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Immunology

The poison on long legs inside the house

Team from Butantan tests new serum and ointment against lesions caused by brown spider bites

Ten years ago, when she was beginning to study brown spiders, the most poisonous species in Brazil, biologist Denise Tambourgi spent three sleepless nights, frightened to death of getting a bite. It was because she had found one of them hidden in the trunk at the foot of her bed, during a collection expedition made on a farm in the municipality of Telêmaco Borba, in the state of Paraná. Even today, she has not become used to these little animals with long, thin legs, sometimes so small to the point of almost not being seen. Even though they are not aggressive and only attack when touched, they caused about 8 thousand cases of poisoning a year in 2003 and 2004 in Brazil, with two deaths, one in the state of Santa Catarina and the other in the state of Goiás.

But Denise has advanced a lot in her research into these spiders in this time. With her team from the Immunochemical Laboratory of the Butantan Institute, she has deciphered the main components of the poison, she has discovered how they act, and developed two new treatments capable of neutralizing the action of the toxins on the human body. One of them is a new serum, now at the stage of testing in vitro and on animals, specifically against the species of brown spider found in Brazil. Produced on the basis of sphingomyelinase, the protein that really causes the damage to the human body, discovered in 1998 by the team from Butantan, the new anti-loxoscelic serum may become an alternative to the one currently used, an antiarachnid serum, prepared only with the poison extracted from one of them, Loxosceles gaucho, and also employed against the bites of scorpions and armed spiders.

The other treatment is an ointment for the lesions caused by the poison of these spiders at the place of the bite and that can take as long as eight months to heal – depending on the extent of necrosis, a skin implant may be necessary. The damage can be even greater: when falling into the bloodstream, a few micrograms of the substance are capable of destroying the red cells of the blood-, impairing the workings of the kidneys and leading to death. The ointment from Butantan contains tetracycline, an antibiotic that acts as an inhibitor of the enzymes activated by the toxins of the spider, and reduced by 75% the development of necrosis of the skin, according to tests carried out on rabbits. Nowadays, the lesion is treated by means of the application of serum and specific medicines like the corticosteroids. “The ideal treatment against the bite of a brown spider will be a very specific and polyvalent serum for neutralizing the action of the poison of several species, added to anti-inflammatories and to a protease inhibitor (an enzyme capable of breaking up proteins)”, Denise explains.

Today’s serums neutralize the toxins in circulation in the human organism, but they are not very effective for treating the lesions on the skin. The reason is that, as the bite of the brown spider is painless and the local reaction does not manifest itself immediately, people only look for help when the lesion in the skin has already set in. Necrosis of the tissues is then no longer a consequence of the poison, but of the reactions of the organism itself. As the team from Butantan found out, one protein from the poison of the brown spider, sphingomyelinase plays a key role on the death of the tissues of the skin. This toxin activates other proteins inside the cells of the skin, which destroy the cells themselves and, consequently, the tissue, according to a study carried out by Danielle Paixão Cavalcante, a member of Denise’s team, published this May in the Journal of Investigative Dermatology.

Nowadays, the antiarachnid serum does not succeed in preventing completely the action of the poison of the three species of brown spider that cause most accidents in Brazil: Loxosceles intermedia, L. laeta and L. gaucho. And there is another problem, which makes it difficult to produce: the quantity of poison extracted from a Loxosceles is very small, around 30 micrograms – to immunize a horse during the preparation of the serum about 20 milligrams has to be applied, a quantity almost 700 times greater. In collaboration with Paulo Lee Ho and Inácio Junqueira, from Butantan’s Biotechnology Center, pharmacist Matheus Fernandes Pedrosa solved this problem in 2002, cloning the gene that contains the recipe for producing the sphingomyelinase protein in L. laeta, a species found in the south of Brazil and in several countries of South, Central and North America, whose poison is the most toxic of all of the genus. The work, published that year in the Biochemical and Biophysical Research Communication, was the first cloning of the sphingomyelinase gene in the world.

Last year, Denise, colleagues from her team and from the Faculty of Medicine of the University of Wales, in Cardiff, United Kingdom, took one more important step towards increasing the production of toxins and serum: they managed to copy the gene of two sphingomyelinase proteins of Loxosceles intermedia, responsible for over 95% of the cases of poisoning in the country, the majority of them in Paraná – in this state, this species is everywhere: in the houses and in the buildings. Described in Molecular Immunology, this process of cloning multiplied the production of poison 5 thousand times: each liter of solution with the genetically modified bacteria, to the genome of which the sphingomyelinase gene was added, yields about 15 milligrams of protein.

Tested on rabbits, the serum made from the proteins from the species of L. laeta and L. intermedia worked. The next step is to get an antidote that covers the sphingomyelinases of various kinds of brown spider. “A polyvalent serum is the most effective way of neutralizing the poison of each one of the species”, Denise explains. Her team, associated with Hisako Higashi’s group, from Butantan’s Production Division, has now begun to compare the degree of efficiency of the new polyvalent serum with the traditional antiarachnid one, produced at Butantan and distributed to the whole country. These are the studies that are going to indicate if it is really worthwhile to replace the current one and make the new compound on a large scale.

Against the red blood cells
It was also with the collaboration of a team from the University of Wales that Denise, Rute de Andrade and Fábio Magnoli, from Butantan, revealed, five years ago, the mechanism by which sphingomyelinase causes the destruction of red cells of the blood. The sphingomyelinase, which had been identified by Denise two years before, makes the red blod cells be perceived as a foreign agent in the organism and, for that reason, be eliminated from the immune system, according to an article published in 2000 in Blood magazine.

When a person is bitten by the brown spider, the sphingomyelinase of the poison binds itself to the red cells, breaks up a lipid – a kind of fat -, called sphingomyelinase, and alters the workings of these cells. This chemical mess puts some enzymes into action, which cut out other proteins, the glycophorins. When they are broken up, the glycophorins lose sialic acid, which makes one of the organism’s defense mechanisms, the so-called complement system, see the red cells as invaders and destroy them. “All the proteins regarded as important in regulating a component of the immune system, the complement system, were intact on the surface of the red cells”, Denise comments. The discovery of this mechanism redimensioned the role of the glycophorins in the hemacytes and in the control of this part of the organism’s defense system.

By understanding the mechanism for the poison to act and cloning the gene of different sphingomyelinases, it became simpler to obtain the molecular structure of this protein, which shows itself in the shape of a barrel. Denise’s team, in collaboration with the group headed up by biophysicist Raghuvir Arni, from the São Paulo State University (Unesp) of São José do Rio Preto, made a crystal from the protein obtained from Loxosceles laeta. Through this work, published this April in the Journal of Biological Chemistry, it may be possible to identify the active points of the protein and, from that, to design other medicines capable of fighting the poison of the brown spider.

The seriousness of the poisoning varies according to the kind of spider and of the victim of the bite. Adult spiders usually caused more serious accidents than the younger ones, because the volume and concentration of toxins in the poison increases with the time of life. The poison of the males has less power of action than the females’, usually more robust. The milder cases are caused by males of the L. gaucho, and the more serious ones by females of L. laeta. On the part of the persons, the degree of poisoning varies according to the age of the victim, his/her genetic constitution, and the place affected. For reasons still not fully understood by specialists, some people show generalized reactions, such as disseminated coagulation and rupture of the red cells, which can result in kidney failure and even death.

In Latin America, the majority of fatal victims is made up of children, particularly those bitten by L. laeta. In Brazil, mortality seems to be lower and is usually caused by the brown spider of the L. intermedia species. “About 80% of the accidents in Paraná come from the city of Curitiba”, says Marcelo Santalucia, from the National Health Foundation (Funasa), an organ of the Ministry of Health that receives the cases notified by the municipalities. “After carrying out the investigation, half the cases are discarded, but the database is not cleaned up, as that is something that only the municipality can do.” Denise Tambourgi, though, believes that the symptoms caused by the bite of the brown spider are often mistaken for those caused by bacterial infections or by allergic reactions. “In many regions”, she says, “the cases of poisoning are probably dealt with as a bacterial infection”.

Differences aside, nobody who knows brown spiders puts in doubt their toxic potential, which the inhabitants of Curitiba and of other cities from Paraná have already learnt to respect. But the tourists and guides at the Upper Ribeira State Tourist Park (Petar), in the south of the state of São Paulo, did not know that those caves were also inhabited by these spiders. The researchers from Butantan did not know whether the poison of Loxosceles adelaida, which lives in rustic environments, like the caves, was as dangerous as that of the other species from urban environments. Biologist Rute de Andrade confirmed that there were indeed spiders of this species in Petar, after covering the 22 caves of the park and finding specimens of L. adelaida in all of them. She and zootechnician Fernando Pretel confirmed that their poison is just as toxic as that of the other spiders of the same genus. It was an important discovery that the researchers from Butantan set about spreading around, with the support, in the institute itself, of the team from the Applied Toxinology Center (CAT), one of the Research, Innovation and Diffusion Centers (Cepids) funded by FAPESP. Last year, the biologists warned of the danger by talking to the tourist guides and distributing leaflets in the city halls, the guest houses and in the hotels of the park itself, whose caves receive 16 thousand tourists a year. “The Loxosceles adelaida has a different behavior from the other brown spiders. They are extremely active and get into rucksacks and clothes”, Denise warns. “It is important to make the scientific work reach society.”

The Project
Molecular mechanisms of hemolysis induced by the poison of the Loxosceles intermedia spider and Loxosceles in the karstic area of the Ribeira Valley: Identification of the fauna, biological and immunochemical characterization of the poisons, study of the action mechanism of the toxins; Coordinator Denise Vilarinho Tambourgi – Butantan Institute; Investiment R$ 162,221.96 and R$ 251,402.01  FAPESP and R$ 500,000 Wellcome Trust

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