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Genomics

At the root of the lupus

Recently discovered genes help to explain autoimmune diseases, which attack the organism itself

At the end of last year, while reading the September edition of Immunity, the molecular biologist Geraldo Passos Junior, a researcher at the University of São Paulo (USP) in Ribeirão Preto, almost fell off his chair. In an article published in the magazine, researchers from Colorado University in the United States, published the fact that the altered expression in a mouse gene, the Ifi202, increased the risk of the animals developing the systemic lupus, a chronic autoimmune disease that provokes lesions on the skin and internal organs. It wasn’t exactly the news of the discovery of the role of this gene – important without a doubt – that caused the surprise in Passos. But, in fact, the coincidence that he and his USP colleagues had just finished obtaining interesting data linked to a human gene of the lupus.

By studying the lymphocytes of patients systemic lupus erythematosus, the Ribeirão Preto researchers had managed to identify the altered expression of the human interferon stimulated gene, exactly the human equivalent of the Ifi202 in mice that the American team had identified by studying the disease in animals. With the help of the technique of DNA chips or microarrays that allows for simultaneously analyzing the expression pattern (use) of thousands of cell genes submitted under the most varied situations or pathological procedures, they produced evidence that the human interferon stimulated gene behaves in a certain manner in lupus patients who have received treatment with immune suppressing drugs and in another sick people who don’t receive treatment .

In non-medicated people, the expression of the gene is higher. It is as if it were more intensely used by the cells of the immune system of these patients. In treated individuals, their expression is much lower. And it is as if the therapy had temporarily neutralized the action of the gene. The comparisons were made with RNA messenger (a molecule of genetic engineering that directs the synthesis of the cell proteins) obtained starting from the lymphocytes of the blood of three treated and three non-treated patients at the Hospital das Clínicas in Ribeirão Preto. “This data is still preliminary”, comments Passos. “We’re still at the beginning of our studies.”

It wasn’t just the behavior of the human equivalent of the Ifi202 which caught the attention of the researchers. The experiments carried out in the microarrays laboratory of the Medical School of Ribeirão Preto indicate that close to sixty other genes seem to express themselves in a more accentuated form during the acute, the more severe phase of lupus. Some of these genes are known and are involved in processes such as apoptosis (the programmed and natural death of cells) and in immunological compatibility (genes of the HLA complex). Others are completely new, of function as yet unknown.

The fact that sixty genes showed themselves to be overexpressed doesn’t mean, in any manner, that all of them are implicated in the origin of the illness. “This data needs to be refined”, emphasizes the rheumatologist Eduardo Donadi, also of the Medical School of Ribeirão Preto, responsible for the sub-project on the theme which is studying the expression of genes in auto-immune self-induced illnesses. They have received this name as they are illnesses, generally chronic, which are started through problems in the cellular defense system, which, instead of attacking elements extraneous to the organism, begin to fight against its own healthy tissues. Besides, Donadi’s team is studying the expression of genes in two other self-generated illnesses, rheumatoid arthritis and diabetes mellitus type 1.

Immunological maturation
With Passos, a team of researchers is attempting to map out in mice expressive genes by way of the thymus, a vital organ in the development of immunological systems, situated above the heart. Produced in bone marrow, the lymphocytes, a type of defense cell, migrate to the thymus, where they reproduce and differentiate themselves into two types of cells T, CD4 or CD8, whose job is fundamental in the defense of the organism. In order to discover the genes linked to this process of differentiation, Passos is studying the expression of 6,000 genes in the thymus of mice fetus, at the crucial moment of the formation of the immunological system: between the fifteenth and sixteenth day of gestation.

Using software that analyzes the experimental results with the DNA chips and also with macroarrays (a technology a little older and ten times less precise that that of microarrays), the researchers managed to identify one hundred and fifty two genes (twenty four with known functions and one hundred and twenty eight whose functions have still to be attributed) whose expression pattern changed radically during this time span. “We put together the genes that had presented a difference of expression of the order of fifty times, for greater or for lesser between the fifteenth and the sixteenth day of gestation” explained Passos.

A third line of research tries to pinpoint genes that are activated or regressed in human lymphocytes exposed to ionized radiation, a process that frequently brings on lesions and breaks in human DNA, which, for example, increases the incidence of lymphomas (malignant tumor that assaults the lymphatic ganglions) and other types of cancer in populations exposed to sources of radiation contamination. In order to understand the changes in genetic expression patterns of lymphocytes exposed to radiation and, consequently an important step in understanding the origin of certain forms of cancer. This thematic sub-project is coordinated by Elza Tiemi Sakamoto-Hojo, who worked with the victims of the cesium-137 contamination that occurred in Goiania in 1987, an episode that entered into the history books as the most serious accident with Brazilian radioactive sources.

In a general manner, the work of the biology team consists of the laboratory cultivation of different types of human tissue – normal cells and genetically modified cells, more sensitive to the effects of radiation – that are submitted to distinct doses of gamma radiation, emitted from a cobalt-60 source. During the process of radioactive contamination, with the help of microarrays technique, the expression pattern of a series of genes – for example, linked to the repair of DNA, to the control of the reproductive cycle and to the death of cells – will be analyzed on samples of the diverse cellular strains under study.

In pilot studies, Elza has carried out experiments with the methodology of macroarrays, in which the analyzed genes are deposited on a membrane made of nylon instead of on a thin glass sheet as occurs with microarrays, making a selection of differentially expressed genes during the irradiation of cultured lymphocytes in vitro. “We are very worried about the cumulative effects of continual exposure to people, above all to health professionals, to small doses of radiation”, comments the researcher. “We believe that our work could have an impact not only on the field of radiobiology, but as well on oncology”.

The Project
Transcriptome Project: Analysis of genetic Expression on a Large Scale Using DNA-Arrays (nº 99/12135-9); Modality Thematic project; Coordinator Geraldo Aleixo da Silva Passos Junior – USP; Investment
R$ 358,461.98 and US$ 386,773.00

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