within this context.
ABSTRACT
We present an analytic solution for the scattering of diffuse photon density waves by spherical inhomogeneities within turbid media. The analytic result is compared to experimental measurements. Close agreement between theory and experiment permits the use of the theory to determine the properties of unknown sphere-like objects embedded in turbid media. The analytic solution is extended to encompass several problems of practical interest in imaging, including the influence of multiple sources, multiple objects, and boundaries on the characterization of spherical inhomogeneities. We also extend the solution to encompass time-domain measurements.