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Modulated defense

Advances against leishmaniasis involve controlling the immune response and the transmitting insect

NÁGILA SECUNDINO E PAULO PIMENTA / FIOCRUZHardly have they crossed through the skin and penetrated our body, foreign microorganisms come across proteins that circulate in the blood and work like a kind of radar. This contact – just like a row of falling dominoes – sets off, in succession, another 30 proteins, and puts the organism’s defense into action. Cells with specific functions arrive at the place of the invasion and start the fight, generating an inflammation. The more intense is this response, the more efficiently and rapidly the enemy is eliminated.

But this reaction, in general beneficial, becomes harmful when it is exaggerated, because not only it destroys the invader, but also damages the tissues of the body itself. It is precisely the hyperactivity of this mechanism that is the cause of the lesions of the skin and mucous membranes that are characteristics of the most common forms of tegumentary leishmaniasis, a tropical disease that occurs in 88 countries, caused by the protozoon (a single cell parasite) Leishmania brasiliensis.

Following this finding, reached in 1997 after over ten years of research, the team led by physician Edgar Marcelino de Carvalho Filho, from the Federal University of Bahia (UFBA), proposed alterations in the treatment of two of the most common forms of tegumentary leishmaniasis (cutaneous and mucocutaneous), which, or so everything indicates, make therapy cheaper and more effective – almost one century after the Scottish physician William Boog Leishman identified the causer of the disease. The new forms of treatment consist of associating the use of the compound traditionally employed – pentavalent antimony, which fights the parasite, but can cause muscular pains and heartbeats alterations – with drugs capable of controlling the level of activity of the defense system. Applied as yet in an experimental manner in cases where the antimony does not produce the desired effect, this therapy has been showing good results, as recent studies by the team from Bahia are showing.

Another encouraging finding is being added to these results. A team from the René Rachou Research Center, from the Oswaldo Cruz Foundation (Fiocruz), in Belo Horizonte, has identified a new species of worm – still unnamed – capable of killing the mosquito that transmits the Leishmania chagasi parasite, a protozoon that causes the most serious form of leishmaniasis: the visceral or kala-azar form, which affects the spleen and the liver and can be fatal when not treated. If the first experiments and field tests are successful, contaminating the insects with this worm may become the first form of biological control of the transmitter of the disease, Lutzomyia longipalpis, nowadays combated with strong pesticides that can contaminate the environment and, in human beings, cause damage to the central nervous system.

Urban problem
Assessed as a whole, the work by the teams from Bahia and Minas Gerais bring important contributions for understanding and fighting this disease, which has been spreading over all the regions of the country since 1980, according to the National Health Foundation (Funasa in the Portuguese acronym). With the devastation of forest areas and the migration of the rural population to the urban centers, both tegumentary leishmaniasis and the visceral form are ceasing to be exclusively rural and are reaching the outskirts of the medium and large sized cities. Between 1980 and 2001, Funasa registered 760,000 cases of tegumentary leishmaniasis, which now affects 41% of the 5,561 Brazilian municipalities, in particular in the northeastern, northern and central-western regions. In this period, 47,000 cases of the visceral form have been detected, above all in Belo Horizonte, in the state of Minas Gerais; Natal, in the state of Rio Grande do Norte; Teresina, in Piauí; Fortaleza, in Ceará; and São Luís, in Maranhão.

Although the disease has not reached the city of São Paulo, its occurrence in the state has been increasing, ever since the first cases appeared in the northwestern region, in 1999, according to epidemiologist Jancintho da Silva, Sucen – Superindent of Endemic Diseases Control, the organ that controls endemic diseases in the state. In a strip that runs from Bauru to the south of Mato Grosso do Sul, the sanitary authorities registered 98 cases of visceral leishmaniasis visceral in humans (with 11 deaths) between 1999 and 2002, besides 9,300 in dogs (5,300 just in Araçatuba). According to Silva, Sucen is testing the use of collars with insecticide as an alternative to putting the dogs down.

A specialist in immunology with parasites like the leishmania, Carvalho, from UFBA, began to suspect that the cause of the lesions of cutaneous and mucocutaneous leishmaniasis was the excessive response of the defense system – and not its below normal activity, as used to be believed – in 1986, when he examined inhabitants from the rural village of Corte de Pedra, in the municipality of Presidente Tancredo Neves. Located 280 kilometers to the south of Salvador, this is one of the regions with the largest numbers of occurrences of tegumentary leishmaniasis in Bahia.

Each year, a thousand cases of the cutaneous form arise – which causes impressive wounds of the skin, in a variety of numbers and sizes (they range from regions like blackheads to ulceration) – and 30 mucocutaneous cases – which destroys the mucous membranes and cartilage of the nose, mouth and throat, and in extreme cases can cause death by asphyxia.

When comparing the immune response of people with cutaneous leishmaniasis with that of victims of the mucocutaneous version, Carvalho noticed something abnormal: in the blood of the individuals with the blander form, the cutaneous one, the levels of cytokines (a kind of protein) against the protozoon were lower than with people with the more aggressive form – the opposite of what was expected. It was the first sign that perhaps the stronger immune response is the cause of the more serious lesions. It runs counter to what is to be seen in the third form of tegumentary leishmaniasis, the diffuse form, and in visceral leishmaniasis, in which the body practically does not produce any immune response against the protozoon.

Another indication that the researchers from Bahia were following the right path was to arise only some years later. On examining victims of the cutaneous and mucocutaneous forms of the disease in Corte de Pedra, Carvalho’s team decided to assess samples of tissue taken from the lesions, instead of testing the blood. To everyone’s surprise, the wounds were free from the protozoa. But the lesions showed a high quantity of two proteins that are fundamental to the defense of the organism: interferon gamma and the tumor necrosis factor alpha. The interferon gamma works like a chemical marker and activates cells called macrophages, which, in turn, fight the protozoon invader, releasing the tumor necrosis factor alpha. High levels of both in the wounds indicate that the immune system is more activated than usual.

Four years later, in 1996, this time in the city of Santo Amaro, 80 kilometers to the north of Salvador, the researchers found individuals who had been contaminated by Leishmania brasiliensis but had not developed the disease. They carried out tests to detect the level of tumor necrosis factor and interferon gamma, produced by other cells of the defense system. On comparing the results from people who did not show any symptoms of leishmaniasis with the victims of the cutaneous and mucocutaneous forms, the team noticed that those who did not have the wounds from leishmaniasis showed an immune response five times less than people with cutaneous leishmaniasis, and 30 times lower than those who had the mucocutaneous form. In the first group, the activity of the defense system increases temporarily, but drops down again 30 days after the infection, while in the other two it remains high.

With this result, Carvalho overturned, at least in the case of cutaneous and mucocutaneous leishmaniasis, the idea that a more intensive action of the mechanisms that combat the parasite is the most desirable. “We saw that a lower immune response was sufficient to combat the protozoon”, comments the researcher, who coordinates the immunology sector of Edgar Santos University Hospital at UFBA. From that point on, the group from Bahia decided to try to control – or, as doctors say, to modulate – this response. In the case of cutaneous leishmaniasis, they managed to regulate this response by adding to the traditional treatment a compound called granulocyte macrophage colony stimulating factor (known as GM-CSF) – a protein that is capable of stimulating the production of defense cells and is also capable of controlling the immune response.

Quicker healing
To verify the effectiveness of this therapy, they treated ten persons with the cutaneous form of the illness with antimony plus injections of GM-CSF in the wounds, and ten volunteers with antimony plus an innocuous solution. The results showed that the alternative treatment reduced the average time for the healing of the wounds, from 110 days to only 40 days, as is shown by a work published in the October 1999 issue of the Journal of Infectious Diseases, carried out with researchers from the Medical College of Cornell University, in the United States. In a similar study, not yet concluded, the researchers from Bahia substituted the injections by the application of GM-CSF directly onto the wounds in the skin. The preliminary details once again indicate that the use of the modulator of the immune system is more effective. The wounds of seven of ten of the individuals treated with antimony and GM-CSF healed in 50 days, while healing occurred in this time in only one person who had used the usual medicine, based on antimony.

The results seemed to be so good that the UFBA team set off for a greater challenge: using this therapy in people that continued to show lesions on the skin for not responding to antimony – the medicine does not work in 10% to 15% of the cases of cutaneous leishmaniasis. The researchers treated ten victims of leishmaniasis with pentavalent antimony along with the topical application of the stimulant factor.

About 60 days later, none of them was showing wounds any more. According to Carvalho, the result came as such a surprise that it is now being applied, as yet in an experimental fashion, in patients who are resistant to the conventional treatment – for it to become the standard therapy, more exams are needed, and the approval of the Ministry of Health. But there is an additional advantage: the association with GM-CSF has reduced the cost of the therapy for cutaneous leishmaniasis that is resistant to antimony to R$ 300.00. Until recently, the alternative was a drug called amphotericin B, which costs R$ 2,500.00 and is more toxic, capable of causing renal insufficiency and heart problems.

As the mucocutaneous form of the disease, where there are lesions of mucous membranes and cartilage, makes it unfeasible to apply shots of GM-CSF, the team from UFBA tried pentoxifylline, a medicine used to treat complications with leprosy that controls the production of the tumor necrosis factor, a protein that the organism in these people produces in excess to combat the protozoon. The scientists treated ten victims of the mucocutaneous form whose wounds had not healed with the antimony. They added to the application of this medicine three doses a day of pentoxifylline over one month, and the lesions of 90% of them healed in 90 days, according to an article published in 2001 in the American Journal of Tropical Medicine and Hygiene.

Another work, at the stage of being concluded, indicates that the use of antimony and pentoxifylline cuts out failures in treatment, which reaches 42% when only the first medicine is used. On the basis of the results of these researches, Carvalho believes it possible even to cut out the use of antimony and treat cutaneous and mucocutaneous leishmaniasis only with modulators of the immune system, which would reduce the side effects of the conventional therapy. “In the great majority of cases, the organism never eliminates the parasites completely, but learns to live with a small quantity of them'” explains Carvalho.

At Fiocruz’s René Rachou Research Center, biologists Paulo Pimenta and Nágila Secundino are doing supplementary work: they are looking into the capacity of mosquitoes for transmitting the leishmania – and other parasites – to human beings. It was there that, five years ago, the team from Minas Gerais saw an apparent sequence of event of bad luck give rise to an important discovery, capable of assisting in the fight against the most serious – and least common – form of leishmaniasis: the visceral form, transmitted in Brazil by the sandfly (Lutzomyia longipalpis).

In 1999, Pimenta and Nágila tried, unsuccessfully, to breed the first colonies of L. longipalpis in the laboratory. Some three or four months after being caught, which took place in the Lapinha Grotto (in Lagoa Santa, 35 kilometers from the capital of Minas Gerais), for some reason, the insects would die before the team could finish the experiments. They only found the cause of death of the mosquitoes when they dissected them and analyzed them under the microscope: their abdomens were full of worms with a cylindrical body (nematodes), of some 1millimeter in length when adult. It was a very rare case of contamination of worms capable of killing phlebotomine flies, the family to which the Lutzomyia longipalpis belongs.

An analysis of the way and cycle of life of the worm made it possible to discover the order to which it belongs (Rhabditida) and the family (Steinernematidae), as the researchers describe, in the June 2002 issue of the Journal of Invertebrate Pathology. At the moment, they are trying to determine the species of the worm that kills the transmitter of visceral leishmaniasis by proliferating in its abdomen and preventing the insect from feeding itself properly.

An analysis of the mosquitoes showed that only 0.5% have the worm inside them, in nature. But when they are raised in the laboratory, contamination is quicker, and half the mosquitoes show the worm in four months – the level of infestation affects the whole colony after one year, something that makes the nematode a candidate to be used in the biological control of visceral leishmaniasis. One of the advantages, according to Pimenta, is that the worm apparently contaminates only the phlebotomine flies.

Pimenta and Nágila now intend to see whether the worm infects other species of the Lutzomyia genus, which transmits tegumentary leishmaniasis, and whether the insects contaminated in the laboratory transmit the worms to other mosquitoes when they are set free in nature.