Orofacial antinociceptive activity of (S)-(-)-perillyl alcohol in mice: a randomized, controlled and triple-blind study.

This study investigated the antinociceptive effects of (S)-(-)-perillyl alcohol (PA) on orofacial nociception in Swiss male mice using formalin-, capsaicin-, and glutamate-induced pain tests. For each test, eight animals per group were pre-treated intraperitoneally by a blinded investigator with PA (50 or 75mg/kg), morphine, or vehicle (saline+0.2% Tween 80). The treatment was performed before the induction of orofacial nociception by injecting formalin, capsaicin, or glutamate solution into the right area of the upper lip. The orofacial nociceptive behaviour was timed in all tests by an investigator who was blinded to the treatments. The statistical analysis was performed using confidence intervals (CI), the effect size, and power. PA blocked the orofacial nociceptive behaviour at both doses tested (P<0.05) similarly to morphine (P>0.05), in all tests. The effect size was high in the phase 1 formalin test for 50mg/kg PA (95% CI 0.48-2.31, power 84.6%) and 75mg/kg PA (95% CI 0.82-2.76, power 96.2%), in phase 2 for 75mg/kg PA (95% CI 0.44-2.26, power 82.3%), and in the glutamate test for 75mg/kg PA (95% CI 1.11-3.16, power 99.2%). These findings show strong evidence for the antinociceptive properties of PA in the orofacial region.

Polyvalent horse F(Ab`)2 snake antivenom: Development of process to produce polyvalent horse F(Ab`)2 antibodies anti-african snake venom

A method to obtain polyvalent anti-Bitis and polyvalent-anti-Naja antibodies was developed by immunizing horses with B. arietans, B. nasicornis, B. rhinoceros, N. melanoleuca and N. mossambicacrude venoms. Antibody production was followed by the ELISA method during the immunization procedure. Once the desired anti-venom antibody titers were attained, horses were bled and theimmunoglobulins were separated from the sera by (NH4)2SO4 precipitation, cleaved with pepsin and filtered through a 30 kDa ultrafiltration membrane. F(ab´)2 fragments were further purified by Q-Fast Flow chromatography, concentrated by molecular ultrafiltration and sterilized by filtration through 0.22 m membranes. The resulting F(ab´)2 preparations were rich in intact L and in pieces of H IgG(T) chains, as demonstrated by electrophoresis and Western blot and exhibited high antibody titers, as assayed bythe ELISA method. In addition, the preparations possess a significant capacity to neutralize the lethalityof venoms, as estimated by ED50 determination in mouse assay and are free of toxic substances, pyrogen and bacterial or fungal contaminations.

Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test.

A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r(2) = 0.95, P < 0.0001) and for diphtheria (r(2) = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test

A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r² = 0.95, P < 0.0001) and for diphtheria (r² = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

Cross reactivity of different specific Micrurus antivenom sera with homologous and heterologous snake venoms.

Coral snakes are the only Elapids in America. They are represented by three genera: Leptomicrurus, Micruroides and Micrurus, of which the latter are the most abundant and diversified group. Little is known about the biochemistry of Micrurus venoms due to low availability. Here, we present a study on the cross reactivity of different specific Micrurus antivenom with homologous and heterologous snake venoms in order to contribute to the generation of more efficient antiserum for therapeutic purposes. The three specific antisera tested, anti-Micrurus corallinus, anti-Micrurus frontalis, and anti-Micrurus spixii, as well as the bivalent anti-elapid venom sera, raised against a mixture (50% each) of Micrurus frontalis and Micrurus corallinus venoms, were assayed by Western Blot against Micrurus and non-Micrurus elapid venoms. An antisera raised against a recombinant alpha-neurotoxin-like protein from Micrurus corallinus venom, only reacted in Western blot with its homologous venom, indicating that this protein is specific for Micrurus corallinus coral snake.

Cross reactivity of different specific Micrurus antivenom sera with homologous and heterologous snake venoms

Coral snakes are the only Elapids in America. They are represented by three genera: Leptomicrurus, Micruroides and Micrurus, of which the latter are the most abundant and diversified group. Little is known about the biochemistry of Micrurus venoms due to low availability. Here, we present a study on the cross reactivity of different specific Micrurus antivenom with homologous and heterologous snake venoms in order to contribute to the generation of more efficient antiserum for therapeutic purposes. The three specific antisera tested, anti-Micrurus corallinus, anti-Micrurus frontalis, and anti-Micrurus spixii, as well as the bivalent anti-elapid venom sera, raised against a mixture (50% each) of Micrurus frontalis and Micrurus corallinus venoms, were assayed by Western Blot against Micrurus and non-Micrurus elapid venoms. An antisera raised against a recombinant á-neurotoxin-like protein from Micrurus corallinus venom, only reacted in Western blot with its homologous venom, indicating that this protein is specific for Micrurus corallinus coral snake.

Neutralization of crotaline snake venoms from Central and South America by antivenoms produced in Brazil and Costa Rica.

A study was performed on the ability of antivenoms, produced in Brazil and Costa Rica, to neutralize lethal, hemorrhagic and coagulant activities of the venoms of 16 species of Central and South American snakes of the subfamily Crotalinae. Neutralization of lethality was studied by two different methods routinely used in the quality control of antivenoms at Instituto Butantan (IB) and Instituto Clodomiro Picado (ICP). Both antivenoms neutralized the majority of the venoms studied, but the values of effective doses 50% (ED(50)) differed markedly depending on the method used. In general, higher potencies were obtained with the method of ICP, where a challenge dose corresponding to 4 LD(50)s is used, than with the method of IB, where a challenge dose of 5 LD(50)s is employed. All venoms induced hemorrhagic activity in the mouse skin test, which was effectively neutralized by the two antivenoms. All venoms, except those of Porthidium nasutum and Bothriechis lateralis, induced coagulation of human plasma in vitro and both antivenoms were effective in the neutralization of this activity. In conclusion, our results provide evidence of an extensive cross reactivity between these antivenoms and Central and South American crotaline snake venoms.

Production of an effective anti-Bothrops-tetanus mixed hyperimmune serum of equine origin

The present investigation reveals the possibility of simultaneous immunization of horses with Bothrops or Crotalus snake venoms and Tetanus antigens for the production of anti-Bothrops-Tetanus or anti-Crotalus-Tetanus mixed serum, with high titers of the respective specific antibodies. Bothrops antivenoms with an average neutralizing titer of 4.16 mg venom/ml were obtained from plasma of horses with titers lower than 0.5 mg venom/ml when Tetanus antigens were not used. This suggests the existence of a synergism between Bothrops venoms and Tetanus antigens in the stimulation of the antibody response. The pooled plasma of the animal had a neutralizing titer of 21.0 mg/ml reference Bothrops venoms and 3,300 IU/ml to Tetanus antigens after purification by enzymatic digestion and ammonium sulphate precipitation. These experiments lead us to conclude that Bothrops envenomation therapy can be successfully performed using Anti-Bothrops-Tetanus serum also serving as Tetanus prophylaxis. anti-Crotalus-Tetanus serum can also be produced, although it is not of medical interest as Crotalus envenomation rarely results in local necrotizing lesions.

Effect of beta-propiolactone treatment on the complement activation mediated by equine antisera.

Reduction of complement activation through an alteration of the Fc fragment of immunoglobulins by beta-propiolactone treatment was carried out in equine antisera raised against rabies virus, Bothrops venoms and diphtherial toxin. Results were evaluated by means of an anaphylactic test performed on guinea-pigs, and compared to the ones obtained with the same sera purified by saline precipitation (ammonium sulfate), followed or not by enzymatic digestion with pepsin. Protein purity levels for antibothropic serum were 184.5 mg/g and 488.5 mg/g in beta-propiolactone treated and pepsin-digested sera, respectively. The recovery of specific activity was 100% and 62.5% when using antibothropic serum treated by beta-propiolactone and pepsin digestion, respectively. The antidiphtherial and anti-rabies sera treated with beta-propiolactone and pepsin presented protein purity levels of 5,698 and 7,179 Lf/g, 16,233 and 6,784 IU/g, respectively. The recovery of specific activity for these antisera were 88.8%, 77.7%, 100% and 36.5%, respectively. beta-propiolactone treatment induced a reduction in complement activation, tested "in vivo", without significant loss of biological activity. This treatment can be used in the preparation of heterologous immunoglobulins for human use.

Antigenic cross-reactivity among components of Brazilian Elapidae snake venoms.

Snake venoms from M. corallinus (LD50 = 7.1 +/- 0.83 micrograms), M. frontalis (LD50 = 19.3 +/- 3.13 micrograms), M. ibiboboca (LD50 = 19.8 +/- 2.07 micrograms) and M. spiixi (LD50 = 6.7 +/- 1.25 micrograms) (family Elapidae, genus Micrurus) injected into horses alone or in combination (M. corallinus with M. frontalis) elicit antibody production, as indicated in vivo by neutralization of venom lethality and in vitro by enzyme-linked immunosorbent assay (ELISA), immunoelectrophoresis (IE) and Western blotting (WB). Venom lethality was efficiently neutralized by the antisera, with the monovalent antivenoms being more efficient than the bivalent antivenom. Antibodies against venom components were detected by all antisera at different titers by ELISA. Upon IE, antisera against M. spiixi and M. frontalis venoms cross-reacted with the four types of venoms studied and recognized several molecular components, the precipitin lines obtained had distinct intensities and electrophoretic motilities, whereas the antivenom against M. corallinus only recognized components of its venom but not of the others. All antivenoms cross-reacted with all the elapid venoms in WB revealing several bands with distinct MWs in M. corallinus and M. spiixi venoms, two very sharp and separate bands in M. corallinus venom and a very sharp band of high MW together with several other smaller and faint bands in M. frontalis venom. The data indicate that snake venoms of the genus Micrurus are good immunogens that contain many cross-reactive molecules, and that their toxic components are neutralized more effectively by monovalent rather than by bivalent antivenom.

Antigenic cross-reactivity among components of Brazilian Elapidae snake venoms

Snake venoms from M. corallinus (LD5=7.1 + or - 0.83 µg), M.frontalis (LD50=19.3 + or - 3.13 µg), M. ibiboboca (LD50=19.8 + or - 2.07 µg) and M. spiixi (LD50=6.7 + or - 1.25 µg) (family Elapidae, genus Micrurus) injected into horses alone or in combination (M. corallinus with M. frontalis) elicit antibody production, as indicated in vivo by neutralization of venom lethality and in vitro by enzyme-linked immunosorbent assay (ELISA), immunoelectrophoresis (IE) and Western blotting (WB). Venom lethality was efficiently neutralized by the antisera, with the monovalent antivenoms being more efficient than the bivalent antivenom. Antibodies against venom components were detected by all artisera at different titers by ELISA. Upon IE, antisera against M. spiixi and M. frontalis venoms cross-reacted with the four types of venoms studied and recognized several molecular components, the precipitin lines obtained had distinct intensities and electrophoretic motilities, whereas the antivenom against M. corallinus only recognized components of its venom but not of the others. All antivenoms cross-reacted with all the elapid venoms in WB revealing several blands with distinct MWs in M. corallinus and M. spiixi venoms, two very sharp and separate bands in M. corallinus venom and a very sharp band of high MW together with several other smaller and faint bands in M. frontalis venom. The data indicate that snake venoms of the genus Micrurus are good immunogens that contain many cross-reactive molecules, and that their toxic components are neutralized more effectively by monovalent rather than by bivalent antivenom

Snake antivenoms from hyperimmunized horses: comparison of the antivenom activity and biological properties of their whole IgG and F(ab')2 fragments.

IgG and F(ab')2 fragments were prepared from horse plasma rich in specific antibodies against Brazilian Bothrops or Crotalus venoms. Both preparations, free of gross contamination with non-immunoglobulin proteins, were able to combine in vitro with their respective antigens, forming immune complexes at antigen excess, equivalence or antibody excess, and activating the C system, through either the classical or the alternative pathways. The IgG preparation was more effective in neutralizing the lethal factors in Bothrops or Crotalus venoms, compared with the F(ab')2 fragments. In contrast, IgG and F(ab')2 anti-Bothrops venom were almost equipotent in neutralizing the haemorrhagic and defibrinating activities in the venom. The method used to purify IgG, precipitation of most non-immunoglobulin plasma proteins with caprylic acid, produced antivenoms richer in specific antibodies, with higher specific activity, recovery and yield, compared with the method commonly used to prepare antivenoms containing F(ab')2.

[Anatomical and clinical features of a case of gall-bladder abnormality. Results of arteriographic and micro-angiographic studies (author's transl)]. / A propos d'une anomalie de la vésicule biliaire humaine. Aspects anatomo-cliniques. Etude artériographique et micro-angiographique.

The authors describe a perplexing clinical picture in which an important factor for diagnosing the lesion in the gall-bladder was hyperselective arteriography of the coeliac trunk, and more especially the hepatic artery. The association of a truc septum in the gall-bladder, without gall-stones, and of another digestive tube lesion, megadolicho-sigmoid and diverticulosis of the sigmoid, would appear to be an extremely rare entity, and this has been the cause of the pain reported by the patient over many years. The cholecystectomy performed appears to be the right choice, and the most effective therapeutic procedure.