The Diversified O-Superfamily in Californiconus californicus Presents a Conotoxin with Antimycobacterial Activity.

Californiconus californicus, previously named Conus californicus, has always been considered a unique species within cone snails, because of its molecular, toxicological and morphological singularities; including the wide range of its diet, since it is capable of preying indifferently on fish, snails, octopus, shrimps, and worms. We report here a new cysteine pattern conotoxin assigned to the O1-superfamily capable of inhibiting the growth of Mycobacterium tuberculosis (Mtb). The conotoxin was tested on a pathogen reference strain (H37Rv) and multidrug-resistant strains, having an inhibition effect on growth with a minimal inhibitory concentration (MIC) range of 3.52⁻0.22 µM, similar concentrations to drugs used in clinics. The peptide was purified from the venom using reverse phase high-performance liquid chromatography (RP-HPLC), a partial sequence was constructed by Edman degradation, completed by RACE and confirmed with venom gland transcriptome. The 32-mer peptide containing eight cysteine residues was named O1_cal29b, according to the current nomenclature for this type of molecule. Moreover, transcriptomic analysis of O-superfamily toxins present in the venom gland of the snail allowed us to assign several signal peptides to O2 and O3 superfamilies not described before in C. californicus, with new conotoxins frameworks.

In silico-designed mutations increase variable new-antigen receptor single-domain antibodies for VEGF165 neutralization.

The stability, binding, and tissue penetration of variable new-antigen receptor (VNAR) single-domain antibodies have been tested as part of an investigation into their ability to serve as novel therapeutics. V13 is a VNAR that recognizes vascular endothelial growth factor 165 (VEGF165). In the present study V13 was used as a parental molecule into which we introduced mutations designed in silico. Two of the designed VNAR mutants were expressed, and their ability to recognize VEGF165 was assessed in vitro and in vivo. One mutation (Pro98Tyr) was designed to increase VEGF165 recognition, while the other (Arg97Ala) was designed to inhibit VEGF165 binding. Compared to parental V13, the Pro98Tyr mutant showed enhanced VEGF165 recognition and neutralization, as indicated by inhibition of angiogenesis and tumor growth. This molecule thus appears to have therapeutic potential for neutralizing VEGF165 in cancer treatment.

Intraocular Penetration of a vNAR: In Vivo and In Vitro VEGF165 Neutralization.

Variable new antigen receptor domain (vNAR) antibodies are novel, naturally occurring antibodies that can be isolated from naïve, immune or synthetic shark libraries. These molecules are very interesting to the biotechnology and pharmaceutical industries because of their unique characteristics related to size and tissue penetrability. There have been some approved anti-angiogenic therapies for ophthalmic conditions, not related to vNAR. This includes biologics and chimeric proteins that neutralize vascular endothelial growth factor (VEGF)165, which are injected intravitreal, causing discomfort and increasing the possibility of infection. In this paper, we present a vNAR antibody against human recombinant VEGF165 (rhVEGF165) that was isolated from an immunized Heterodontus francisci shark. A vNAR called V13, neutralizes VEGF165 cytokine starting at 75 µg/mL in an in vitro assay based on co-culture of normal human dermal fibroblasts (NHDFs) and green fluorescence protein (GFP)-labeled human umbilical vein endothelial cells (HUVECs) cells. In the oxygen-induced retinopathy model in C57BL/6:Hsd mice, we demonstrate an endothelial cell count decrease. Further, we demonstrate the intraocular penetration after topical administration of 0.1 µg/mL of vNAR V13 by its detection in aqueous humor in New Zealand rabbits with healthy eyes after 3 h of application. These findings demonstrate the potential of topical application of vNAR V13 as a possible new drug candidate for vascular eye diseases.

Novel Imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxides as antiproliferative trypanosoma cruzi drugs: Computational screening from neural network, synthesis and in vivo biological properties.

A new family of imidazo[4,5-c][1,2,6]thiadiazine 2,2-dioxide with antiproliferative Trypanosoma cruzi properties was identified from a neural network model published by our group. The synthesis and evaluation of this new class of trypanocidal agents are described. These compounds inhibit the growth of Trypanosoma cruzi, comparable with benznidazole or nifurtimox. In vitro assays were performed to study their effects on the growth of the epimastigote form of the Tulahuen 2 strain, as well as the epimastigote and amastigote forms of CL clone B5 of Trypanosoma cruzi. To verify selectivity towards parasite cells, the non-specific cytotoxicity of the most relevant compounds was studied in mammalian cells, i.e. J774 murine macrophages and NCTC clone 929 fibroblasts. Furthermore, these compounds were assayed regarding the inhibition of cruzipain. In vivo studies revealed that one of the compounds, 19, showed interesting trypanocidal activity, and could be a very promising candidate for the treatment of Chagas disease.

Antivenom Evaluation by Electrophysiological Analysis.

Scorpion stings on humans are medically relevant because they may contain toxins that specifically target ion channels. During antivenom production, pharmaceutical companies must use a large number of experimental animals to ensure the antivenom's efficacy according to pharmacopeia methods. Here we present an electrophysiological alternative for the evaluation of horse antivenoms produced against two species of Moroccan scorpions: Buthus mardochei and Androctonus mauretanicus. Human sodium and potassium channels and acetylcholine nicotinic receptors were analyzed by standard patch-clamp techniques. The results showed that the antivenom is capable of reversing ion current disruption caused by the venom application. We propose the use of this in vitro technique for antivenom evaluation as an alternative to using a large number of live animals.

Anti-tumor necrosis factor VNAR single domains reduce lethality and regulate underlying inflammatory response in a murine model of endotoxic shock.

BACKGROUND: In sepsis, tumor necrosis factor (TNF) is the key factor triggering respiratory burst, tissue injury and disseminated coagulation. Anti-TNF strategies based on monoclonal antibodies or F(ab')2 fragments have been used in sepsis with contradictory results. Immunoglobulin new antigen receptors (IgNAR) are a unique subset of antibodies consisting of five constant (CNAR) and one variable domains (VNAR). VNAR domains are the smallest, naturally occurring, antibody-based immune recognition units, having potential use as therapy. Our aim was to explore the impact of an anti-TNF VNAR on survival in an experimental model of endotoxic shock. Also, mRNA expression and serum protein of several inflammatory molecules were measured. RESULTS: Endotoxic shock was induced by lipopolysaccharide (LPS) in male Balb/c mice. Animals were treated with anti-TNF VNAR domains, F(ab')2 antibody fragments, or saline solution 15 minutes before, 2 h and 24 h after lethal dose100 (LD100) LPS administration. TNF blockade with either VNAR domains or F(ab')2 fragments were associated with lower mortality (60% and 75%, respectively) compared to LD100. Challenge with LPS induced significant production of serum TNF and interleukins -10 and -6 at 3 h. After that, significant reduction of IL-6 at 24 h (vs 3 h) was shown only in the VNAR group. Nitrites level also increased in response to LPS. In liver, TNF and IL-10 mRNA expression showed a pro-inflammatory imbalance in response to LPS. Blocking TNF was associated with a shift towards an anti-inflammatory status; however, polarization was more pronounced in animals receiving F(ab')2 fragments than in those with VNAR therapy. With regard to IL-6, gene expression was increased at 3 h in all groups. TNF blockade was associated with rapid and sustained suppression of IL-6 expression, even more evident in the VNAR group. Finally, expression of inducible-nitric oxide synthase (iNOS) increased in response to LPS at 3 h, but this was decreased at 24 h only in the anti-TNF VNAR group. CONCLUSIONS: Anti-TNF VNAR single domains improved survival in a murine model of endotoxic shock. Protection was associated with regulation in the TNF/IL-10 balance, attenuation of IL-6 and iNOS gene expression in the liver as well as decreased serum IL-6 concentration.

Antimicrobial peptides from arachnid venoms and their microbicidal activity in the presence of commercial antibiotics.

Two antimicrobial peptides (AMPs), named La47 and Css54, were isolated from the venom of the spider Lachesana sp. and from the scorpion Centruroides suffusus suffusus, respectively. The primary structures of both La47 and Css54 were determined using N-terminal sequencing and mass spectrometry. La47 is identical to the AMP latarcin 3a obtained previously from the venom of the spider Lachesana tarabaevi, but the primary structure of Css54 is unique having 60% identities to the AMP ponericin-W2 from the venom of the ant Pachycondyla goeldii. Both La47 and Css54 have typical α-helix secondary structures in hydrophobic mimicking environments. The biological activities of both La47 and Css54 were compared with the AMP Pin2 isolated from the venom of the scorpion Pandinus imperator. La47 has lower antimicrobial and hemolytic activities compared with Css54 and Pin2. In addition, La47 and Pin2 were evaluated in the presence of the commercial antibiotics, chloramphenicol, ampicillin, novobiocin, streptomycin and kanamycin. Interestingly, the best antimicrobial combinations were obtained with mixtures of La47 and Pin2 with the antibiotics chloramphenicol, streptomycin and kanamycin, respectively. Furthermore, the novel peptide Css54 was evaluated in the presence of antibiotics used for the treatment of tuberculosis, isoniazid, rifampicin, pyrazinamide and ethambutol. Although the mixtures of Css54 with isoniazid, pyrazinamide or ethambutol inhibit the growth of Staphylococcus aureus, the best effect was found with rifampicin. Overall, these data show a motivating outlook for potential clinical treatments of bacterial infections using AMPs and commercial antibiotics.

Human TNF cytokine neutralization with a vNAR from Heterodontus francisci shark: a potential therapeutic use.

The therapeutic use of single domain antibodies (sdAbs) is a promising new approach because these small antibodies maintain antigen recognition and neutralization capacity, have thermal and chemical stability and have good solubility. In this study, using phage display technology, we isolated a variable domain of a IgNAR (vNAR) from a Heterodontus francisci shark immunized against the recombinant human cytokine TNFα (rhTNFα). One clone T43, which expresses the vNAR protein in the periplasmic space, was isolated from the fourth round of panning. T43 had the capacity to recognize rhTNF and neutralize it in vitro, indicating that T43 has potential as a therapeutic that can be used for diseases in which this pro-inflammatory cytokine needs to be controlled.

Pharmacokinetics of a F(ab')2 scorpion antivenom administered intramuscularly in healthy human volunteers.

This paper presents the first study of F(ab')(2) scorpion antivenom pharmacokinetics in humans after intramuscular (im) administration. The specific anti-Centruroides scorpion antivenom was used in 6 human healthy volunteers. The fabotherapeutic was administered as a 47.5mg im bolus. Blood samples were drawn at 0, 5, 15, 30, 45, 60 , 90, 120, and 180 min, 6h and at 1, 2, 3, 4, 10 and 21 days after antivenom administration. We measured antivenom concentrations in serum using a specific high sensitivity ELISA method for F(ab')(2). Antivenom concentration in serum was fit to a 3 compartment model (inoculation site, plasma and extra vascular extracellular space), it was assumed that the venom may also be irreversibly removed from plasma. Calculated time course of antivenom content shows that at any time no more that 16.6 (5.3, 31.9)% (median and 95% confidence interval) of the antivenom bolus is present in plasma. The time to peak plasma [F(ab')(2)] was 45 (33, 74) h. The most significant antivenom pharmacokinetic parameters determined were: AUC(im,∞)=803 (605, 1463) mg·h·L(-1); V(c)=8.8 (2.8, 23.6) L; V(ss,im)=55 (47, 64) L; MRT(im)=776(326, 1335) h; CL(t)=3.7 (0.6, 1.9) mL·min(-1); f(im,)V(ss)=0.300 (0.153, 0.466). Comparing these parameters with the ones obtained intravenously by Vázquez et al., the parameters were more disperse between subjects, determined with more uncertainty in each individual subject, and the peak F(ab')(2) in plasma occurred with considerable delay; all indicating that the IM route should not be used to administer the antivenom, with the possible exception of cases occurring very far from hospitals, as an extreme means to provide some protection before the IV route becomes available.

Characterization of a new polyvalent antivenom (Antivipmyn Africa) against African vipers and elapids.

As a response to the antivenom shortage in Sub-Saharan Africa, evident for well over a decade, we developed a new polyvalent anti-ophidian antivenom (Antivipmyn((R)) Africa) designed for use in the region. We report a detailed characterization of its biochemical composition (protein content and profiling by size-exclusion chromatography and electrophoresis) as well as the specific and para-specific neutralization potencies (as median effective dose in the mouse lethality test). Additionally, we studied the neutralization of hemorrhagic, anti-hemostatic and necrotic activities of Echis ocellatus venom, responsible for a majority of severe envenomations in the continent according to existing epidemiological data. The antivenom is currently under production and has already been employed in the field in a pragmatic Phase III clinical trial in the Republic of Benin. It is a purified lyophilized polyvalent equine F(ab')(2)-based product obtained by immunization with the venoms of eleven species of African snakes of the Genera Echis, Bitis, Naja and Dendroaspis. The criteria for its design are discussed, particularly in terms of the implementation of realistic public health policies targeting mostly rural populations in the continent.

North and South American Loxosceles spiders: development of a polyvalent antivenom with recombinant sphingomyelinases D as antigens.

We report the cloning of sphingomyelinase D (SMD) cDNA from Loxosceles reclusa, Loxosceles boneti and Loxosceles laeta into bacterial expression systems, as well as optimization of expression conditions so as to obtain soluble and active recombinant enzymes. The recombinant mature SMDs, tagged with a histidine tail at the N- or C-termini, were compared in terms of toxicity and enzymatic activity, and were used as immunogens for the production of monovalent antisera in rabbits and F(ab')(2) preparations in animals used for commercial antivenom production (horses). We performed studies on in vitro inhibition of enzymatic activity of natural venom preparations by antibodies generated against the tagged proteins. We also present and discuss the results of studies on the specific and para-specific in vivo protective potential of the rabbit and equine antibody preparations against the recombinant proteins themselves and natural venom preparations. Our conclusions support the feasibility of using recombinant SMDs for production and evaluation of polyvalent anti-Loxosceles antivenoms, and we offer data on the potential of paraspecific neutralization in the context of the antigenic groupings and the molecular phylogeny of those active SMDs for which amino acid sequence information is available.

[Poisoning by bee sting]. / Envenenamiento por picaduras de abeja.

Among the human pathologies produced by venomous animals, bee stings constitute the largest number of accidents in several countries, exceeding the mortality rate caused by other venomous animals such as snakes, spiders or scorpions. The clinical picture after the bee sting may include anaphylaxis or poisoning. The latter is produced by massive attacks and is a serious problem that may put the patient's life at risk. People that are poisoned display hemolysis, rhabdomiolysis and acute renal failure that together with other systemic failures can bring about death. The knowledge of the physiopathological mechanisms involved in the massive attack of bees is crucial for health care professionals as to date we do not have antivenoms with proven clinical efficacy. In this review we include the bee's biological aspects, venom composition and its relation with the occurrence and severity of accidents as well as epidemiological data that can be useful for this type of accidents.

Envenenamiento por picaduras de abeja / Poisoning by bee sting

Dentro de las patologías humanas producidas por animales con la capacidad de inocular veneno, las picaduras de abeja producen el mayor número de accidentes por animales en muchos países, superando a menudo en mortalidad a los producidos por serpientes, escorpiones y arañas. El cuadro clínico por la picadura de estos himenópteros puede consistir en fenómenos alérgicos o en cuadros de envenenamiento. Estos últimos se producen por el ataque de enjambres constituyendo un hecho grave que puede comprometer la vida. En el sujeto envenenado pueden observarse hemólisis, rabdomiólisis e insuficiencia renal, que junto a otras alteraciones sistémicas pueden conducir a la muerte. El conocimiento de los acontecimientos fisiopatológicos que se producen ante los ataques masivos de abejas es de suma importancia para el personal de salud dado que hasta la fecha no existen antivenenos que hayan demostrado tener eficacia clínica comprobada. En esta revisión se consideran los aspectos biológicos de las abejas y de la composición de su veneno relacionado con la ocurrencia y severidad de los accidentes, así como datos epidemiológicos de utilidad para enfrentarse a este tipo de cuadro.

Among the human pathologies produced by venomous animals, bee stings constitute the largest number of accidents in several countries, exceeding the mortality rate caused by other venomous animals such as snakes, spiders or scorpions. The clinical picture after the bee sting may include anaphylaxis or poisoning. The latter is produced by massive attacks and is a serious problem that may put the patient's life at risk. People that are poisoned display hemolysis, rhabdomiolysis and acute renal failure that together with other systemic failures can bring about death. The knowledge of the physiopathological mechanisms involved in the massive attack of bees is crucial for health care professionals as to date we do not ha ve antivenoms with proven clinical efficacy. In this review we include the bee's biological aspects, venom composition and its relation with the occurrence and severity of accidents as well as epidemiological data that can be useful for this type of accidents.

[Toxicity of venoms from snakes of medical importance in México]. / Toxicidad de venenos de serpientes de importancia médica en México.

The characterization of the toxic activities of snake venoms is necessary to understand the physiopathology of the envenomation and to test the potency of the antivenoms used to treat this pathology. Because of the lack of data on the toxic activities of venoms from Mexican snakes of medical importance, we studied the venoms from Bothrops asper, Athropoides nummifrr, Agkistrodon billineatus, Crotalus durissus durissus, Crotalus basiliscus, Crotalus scutulatus, Crotalus atrox and Micrurus nigrocinctus. The studies performed were: SDS-PAOE, determination of lethal potency, hemorrhagic, necrotizing, coagulation on plasma and fibrinogen, phospholipasic and fibri(noge)nolytic activities. In addition we studied the neutralizing capacity of the toxic activities of an antivenom currently used for the treatment of snakebites in Mexico. The venom from viperids showed important hemorrhagic, necrotizing, coagulative on plasma, prothrombinic, fibrinogenolytic and phospholipase activities. The venoms with the highest lethal potency were those of Micrurus nigrocinctus and Crotalus scutulatus; however, the viperine venom that globally displayed the most potent toxic activities was from Bothrops asper. All the venoms showed toxic activities of similar range to those described for other American venomous snakes. The activity on plasma or fibrinogen varied widely among the different venoms but all displayed capacity to act on the coagulation system. The antivenom tested not only neutralized the lethality B. asper venom but also its other toxic activities.

Toxicidad de venenos de serpientes de importancia médica en México / Toxicity of venoms from snakes of medical importance in Mexico

La caracterización de las actividades tóxicas de los venenos de serpientes es necesaria para el cabal entendimiento de los procesos fisiopatológicos que se producen ante su mordedura, como también para evaluar la potencia neutralizante de los antivenenos utilizados para tratar estos envenenamientos. A causa de los pocos datos disponibles sobre la toxicidad del veneno de serpientes con importancia sanitaria en México, estudiamos las actividades tóxicas de los venenos de Bothrops asper, Athropoides nummifer, Agkistrodon billineatus> Crotalus durissus durissus, Crotalus basiliscus, Crotalus scutulatus, Crotalus atrox y Micrurus nigrocinctus. A los venenos se les realizaron los siguientes estudios: SDS-PAUE, determinación de la potencia letal, y de las actividades hemorrágica, necrotizante, coagulante en plasma y fibrinógeno, fosfolipásica y fibrinogenolítica. Se estudió además la capacidad neutralizante de un antiveneno de uso corriente para la terapéutica de las mordeduras de serpientes venenosas en México, sobre varias de estas actividades. Los venenos de vipéridos mostraron actividades hemorrágicas, necrotizante, coagulante sobre plasma, protrombínica, fibrinogenolítica y fosfolipásica importantes. Los venenos de mayor potencia letal fueron los de Micrurus nigrocinctus y Crotalus scutulatus, sin embargo el veneno que presentó en general potencias tóxicas mayores fue el de Bothrops asper. Las diferentes potencias tóxicas halladas se encontraron dentro de los márgenes descritos para especies de vipéridos y elápidos de Sudamérica. La actividad sobre el plasma y el fibrinógeno fue muy diferente en los diferentes venenos viperinos, sin embargo todos mostraron ser capaces de afectar componentes del sistema de la coagulación. El antiveneno probado no sólo neutralizó la letalidad del veneno sino también sus actividades tóxicas.

The characterization of the toxic activities of snake venoms is necessary to understand the physiopathology of the envenomation and to test the potency of the antivenoms used to treat this pathology. Because of the lack of data on the toxic activities of venoms from Mexican snakes of medical importance, we studied the venoms from Bothrops asper, Athropoides nummifer, Agkistrodon billineatus, Crotalus durissus durissus, Crotalus basiliscus, Crotalus scutulatus, Crotalus atrox and Micrurus nigrocinctus. The studies performed were : SDS-PAOE, determination of lethal potency, hemorrhagic, necrotizing, coagulation on plasma and fibrinogen, phospholipasic and fibrinogenolytic activities. In addition we studied the neutralizing capacity of the toxic activities of an antivenom currently used for the treatment of snakebites in Mexico. The venom from viperids showed important hemorrhagic, necrotizing, coagulative on plasma, prothrombinic, fibrinogenolytic and phospholipase activities. The venoms with the highest lethal potency were those of Micrurus nigrocinctus and Crotalus scutulatus; however, the viperine venom that globally displayed the most potent toxic activities was from Bothrops asper. All the venoms showed toxic activities of similar range to those described for other American venomous snakes. The activity on plasma or fibrinogen varied widely among the different venoms but all displayed capacity to act on the coagulation system. The antivenom tested not only neutralized the lethalityB. asper venom but also its other toxic activities.

Genetic and enzymatic characterization of sphingomyelinase D isoforms from the North American fiddleback spiders Loxosceles boneti and Loxosceles reclusa.

In this study we report the isolation and characterization of several sphingomyelinase D isoforms from the venoms of the North American spiders Loxosceles boneti and Loxosceles reclusa, from Mexico and the United States, respectively. We have measured their enzymatic activity, their capacity to induce necrotic lesions in rabbits, cloned the cDNAs coding for the mature forms of two of the isoforms from L. boneti and two of L. reclusa based on N-terminal sequence information of the purified proteins, and performed a comprehensive comparison of the sequence data generated by us with that reported for other sphingomyelinase genes to date.

Effectiveness of two common antivenoms for North, Central, and South American Micrurus envenomations.

Micrurus snakes (coral snakes) may produce severe envenomation that can lead to death by peripheral respiratory paralysis. Only few laboratories produce specific antivenoms, and despite the cross-reactivity found in some Micrurus species venoms, the treatment is not always effective. To test two therapeutic antivenoms against the venom of four species of Micrurus from Southern America, North of South America, Central America, and North America, the determination of the lethal potency of the venoms, the study of some biochemical and immunochemical characteristics, and the determination of the neutralizing activity of both antivenoms were studied. North American and South American antivenoms neutralized well venoms from Micrurus species of the corresponding hemisphere but displayed lower effectiveness against venoms of species from different hemispheres. It was concluded that the neutralization of Micrurus venoms by regional antivenoms could be useful to treat the envenomation by some Micrurus snakes but is necessary to evaluate carefully the antivenoms to be used with the venoms from the snakes of the region. Also, considering the difficulties for coral snake antivenom production, the development of a polyvalent antivenom is useful to treat the envenomation by coral snakes from different regions is necessary.

DIG-ELISA test in onchocerciasis: serum antibodies and probability of infection

An immunoenzymatic test (DIG-ELISA) was serologically evaluated for the serodiagnosis of onchocerciasis. Control and infected sera from the onchocerciasis endemic area of Mexico was collected and the donors assessed for onchocerciasis according to parasitological, clinical, and epidemiological data. The sera were submitted to the DIG-ELISA test using a crude extract prepared from O. volvulus adults woems which had been preserved in nodules in 67 percent glycerol. The test showed a 100 percent sensitivity with sera from 38 microfilariae carriers and 96 percent specificity with sera from 133 non-infected people living outside the endemic zone. In addition, seropositivity was 52.9 percent with samples from nononchocercotic people living indide the endemic area, while 82.5 and 90.5 percent of sera from patients with clinical symptoms and subcutaneous nodules, respectively, were positive. A high rate (30 percent) of cross-reactivity with serum samples from people infected with Wuchereria bancrofti or Brugia timori was obtained, which is in contrast with the low seropositivity rates (7.4 percent) obtained with sera from patients infected with other parasites. These results suggest that DIG-ELISA test may be a useful serological test for antibody detection in onchocerciasis, especially for epidemiological surveillance of disease, but because of the high cross-reactivity observed, its use must be limited to areas where this parasitic infection does not coexist with other human filiariasis. Finally, the arrangement of sera in groups according to the relative likelihood to have onchocerciasis seems to be a useful procedure for evaluation of serologicales tests