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IMMUNOLOGY

Rattlesnake antidote gets lighter

New immunotherapy to permit less aggressive treatment against snake’s poison

The rattlesnake is the snake that causes most deaths in Brazil.  Two surveys by the Butantan Institute (São Paulo) show that it is possible to use fragment of the rattlesnake’s anti-poison antibodies to act specifically against the venom, reducing the serious collateral effects of serum therapy. This possibility of a treatment that is less aggressive than the conventional one – which was taken up over 100 years ago and can often be jeopardized by adverse reactions when applied – has shown good results in the laboratory, in tests on mice.

The rattlesnake poison (Crotalus durissus terrificus) is 65% crotoxin, a protein that is its main active component. It inhibits the movement of the muscles: for this reason, the victim can even stop breathing, if suitable assistance is not received in time, somewhere between 2 and 6 hours. The consequences of poisoning are well known, but little is known about the effects of the poison on the cells and on the development of the immune response by the organism.  It was precisely this that was the focus of the project Effect of the Venom of Crotalus durissus terrificus (rattlesnake) on the Immune System, run by Professor Ivan da Mota e Albuquerque, the Scientific Leadership of the Immunopathology Laboratory of the Butantan Institute.

Crotoxin acts in the region of contact between the nerve and the muscle, inhibiting the release of acetyl colina at the level of the neuromuscular joints. It thereby prevents the transmission of the nervous stimulus for the muscle to contract: “One of the first signs that the venom is acting is the ptosis of the eyelid – the person cannot lift his eyelids”, says Professor Ivan.

When the organism perceives the entry of strange proteins, it reacts against them by producing antibodies, made up of blood cells called lymphocytes. The B-lymphocytes produce antibodies that neutralize the toxic effects of the poison. Earlier results achieved at the Immunopathology Laboratory showed that the crotoxin in the rattlesnake’s venom diminishes considerably the immune response, so that the victim’s immunological system produces fewer antibodies, in particular anti-crotoxin, which neutralize the effects of the crotoxin.

As the response of the B lymphocytes is strongly influenced by the cytosines (substances that are also produced by them), researchers Diva Ferreira Cardoso, Ivan da Mota and his team decided to inject into mice (Mus musculus) both the rattlesnake’s poison  and the crotoxin on its own. They then monitored the animal’s production of cytosine and antibodies, to study the action of the venom on the immunological system. They found out that both the venom and the crotoxin on its own inhibit the production of cytosines and antibodies, but that this suppressive effect on the immunological system occurs only with the B-lymphocytes (which produce antibodies), and not with the T-lymphocytes (which are responsible for the immunity of the cells).

The project, which started in February 1997 and ended in 1999, received funding for FAPESP and resulted in the thesis for Diva Cardoso’s doctorate, as well as an article in the most important scientific magazine on animal venom, Toxicon (Vol. 35: 607, 1997), signed by Mota and Diva.

From then onwards, they both decided to continue their studies on the poison, as an alternative to the anti-crotalinae serum produced in horses or sheep. Efficient in neutralizing the toxic components of the poison, this serum , taken from the animals that have been immunized with the complete venom, causes undesirable reactions in between 30% and 84% of the cases, which range from the so-called serum disease to anaphylactic shock. The serum disease produces a skin rash that, in more serious cases, causes a breakdown of the kidneys. In addition, there is a decrease in the immunity caused by the poison itself, which results in a situation that is complicated to resolve.

Regarding the mechanism that makes the venom act, the researchers also wanted to answer further questions. One of them was to discover what was the dose of cytosines produced by the B-lymphocytes, and what effect crotoxin had on the production of cytosines by these lymphocytes.

In the report on his project, Mota already indicated, as a future objective, the obtaining of “monoclonal antibodies of human origin, for therapeutic purposes”, which would inhibit the collateral effects. And so his team started a new project, in the course of which the researcher Diva Cardoso succeeded in making great progress: she synthesized a fragment of human antibody, exactly the part that is connected to the antigen (the venom). This piece of the antibody, with specificity for the venom (it recognizes the venom), is called recombinant human antibody, or scFv.  “Diva showed that the scFv anti-crotoxin molecules are potentially capable of neutralizing, in vitro and in vivo, the enzymatic and lethal activity of the crotoxin, with animals in the experiment”, reveals Ivan da Mota.

The new project – Characterization of the Potential for Neutralization of the Anti-Crotoxin Recombinant Human Antibodies (scFv) on the Venom of the C. d. terrificus, compared with the Crotoxin of C. d. collineatus and C. d. cascavella – was the theme of Diva’s thesis, and continues to be carried out by biomedic Edna Cristina dos Santos, who has a FAPESP scholarship for her master’s thesis, under the supervision of Ivan da Mota, and with the collaboration of Hybridolab, of the Pasteur Institute, in France. The researchers have a long task ahead of them, which includes more tests with animals and humans, until scFv can be used in routine attendance.

Profile:
• Ivan da Mota e Albuquerque, 79 years old, graduated from the School of Medicine at the Federal University of Pernambuco. He was a professor of Histology at USP’s School  of Medicine, and of Immunology at USP’s Institute of Biomedical Sciences. He has been working since 1970 at the Laboratory of Immunopathology, of the Butantan Institute.
Projects:
1. Effect of the Venom of Crotalus durissus terrificus (rattlesnake) on the Immune System.
Investment: R$ 12,091.76 and US$ 36,200.37
2. Characterization of Crossed Reactivity and of the Potential for Neutralization of Anti-Crotoxin Recombinant Human Antibodies (scFv) on the Venom of Crotalus durissus terrificus compared with the Crotoxin of other Crotalinae Venoms.

 

An important legacy
Graduated in Sciences (1982) from São Paulo’s Mackenzie University, Diva Cardoso took her master’s degree in Microbiology and Immunology (1992), at the former São Paulo School of Medicine (now the Federal University of São Paulo), and her doctorate in Immunology (1999) at the Institute of Biomedical Sciences at the University of São Paulo. Since 1987, she has been a scientific researcher at the Laboratory of Immunopathology, of the Butantan Institute. She has signed ten papers in international publications, particularly on the venom of rattlesnakes.

She was the main responsible for obtaining a fragment of the antibody that may mean the difference between a more or a less aggressive treatment for the victims of rattlesnake bites. Dr. Diva’s important work, interrupted by her tragic death, in December 1999, has been resumed by the person whom she supervised for a master’s degree, Edna Cristina dos Santos, under the guidance of Professor Ivan Mota.  According to the presentation of the project, “the proposed alternative to an improvement in serum therapy is the possible use of homologous antibodies (of human origin), specific to the main toxins in the poison, which may avoid adverse reactions. Recombinant human antibodies are already being used successfully in the treatment of tumors, rejection of transplants, rheumatoid arthritis and allergies, suggesting that their use is also possible in the treatment of rattlesnake accidents.”

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