An objective approach to antivenom therapy and assessment of first-aid measures in snake bite

Treatment of systemic envenoming in snake-bite victims has, in the past, depended almost entirely on the individual clinician's experience in assessing the severity of envenoming. The efficacy of treatment is obviously related to the neutralizing potency of the antivenom used, the route by which it is administered and the dose. The development of enzyme immunoassays has permitted a more scientific appraisal, allowing estimation of circulating specific venom and antivenom concentrations at any time after the bite in the patient's blood. It is therefore possible to measure accurately the efficacy of antivenom in the neutralization and clearance of venom antigen. In Brazil, it appears that clinicians treat patients with excessive amounts of highly efficient antivenoms and this results in an unacceptably high incidence of reactions. In Sri Lanka, the use of imported, Indian antivenom is relatively ineffective in neutralizing the venoms of Sri Lankan snakes, demonstrating the real problem of venom variability within individual species. In West Africa, the improved clearance of venom following treatment of Echis victims with a monospecific as opposed to a polyspecific antivenom has been demonstrated, and new, smaller fragment, Fab antivenoms have been developed and are now under clinical assessment. Such clinically-based immunological studies should result in more efficient and controlled use of expensive antivenoms for treatment of systemic envenoming and the accurate assessment of newly designed products. Such studies also emphasise the importance of individual countries producing their own antivenoms for treatment of systemic envenoming. Likewise, the use of such objective systems now enables the use of first-aid measures such as tourniquets to be properly assessed.     

Serum and egg yolk IgG antibody titers from laying chickens vaccinated with Pasteurella multocida

Through determining the serum and egg yolk antibody titers in immunized laying hens to Pasteurella multocida regularly, the growth-decline trend of the egg yolk antibody levels was found to be similar to that of the serum antibody levels (r = 0.94), but the growth and decline of the egg yolk antibody seemed to be delayed 3-6 days compared with that of the serum antibody, and the egg yolk antibody titers were generally lower than those of the serum antibody (P < 0.01). Serum and egg yolk antibody levels declined 3 and 6 days, respectively, after booster immunizations. The higher the antibody levels were before booster immunization, the more they declined.     

Preparation of compound-specific and group-specific antibodies to 7-methylguanine and related 7-alkylguanines and their use in immunoaffinity purification

The preparation and characteristics of compound-specific and group-specific antibodies against 7-alkylguanines (7-alkGua) are described. A compound-specific antibody against 7-methylguanine was prepared using a hapten bound to carrier protein through the N2 position. In a competitive enzyme-linked immunosorbent assay (ELISA) 7-methylguanine (7-MeGua) showed 50% inhibition (I50%) at 10 pmol/well at room temperature, but the inhibition was found to be 40 times better at 4 degrees C (I50% at 250 fmol/well). When the antibody was bound to protein A-Sepharose CL4B 7-MeGua was retained in immunoaffinity columns. A group-specific antibody to 7-alkGua was prepared using 7-(2-carboxyethyl)guanine (7-CEGua) bound to carrier protein via the carboxyl group. In a competitive ELISA, this antibody cross-reacted well with 7-CEGua, 7-ethylguanine (7-EtGua), 7-(2-hydroxyethyl)guanine (7-HOEtGua) and 7-(2',3'-dihydroxy)-propylguanine (7-DHPGua) and some inhibition was seen with 7-MeGua. Immunoaffinity columns prepared from this antibody retained a number of 7-alkGua of diverse structure. 7-EtGua in calf thymus DNA treated with diethylsulphate and ethylnitrosourea was isolated by immunoaffinity purification and quantified by HPLC-fluorescence. These results illustrate the potential of immunoaffinity purification for both individual DNA adducts and groups of adducts.     

Optimisation of immunoaffinity purification of Wuchereria bancrofti specific antibodies from human sera

Immunoaffinity column using Setaria digitata antigens coupled to cyanogen bromide activated Sepharose 4B beads were developed to purify antibodies from sera of filarial patients. Chaotropic (KSCN) ion elution was more efficient for purifying specific antibodies from the column in comparison to ]c elution. Dot blot analysis indicated that purified antibodies showed a high degree of reactivity with cattle filarial antigen and recombinant filarial protein but not with bacterial proteins of E. coli suggesting that the antibodies are specific.     

Envenoming and antivenom use in Australia

Australia has a diverse and rich venomous fauna, both terrestrial and marine, including some of the most venomous species in each class. Antivenom is the principal therapy for the majority of medically significant envenomings and is currently supplied through a single source, CSL, Melbourne. Cases of envenoming reported to Australian poisons information centers (PICs) are dominated by spiderbite and insect stings, respectively accounting for 53.7% and 39.3% of all bite/sting calls. Marine animal bites/stings/poisonings account for only 4% of PIC calls in this category, while snakebites account for a mere 3% (still at least 400 calls/yr). Because most PIC calls are from the public, not doctors/hospitals, snakebite in particular is under-represented. The author has recently reviewed antivenom usage in Australia. Snakebite affects between 1,000 to 3,000 people per year, with an average annual mortality of about 2 cases. Brown snakes (genus Pseudonaja) cause the majority of deaths (and bites), with tiger snakes (genus Notechis) and taipans (genus Oxyuranus) accounting for nearly all other fatalities. Up to 500 cases require snake antivenoms each year, the majority of cases coming from rural areas of Queensland, Western Australia, New South Wales and Victoria, these being the most populated states. The wide availability of snake venom detection kits has allowed specific antivenom to be used more often, rather than polyvalent antivenom, but the latter is still used in nearly 30% of cases, suggesting underutilization of venom detection. The issue of premedication prior to antivenom remains unresolved. Antivenom usage and complication data for 1995 and 1996 will be presented. Red back spider antivenom is the most commonly used antivenom, with reports of usage being greater than for all other antivenoms combined. It is reported as being therapeutically efficacious in 94% of cases, with a single ampoule being used in 76% of cases, 2 ampoules in 18% of cases, and 3 or more ampoules in 6% of cases. Clinical experience suggests only 20% of red back spider bites require antivenom therapy. It is likely that between 5-10,000 bites occur annually.     

Deposition of genetically engineered human antibodies into the egg yolk of hens

This is an exploratory study that investigated factors influencing the psychosocial adjustment of Central American immigrants with disabilities. The relationships between stress, and perception of disability severity and (a) depression and (b) anxiety were assessed. Furthermore, this study investigated whether social support moderated the impact of stress and severity of disability on depression and anxiety. Stress, severity of disability, and social support explained a high percentage (54%) of the variance in depression. High levels of stress, increased perceptions of severity of disability, and low social support were associated with increased depression. The interactions between support and stress and between support and disability severity did not significantly add to the original model which predicted depression. Main effects were found for stress, disability severity, and the interaction between support and disability severity. Stress and social support significantly accounted for 31% of the variance in anxiety. Increased stress and decreased social support were associated with greater levels of anxiety. The interaction between support and stress did not significantly predict anxiety. Implications of the study in terms of future research and intervention programs targeting mental health outcomes for Latino immigrants with disabilities are discussed.     

The relationship between parental yolk cholesterol and yolk fat concentration to abdominal fat content and feed conversion ratio of their respective offspring

The correlation of yolk cholesterol and yolk fat concentrations of egg from the pedigreed Athens-Canadian Randombred control population with the percentage of abdominal fat (AF) and feed conversion ratio (FCR) of their progeny were studied. The average yolk cholesterol, yolk fat, and AF were 20.3 mg/g yolk, 244 mg/g yolk, and 1.64%, respectively. The phenotypic correlation of both yolk cholesterol and yolk fat content of eggs from the parental population with AF or FCR of their progeny were low and nonsignificant.     

Development of an ELISA to assess the potency of horse therapeutic polyvalent antibothropic antivenom

The objective of this study was the search for a suitable venom antigen to be used in an in vitro alternative immunoassay, to the standard antivenom neutralization assay using mice. Bothrops jararaca venom was fractionated in DEAE-Sephacel columns and the fractions were tested for a correlation between antibody capture enzyme linked immunosorbent assay (ELISA) absorbance values and the 'in vivo' antivenom potency. Individual antivenoms from 14 horses and 15 separate FUNED polyspecific Bothrops ampouled antivenoms (final product) were used. Fractions showing the higher correlations were further chromatographed in a Sephadex G-75 column and again tested for the correlation. Two fractions with haemorrhagic activity displayed a correlation of r = 0.77 and r = 0.8 against the individual horse antivenom sera and of r = 0.79 and r = 0.8 for the ampouled antivenom. For all results p < 0.001. Two other fractions with phospholipase A2 activity showed a correlation of r = 0.66 (p < 0.01) and r = 0.56 (p < 0.03) against the individual horse antivenom sera. Electrophoresis results show a similar composition for both antigens with haemorrhagic activity. Results indicate that the fractions purified would be suitable for the desired objective of this study.     

Comparative studies of phosvitin from chicken and salmon egg yolk

1. A method developed for the isolation of phosvitin from chicken egg yolk was successfully applied to the isolation of phosvitin from salmon eggs. 2. Salmon roe phosvitin is smaller in molecular size than chicken egg phosvitin. 3. Circular dichroism spectra of all phosvitins investigated displayed good similarities with spectra showing characteristics of unordered and beta-sheet secondary structure. 4. The main component in the Fourier transform infrared spectra of chicken egg phosvitin is indicative of unordered conformation, whereas the Fourier infrared data of the salmon egg phosvitin are consistent with more of beta-sheet structure compared to the chicken egg phosvitin.     

Development of human mast cells from their progenitors

Two types of human mast cells, which are morphologically similar to skin mast cells and lung mast cells, respectively, can be developed from pluripotent stem cells under different culture conditions. The major growth factor for mast-cell development is c-kit ligand, which induces mastocytosis in vivo. However, this cytokine is not sufficient for full maturation of the cells.     

Development of responsiveness to suprathreshold acoustic stimulation in chickens.

Observed developmental changes in a UCR to acoustic stimulation in young Hubbard?×?Hubbard chickens. Specifically, durations of distress call (peep) suppression were measured after the onsets of tones that differed in intensity and frequency in 384 newly hatched and 4-day-old chicks. Resuppression was also measured after a 6% change in the frequency of these tones, once Ss had habituated to the original tone. Data show that the suppression varied systematically as a function of age, intensity, and frequency: (a) the duration of suppression increased with increasing stimulus intensity; (b) responsiveness to high frequencies grew more rapidly over the 1st 4 days than responsiveness to low frequencies, an effect indicating a developmental gradient across frequencies with age; (c) resuppression to the 6% change in frequency increased in duration with age; and (d) young Ss suppressed vocalizations longer to loud tones in the range of their species' maternal assembly call than to other frequency–intensity combinations. These developmental trends indicate rapid changes in perceived loudness and perceptual sharpening over the first few days of postnatal life. (51 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Absorption and elimination of viper venom after antivenom administration

The mechanisms by which antivenom neutralizes the venom are still poorly understood. In the present work, we studied the effects of antivenom, constituted with either F(ab')2 or Fab, on the processes of absorption and elimination of Vipera aspis venom in experimentally envenomed rabbits. We first concluded from this study that during the few hours after intramuscular injection, the venom rapidly disappeared from the site of injection but did not immediately reach the vascular system, suggesting that it is partly absorbed via the lymphatic circulation. Concerning the elimination process of the venom in the presence of antivenom, we observed that the elimination of F(ab')2/venom complexes is slower than that of free venom in the absence of antivenom but faster than that of free F(ab')2, suggesting that F(ab')2/venom complexes are eliminated by phagocytosis. The Fab/venom complexes, on the other hand, are eliminated more slowly than free Fab. These complexes are not eliminated through the renal route in agreement with their high molecular weight. In addition, we observed that the treatment of envenomed rabbits with antivenom made of Fab, but not F(ab')2, is responsible for an oliguria that could be responsible for clinical problems.     

Inhibition of hemorrhagic activities of various snake venoms by purified antihemorrhagic factor obtained from Japanese Habu snake

The ability of purified antihemorrhagic factor isolated from the serum of Japanese Habu (Trimeresurus flavoviridis) was tested on 17 snake venoms to inhibit their hemorrhagic activity. The factor strongly inhibited that of the venoms of Crotalus horridus horridus and Vipera latastei gaditana in addition to that of the homologous (T. flavoviridis) venom. Hemorrhagic activity of Agkistrodon halys blomhoffi, Agkistrodon contortrix contortrix, Bothrops atrox asper and Crotalus atrox venoms was also remarkably inhibited, but that of Vipera mauretanica to less extent. The hemorrhagic activities of nine other snake venoms were not inhibited.     

Techniques for purification of rabbit osteoclasts and analysis of their functions

Autoantibodies to the human thyrotropin receptor (TSH-R) are pathogenic in a number of autoimmune thyroid diseases including Graves' disease. We have characterised polyclonal antisera to TSH-R for antibodies which may mimic those present in autoimmune thyroid disease. For immunisations, recombinant extracellular region of human TSH-R which does not interact with its ligand TSH was used. The induced antibodies react with the full length membrane receptor in transfected mammalian cells by flow cytometry showing the presence of antibody capable of recognising the native functional receptor. The properties of the generated antibodies have been compared after two injections or following a multiple immunisation protocol with the receptor in adjuvant. High titre antisera were readily generated after the short injection protocol and further immunisations did not lead to any change in antibody titers. Analysis of the epitopes recognised using synthetic peptides confirmed previous observations that the immunodominant determinants localise to the amino and the carboxyl terminal part of the extracellular region of the receptor. Antisera from both rabbits contain TSH blocking antibody as assessed by inhibition of TSH mediated cAMP stimulation. There was an increase in TSH binding inhibitory immunoglobulin (TBII) activity with multiple injections. Furthermore, the increase in TBII activity was not related to spreading of the antibody response to new determinants on TSH-R. Our results support previous observations on the difficulties in reproducing, by adjuvant immunisation with recombinant TSH-R preparations, the fine specificity of antibodies to TSH-R present in autoimmune disorders such as Graves' disease or primary myxoedema.     

A method for high yield isolation and purification of anti-native DNA antibodies present in lupus sera

Antibodies to nucleic acids may serve as biochemical tools or as probes of cellular function. Particularly important, but also particularly difficult to obtain, is antibody which reacts exclusively with double stranded DNA. We describe here a method for the separation of antibodies to double stranded DNA from SLE serum, using hydroxyapatite to which DNA is adsorbed at a low molarity of phosphate buffer. Having applied the serum to the column we passed it through a continuous gradient of phosphate buffer ranging from 0.005 to 0.5 M. Deoxyribonuclease and magnesium ions were added when the gradient had reached the molarity at which single stranded DNA had already been desorbed and double stranded DNA began to be eluted. The antibody to native DNA that we obtained reacted in complement fixation, counterimmunoelectrophoresis and Farr's assay with native DNA and did not react with single stranded DNA, single and double stranded RNA or with a panel of 24 protein-coupled nucleosides, nucleotides and dinucleotides.     

Expression and purification of large nebulin fragments and their interaction with actin

cDNA clones encoding mouse skeletal muscle nebulin were expressed in Escherichia coli as thioredoxin fusion proteins and purified in the presence of 6 M urea. These fragments, called 7a and 8c, contain 28 and 19 of the weakly repeating approximately 35-residue nebulin modules, respectively. The nebulin fragments are soluble at extremely high pH, but aggregate when dialyzed to neutral pH, as assayed by centrifugation at 16,000 x g. However, when mixed with varying amounts of G-actin at pH 12 and then dialyzed to neutral pH, the nebulin fragments are solubilized in a concentration-dependent manner, remaining in the supernatant along with the monomeric actin. These results show that interaction with G-actin allows the separation of insoluble nebulin aggregates from soluble actin-nebulin complexes by centrifugation. We used this property to assay the incorporation of nebulin fragments into preformed actin filaments. Varying amounts of aggregated nebulin were mixed with a constant amount of F-actin at pH 7.0. The nebulin aggregates were pelleted by centrifugation at 5200 x g, whereas the actin filaments, including incorporated nebulin fragments, remained in the supernatant. Using this assay, we found that nebulin fragments 7a and 8c bound to actin filaments with high affinity. Immunofluorescence and electron microscopy of the actin-nebulin complexes verified that the nebulin fragments were reorganized from punctate aggregates to a filamentous form upon interaction with F-actin. In addition, we found that fragment 7a binds to F-actin with a stoichiometry of one nebulin module per actin monomer, the same stoichiometry we found in vivo. In contrast, 8c binds to F-actin with a stoichiometry of one module per two actin monomers. These data indicate that 7a can be incorporated into actin filaments to the same extent found in vivo, and suggest that shorter fragments may not bind actin filaments in the same way as the native nebulin molecule.     

Rapid and convenient purification of tonin from rat submandibular gland. Comparison of tonin and tissue kallikrein in their interactions with inhibitors

Tonin was isolated from rat submandibular glands by a very convenient procedure consisting of sequential anion-exchange, hydrophobic interaction and gel filtration chromatographies. The method is superior to earlier purifications as it consists of fewer stages, resulting in a much higher recovery (41%) of tonin. The final preparation was seen to be pure on SDS gel electrophoresis (M(r) 32,800) and on gel isoelectric focusing (pI 6.15). The stability of tonin and its interaction with various inhibitors were investigated, and compared with the corresponding behavior of rat tissue kallikrein.     

Survey of antitrypanosome antibodies and studies of their specificity and autoimmunity

As a part of investigations to characterize trypanosome infections in Taiwan, sera collected from patients admitted to Veterans General Hospital, Taipei were tested for antitrypanosome antibodies. A Trypanosoma cruzi extract-based enzyme-linked immunosorbent assay (ELISA) was used to screen and titrate 1,297 patient sera. High antitrypanosome titers were detected in 166 (12.8%) of these sera. Retitration of random samples of the high titer (HT) sera indicated a 5.4% false positive. Thirteen donors with high antitrypanosome ELISA titers were followed up. Twelve of then remained high serum titers also showed high ELISA titers against an extract of Trypanosoma conorhini. Hemocultures conducted on freshly drawn blood specimens of the 13 subjects did not provide any evidence of trypanosome infections. Electrophoretic analyses of sera from HT and low titer (LT) patients suggested differences between serum proteins of the subjects in each of the groups. Atypical reactions were observed in immunodiffusion tests performed with HT and LT sera and trypanosome extracts, while western blot analyses revealed a complex pattern of binding by both sera. The qualitative and quantitative differences in these tests suggested interactions of T. cruzi antigens with donor antibodies against unrelated antigens and/or with autoantibodies. Subsequent analyses did not indicate any association between rheumatoid factor and the reactivities of the HT sera with the parasites. However, antinuclear antibodies were detected with an indirect fluorescent antibody test (IFAT) in 50% of the HT sera and 22% of the LT sera. No differences were found between the levels of antilaminin activity of the two groups. The IFAT employing T. cruzi epimastigotes was positive for 100% of the HT sera and 22% of the LT sera. The data indicate that the high seropositivity recognized in this study is due in part to the activities of cross-reacting antibodies and/or autoantibodies in the sample population.     

Snakes and snake bite in Nepal

In this paper the author shows the basic concepts of set theory, which ones are necessary to understand biometric methods. The sets, the most important operations, the power of sets and the types of the used variables are presented.