Venom of the neotropical rattlesnake, Crotalus culminatus: Intraspecific variation, neutralization by antivenoms, and immunogenicity in rabbits.

Crotalus culminatus is a medically significant species of rattlesnake in Mexico [1]. While the proteomic composition of its venom has been previously reported for both juvenile and adult specimens, there has been limited research into its functional properties, with only a few studies, including one focusing on coagulotoxicity mechanisms. In this study, we aimed to compare the biochemical and biological activities of the venom of juvenile and adult snakes. Additionally, we assessed antibody production using the venoms of juveniles and adults as immunogens in rabbits. Our findings reveal lethality and proteolytic activity differences between the venoms of juveniles and adults. Notably, juvenile venoms exhibited high proportions of crotamine, while adult venoms displayed a reduction of this component. A commercially available antivenom demonstrated effective neutralization of lethality of both juvenile and adult venoms in mice. However, it failed to neutralize the paralytic activity induced by crotamine, which, in contrast, was successfully inhibited by antibodies obtained from hyperimmunized rabbits. These results suggest the potential inclusion of C. culminatus venom from juveniles in commercial antivenom immunization schemes to generate antibodies targeting this small myotoxin.

Neutralization of black widow spider (Latrodectus mactans) venom with rabbit polyclonal serum hyperimmunized with recombinant alpha-latrotoxin fragments.

Alpha-latrotoxin (ɑLTx) is the component responsible for causing the pathophysiology in patients bitten by spiders from the genus Latrodectus, commonly known as black widow spiders. The current antivenom used to treat these envenomations in Mexico is produced using the venom of thousands of spiders, obtained through electrical stimulation. This work aimed to produce this protein as well as two of its fragments in a bacterial model, to evaluate their use as immunogens to produce neutralizing hyperimmune sera, in rabbits. ɑLTx is a 130 kDa protein which has not yet been obtained in a soluble active form using bacterial models. In the present work, ɑLTx and two of its fragments, ankyrin domain and amino terminal domain (LTxAnk and LTxNT) were produced in bacteria and solubilized from inclusion bodies using N-lauroyl sarcosine. These three proteins were used for hyperimmunization in order to evaluate their potential as immunogens for the production of neutralizing hyperimmune sera against the complete venom of Latrodectus mactans. The hyperimmune sera obtained using the complete ɑLTx as well as the LTxNT, was capable of preventing death of mice envenomated with 3 LD50s of venom, both in preincubation and rescue experiments. Conversely, the serum obtained using the LTxAnk fragment, generated only partial protection and a delay in the time of death, even with a maximum dose of 450 µL. We therefore conclude that the produced proteins show great potential for their use as immunogens and should be further tested in large animals, such as horses.

Characterization of the venom and external morphology of a natural hybrid between Crotalus atrox and Crotalus mictlantecuhtli.

Here we report, for the first time, a natural hybrid between Crotalus atrox and C. mictlantecuhtli based on intermediate characteristics of the external morphology and venom. Morphologically, the individual had characteristics of both parent species. The hybrid's venom exhibited an intermediate composition including the presence of crotoxin which has never been documented in C. atrox but is well documented in C. mictlantecuhtli. The hybrid's venom was highly toxic and showed an intermediate proteolytic activity between the parental species. The two Mexican antivenoms were able to neutralize the hybrid's venom's lethality.

Neutralization of crotamine by polyclonal antibodies generated against two whole rattlesnake venoms and a novel recombinant fusion protein.

Crotamine is a paralyzing toxin (MW: ~5 kDa) found in different proportions in some rattlesnake venoms (up to 62%). Mexican pit viper antivenoms have shown low immunoreactivity against crotamine, which is an urgent quality to be improved. The objective of this work was to evaluate the ability of a novel recombinant fusion protein composed of sphingomyelinase D and crotamine, and two whole venoms from Crotalus molossus nigrescens and C. oreganus helleri to produce neutralizing antibodies against crotamine. These immunogens were separately used for immunization procedures in rabbits. Then, we generated three experimental antivenoms to test their cross-reactivity via western-blot against crotamine from 7 species (C. m. nigrescens, C. o. helleri, C. durissus terrificus, C. scutulatus salvini, C. basiliscus, C. culminatus and C. tzabcan). We also performed pre-incubation neutralization experiments in mice to measure the neutralizing potency of each antivenom against crotamine induced hind limb paralysis. Our antivenoms showed broad recognition across crotamine from most of the tested species. Also, neutralization against crotamine paralysis symptom was successfully achieved by our three antivenoms, albeit with different efficiencies. Our results highlight the use of crotamine enriched venoms and our novel recombinant fusion protein as promising immunogens to improve the neutralizing potency against crotamine for the improvement of Mexican antivenoms.

TIE2 Induces Breast Cancer Cell Dormancy and Inhibits the Development of Osteolytic Bone Metastases.

Breast cancer (BCa) cells disseminating to the bone can remain dormant and resistant to treatments for many years until relapsing as bone metastases. The tyrosine kinase receptor TIE2 induces the dormancy of hematopoietic stem cells, and could also induce the dormancy of BCa cells. However, TIE2 is also a target for anti-angiogenic treatments in ongoing clinical trials, and its inhibition could then restart the proliferation of dormant BCa cells in bone. In this study, we used a combination of patient data, in vitro, and in vivo models to investigate the effect of TIE2 in the dormancy of bone metastases. In BCa patients, we found that a higher TIE2 expression is associated with an increased time to metastases and survival. In vitro, TIE2 decreased cell proliferation as it increased the expression of cyclin-dependent kinase inhibitors CDKN1A and CDKN1B and arrested cells in the G0/G1 phase. Expression of TIE2 also increased the resistance to the chemotherapeutic 5-Fluorouracil. In mice, TIE2 expression reduced tumor growth and the formation of osteolytic bone metastasis. Together, these results show that TIE2 is sufficient to induce dormancy in vitro and in vivo, and could be a useful prognostic marker for patients. Our data also suggest being cautious when using TIE2 inhibitors in the clinic, as they could awaken dormant disseminated tumor cells.