An advance against malaria

Attack of the erythrocyte protozoan parasite is related to the level of melatonin

The parasite responsible for malaria is transmitted to the human being by the bite of a mosquito. The protozoan parasite has been known for more than a century and is more and more resistant to the classic medicines such as chloroquine. To develop new chemotherapeutics, it is essential to understand the mechanisms which regulate the cycle of life of the protozoa. Recently, the research of a team from the Department of Physiology of the Institute of Biosciences (IB) of the University of São Paulo (USP) found a solid clue to these mechanisms.

By studying the cells of mice and of humans infected with the protozoan parasite, the team – coordinated by the biochemist Celia Garcia, with the collaboration of the pharmacologist Regina Markus – showed that the hormone melatonin contained in the host, appears to be responsible for the fine synchrony of the life cycle of the parasite within the organism.

A frightening synchrony
The work, which was the headline in a special publication of the English magazine Nature, was developed during the thematic project Cellular and Molecular Biology of Plasmodium: Comparative Aspects of the Study of Transduction Indication in the Relationship Parasite-Host, financed by FAPESP. This unicellular organism of the genre plasmodium houses itself in the salivary glands of the mosquito Anopheles and penetrates into the organism through the bite of the insect. Within the host – as well as man, the parasite infects other mammals, birds and amphibians -, the protozoan parasite makes its way to the cells of the liver and afterwards to the erythrocytes – the red globules of blood.

In fact, it is in the erythrocytes that the protozoa reveals a peculiarity of its lifecycle which makes the control of the infection very difficult. Obeying a frightening synchrony, the parasites, practically all of them at the same time, mature and multiply, attack and breakdown the infected cellular membrane, opening the way to simultaneously enter the blood stream and toinvade cells which are still healthy. What was needed was to disclose the mechanism which governs the orchestrated advance of the parasites, to arrive at a discovery which would help in the development of more efficient medicines against malaria an illness which each year kills 1 million people in the world.

It is here that we put in the good news from the research team. By way of studies with the cells of mice and of humans infected with the protozoan parasite, the team made clear the relationship between the quantity of the hormone melatonin present in the cells and the synchrony verified in all the phases of the life cycle of the parasite.

Counter attack
Melatonin is a hormone produced during the night by the pineal gland, in a daily cycle – repeated every 24 hours. It is already known that this hormone synchronizes various functions of the immunological system, nerves and endocrine. More was discovered: “Both in vitro and in vivo, the melatonin is capable of modifying the cycle of the parasite. We took a step to reveal the regulation of the synchronism of the protozoan parasite in the cells of the host. We are pioneers in this work.” reassured Dr. Celia, a graduate in chemistry from the Federal University of Rio de Janeiro (UFRJ), where she did her masters and doctorate. Her research found its way onto the pages of the July edition of Nature Cell Biology, one of the publications of the renowned English scientific magazine Nature.

The team also revealed that the synchrony of the attack of the protozoan can be broken if the rhythm of the production of melatonin was to be altered – which, in theory, leaves the infection more vulnerable and easier to be overcome. When in mice the secretion of this hormone was impeded – by way of the removal of the pineal gland – or there was administered to them drugs which impede the protozoan from detecting the presence of melatonin, the life cycle of the protozoan was totally altered.

Biochemical action
In general lines, one can say that the increase in the concentration of melatonin in the host allows the protozoa parasites which invade red blood cells to evolve simultaneously from their initial stages, called ring and trophozont, for the more mature stage of schizont. At the end of this stage, after they have reproduces asexually, the parasites break down the host cell, enter into the blood stream and reinitiate the process of the infection. When they pass from the stage of trophozont for that of schizont each parasite generates between 18 and 35 new protozoan parasites inside the invaded red blood cell – an army which is put in charge of destroying it.

In the proposed model, it is believed that the plasmatic membrane of the parasite should be allowed to be a receptor of melatonin which, on entering in contact with this hormone of the host, detonates a series of chemical reactions. The team have already demonstrated that the protozoan parasite is endowed with equipment for signaling with calcium, similar to higher organisms, in a way that it can also use calcium as a signaler of its cellular processes. Using fluorescent indicators which permit the quantification of the calcium ion, the team showed that the melatonin is capable of causing the liberation of calcium contained in the intercellular deposits of the protozoa.

The calcium is extremely important, as it determines various processes, especially cell division. Consequently, with the melatonin of the host active to the liberation of calcium within the cell of the parasite, this calcium could activate the process which provokes the maturation and the cellular multiplication of the protozoan parasite itself.

To arrive at these conditions, the researchers carried out live experiments with mice and in test tubes with cell cultures of rats and of humans. Firstly, they added melatonin to a culture of cells of mice of the species called Balb/C infected with Plasmodium chabaudi – the protozoan malaria which infects this animal. Seventeen hours later, they verified that there had been an increase in the number of red blood cells invaded by parasites, proportional to the quantity of the hormone injected. The result indicated that the melatonin accelerates the reproduction of the parasite.

In order to prove that the melatonin really accelerates the infection, the team carried out similar experiments with a culture of human cells. Contaminated with Plasmodium falciparum – the species of parasite of malaria which most commonly attacks man – , this culture also received gradual doses of melatonin. In parallel, they kept as a control group, a culture of human red blood cells contaminated with the same species of the protozoan parasite, but not submitted to doses of melatonin.

The result was exactly as expected: 24 hours after the start of the treatment with melatonin, the red blood cells infected with Plasmodium falciparum had a much higher quantity of parasites in the final stage of schozont than that of the control group – proof that the melatonin had accelerated the development of the parasite in the red blood cells. Nevertheless, in the two groups the number of infected cells of the host remained the same as at the beginning of the experiment – that could appear strange, but was also expected since there was not enough time for this number to change as the life cycle of Plasmodium falciparum within the red blood cells is of 48 hours – 16 in the ring phase, 20 in the trophozont and 12 in the schizont.

Or that is to say, this species of parasite needs two days to pass through the three phases – develop, mature and multiply – before it breaks down the invaded red blood cell and is ready to attack new cells. “The life cycle of all the protozoan parasites is always a multiple of 24 hours.” emphasized Dr. Celia. After having put together evidence that the hormone melatonin dictated the rhythm of the maturing of the parasite in cultures within the laboratory, the researchers began live experiments with mice. The objective was to determine of the absence of the production of melatonin by the host or the perception of it by the parasite would also have some effect on the life cycle of the development of malaria.

Therefore, they removed the pineal gland – which stopped the complete production of the hormone – from the mice of species Balb/C and infected them with a population of Plasmodium chabaudi, whose life cycle in the host is 24 hours. Seventy two hours after the start of the infection, the quantity of parasites in the initial ring stage was twice as high in the mice which had not produced melatonin than of the control group which had remained with the pineal gland. Also, it was revealed that the quantity of parasites in the phase of trophozont was a lot less in the animals incapable of secreting the hormone than in the control group. That is to say, without the melatonin of the host, the life cycle of the parasite losts its synchrony.

They carried out another study in which they changed the focus – from the host to the parasite. Working with two types of mice, Balb/C and C3H, they injected them with the drug luzindole – an inhibitor of the receptors of melatonin – and, at the same time, infected them with Plasmodium chabaudi. In a general manner, the inhibitor reduces the capacity of the parasite to identify the melatonin of the host and to react to it, which alters all of the chemical process which gives it its changes of phase and its cell divisions. In this situation, the host continues producing normally the melatonin, but the parasite isn’t completely able to perceive this. The result of the experiment was similar to that carried out on the animals who had had their pineal gland removed: more rings and less trophozont in the mice who received the dose of luzindole than in the control group.

The newest front of research by Dr. Celia Garcia is the cycle of the malaria parasite in lizards. Motive: half of the close to 170 species of protozoan parasites in existence infect lizards, which, strangely enough, have a very low indices of mortality due to this infection. Why do the lizards resist so well the protozoan parasite? The researchers are looking for an answer by studying the life cycle of the parasites within the organism of these reptiles. This could point in some direction in the sense of avoiding the deadliness of malaria in other species, above all in humans. There are two species of lizards of the Amazon which are under study in the laboratories of Biosciences of USP, the Ameiva and the Tupinambis meriane .

The scourge of the tropics

Malaria causes high fever over regular intervals – more or less every 24 hours, the moment at which the parasites break down the infected red blood cells and again reach the blood stream in order to invade more red blood cells. Other symptoms are vomiting headaches and pains in the joints. The more serious cases evolve into a state of coma and death. There are no vaccines approved by the scientific community – some are under test – however, though sometimes it resists medicines, the infection can be weakened if it is treated rapidly and adequately. Nevertheless, the numbers go on impressing. The scourge of malaria kills each year between two and three million people in the whole world, above all the young – it is estimated that a child dies from malaria every 30 seconds, almost three thousand per day.

Maintaining the vicious circle, the transmitter mosquito Anopheles bites between 300 and 500 million people per year and into them injects the protozoan parasite which unleashes the illness in new victims. Of every ten cases, nine occur in the portion of Africa below the Sahara. Of the remainder, six countries concentrate two thirds – among them is Brazil. It is calculated that close to 40% of the population of the world live in areas with a risk of transmission. For the World Health Organization, malaria is the most serious tropical illness caused by a parasite.

The latest results
Called by many as “the Queen of the diseases” because of these high indices of incidence and mortality, it continues to challenge the world. To combat it in Brazil, close to 300 specialists participated from the 26th until the 29th of November in Rio de Janeiro, at the 7th National Meeting of Malaria Research. The meeting – which had the support of FAPESP in financing the travelling expenses of the researchers – brought the latest results in the areas of vaccines, diagnosis, transmission control and National data.

The principal theme was the intensification of the disease in the Amazon, above all in the areas of the mining camps and settlements. The tendency of the growth is worrying: according to the National Health Foundation, there were 405,000 cases in 1997 and a record 632,000 in 1999 – 34% more than in the previous year. In this last year, 99% of the cases were in the Amazon. The State of Amazonas and Pará concentrate 66% of the victims, but Acre and Maranhão had in 1999 the highest percentages of annual an increase: 143% and 87% respectively. The spreading of mining, extraction of wood, the construction of dams and rural settlements were pointed to as the principal reasons for the increase in the Amazon basin.

“The movement of people throughout the region and the disorderly occupation of space, principally over the last three decades, has made the situation worse.” said Wilson Alecrim, of the Foundation of tropical Medicine of the State of Amazonas. The migration of part of the population from rural zones to the cities has also worsened the situation. “The social problems determine the increase of malaria in the Amazon.” emphasized Alecrim. A group from the Federal University of São Paulo (Unifesp), coordinated by the immunologist Mauricio Rodrigues, is studying the immunizing action of the protein MSP 1 of Plasmodium vivax, the most common species of the parasite which causes malaria in the country. In tests with guinea pigs this protein was capable of stimulating an immunological response of up to 90% among the guinea pigs infected.

“The results lead us to think about developing a vaccine.” revealed Dr. Rodrigues. In 2001 tests of the protein will begin in saimiris monkeys at the National center of Primates of Belém. “Let’s check out the immunological response of these monkeys, in which we could reproduce the malaria caused by the vivax, since they are animals closely related to man.” he explained.

A transgenic mosquito
The development of a mosquito which would be resistant to the infections of parasites is another alternative. The insect physiologist Dr. Margareth Capurro, of Unifesp, explained that the objective of the research is the introduction into the mosquito which is the transmitter of malaria, the Anopheles, of a gene which interferes with the development of the parasite. In this way, the mosquito will become a poor transmitter of malaria or stop transmitting it at all, breaking the cycle of the disease.

“We are studying the blocking of the parasite in the salivary glands of the mosquitoes.” said Margareth. She highlighted some challenges in order to arrive at the transgenic mosquito: to liberate this mosquito into the wild with an assurance that it will become dominant, in a way that it will pass its new genes to the wild population of Anopheles. Dr. Denise Vale, of Oswaldo Cruz Foundation, is also studying the blocking of the parasite in the digestive tube of the mosquito.

Edmund Christian, of the Pan-American Health Organization (Opas), remembered that the walls of the mud brick houses and brickwork of the endemic regions of the disease absorb many times the insecticides used in the control of the mosquito, reducing its efficiency. He believes that “it is necessary to test other frameworks for distributing the insecticides to increase their efficiency.” He also cited alternative methods of prevention such as a soap with repellant compounds to the malaria transmitter which is being tested in Colombia and in Venezuela.

As far as the diagnostic methods are concerned, Dr. Cor Jesus Fontes, of the Federal University of Mato Grosso, believes that it is necessary to perfect diagnostic kits which are more modern and cheaper. He analyzed blood samples from 630 people of Mato Grosso and discovered that the actual methods have a detection rate of 80% for the Plasmodium falciparum – for the species linked to the highest number of serious cases and those causing mortality -, an indices considered insufficient.

Plan of attack
In spite of everything, the deadliness of the disease in Brazil has gone down a lot: according to the National Health Foundation, close to 150 people died from malaria during 1998, five times less than at the beginning of the 90s. At the national reunion it was estimated that the increase in the incidences could be reverted as from 2001 with the implementation of the Plan of Intensification of the Control of Malaria. The training of personnel and the acquisition of microscopes for diagnosis are among the strategies of the plan which will invest R$ 145 million over the next few years to reduce by 50% the incidence of malaria in the Amazon.

The project
The Cellular and Molecular Biology of the Plasmodium: Comparative Aspects of the Study of Transduction of the Signal in Relation: Parasite – Host (nº 98/00410-2); Modality Thematic Project; Coordinator
Dr. Celia Regina da Silva Garcia – Institute of Biosciences (IB) of the University of São Paulo (USP); Investment R$ 220,000.00