O sistema imune inato desempenha papel central na reprodução, tendo as células NK participação marcante. Durante a gravidez, seu comportamento pode esclarecer pontos cruciais na patogênese das complicações que podem ocorrer em gestantes com LES. OBJETIVO: Quantificar as células NK circulantes e sua viabilidade em gestantes com LES. MATERIAL E MÉTODOS: Avaliaram-se amostras de sangue de quatro grupos de dez pacientes cada: 1 GLES: Gestantes com LES; 2 PLES: Pacientes com LES não gestantes; 3 Gcontroles: Gestantes controles; 4 Controles: Mulheres não gestantes saudáveis. Em todas as pacientes, a quantidade e a viabilidade das células NK foram medidas por citometria de fluxo, assim como por apoptose total por coloração para anexina V e iodeto de propidium. RESULTADOS: Devido à variabilidade dos resultados, a mediana de cada grupo foi utilizada para avaliar: porcentagem CD56+ [GLES (0,10), PLES (0,12), Gcontroles (0,15), Controles (0,08)]; apoptose total [GLES (0,06), PLES (0,04), Gcontroles (0,11), Controles (0,11)]. Os resultados da contagem de células vivas tiveram baixa variabilidade, por isso média e desvio-padrão foram utilizados para comparação: [GLES (0,91 ± 0,06), PLES (0,95 ± 0,03), Gcontroles (0,86 ± 0,11), Controles (0,88 ± 0,08). CONCLUSÃO: Apesar de não terem alcançado valor de significância estatística, o percentual de apoptose total nos grupos com LES foi menor que o dos controles, e a porcentagem de células vivas foi maior. Isso sugere que, em pacientes com LES, grávidas ou não, as células NK têm vida útil prolongada (ou tem turnover menor/diferente), o que indica um maior estímulo imune, fazendo com que as células NK levem mais tempo para ativar o processo de apoptose.
gravidez; lúpus eritematoso sistêmico; células NK
The innate immune system plays an important role in reproduction, with marked involvement of NK cells. These cells behavior during pregnancy may clarify crucial points in the pathogenesis of complications that may occur in pregnant women with SLE. OBJECTIVE: To measure the amount of circulating NK cells and their viability in pregnant SLE patients. MATERIALS AND METHODS: Blood samples from four groups of ten patients each were evaluated: 1. GLES: Pregnant SLE patients; 2. PLES: Non-pregnant SLE patients; 3. Gcontrols: Pregnant controls; 4. Controls: Healthy non-pregnant women. In all patients the amount and viability of NK cells was measured by flow cytometry, as well as the total apoptosis by annexin V and propidium iodite staining. RESULTS: Due to the great variability, median of each group was used for evaluation: CD56+ count [GLES (0.10), PLES (0.12), Gcontrols (0.15), Controls (0.08)]; total apoptosis (addition of initial and late apoptosis to total number of dead cells) [GLES (0.06), PLES (0.04), Gcontrols (0.11), Controls (0.11)]. The results for live cells count had low variability, so the averages and standard deviations were used for comparisons: [GLES (0.91±0.06), PLES (0.95±0.03), Gcontrols (0.86±0.11), Controls (0.88+0.08)]. CONCLUSION: Although not statistically significant, the total apoptosis in the SLE groups was lower than in the control groups, and the live cell count was higher. This suggests that in SLE patients, pregnant or not, the NK cells have a prolonged life cycle (or have a lower/different turnover), and that there may be a higher immune stimulus making the NK cells take longer to activate the apoptosis process.
pregnancy; systemic lupus erythematosus; NK cells
IAssistant Professor, Discipline of Rheumatology, School of Medicine (UNIGRANRIO) and master of Medical Sciences (PGCM-UERJ)
IIBiologist Professor, School of Medical Sciences, Department of General Pathology, Laboratory of Immunopathology (UERJ)
IIIAssistant Professor, Discipline of Obstetrics, School of Medical Sciences (UERJ)
IVResident of the Discipline of Ophthalmology, Pedro Ernesto University Hospital (UERJ)
VProfessor, Discipline of Biostatistics (Universidade Estácio de Sá)
VIAssociate Professor, Discipline of Rheumatology, School of Medical Sciences (UERJ)
The innate immune system plays an important role in reproduction, with marked involvement of NK cells. These cells behavior during pregnancy may clarify crucial points in the pathogenesis of complications that may occur in pregnant women with SLE.
OBJECTIVE: To measure the amount of circulating NK cells and their viability in pregnant SLE patients.
MATERIALS AND METHODS: Blood samples from four groups of ten patients each were evaluated: 1. GLES: Pregnant SLE patients; 2. PLES: Non-pregnant SLE patients; 3. Gcontrols: Pregnant controls; 4. Controls: Healthy non-pregnant women. In all patients the amount and viability of NK cells was measured by flow cytometry, as well as the total apoptosis by annexin V and propidium iodite staining.
RESULTS: Due to the great variability, median of each group was used for evaluation: CD56+ count [GLES (0.10), PLES (0.12), Gcontrols (0.15), Controls (0.08)]; total apoptosis (addition of initial and late apoptosis to total number of dead cells) [GLES (0.06), PLES (0.04), Gcontrols (0.11), Controls (0.11)]. The results for live cells count had low variability, so the averages and standard deviations were used for comparisons: [GLES (0.91±0.06), PLES (0.95±0.03), Gcontrols (0.86±0.11), Controls (0.88+0.08)].
CONCLUSION: Although not statistically significant, the total apoptosis in the SLE groups was lower than in the control groups, and the live cell count was higher. This suggests that in SLE patients, pregnant or not, the NK cells have a prolonged life cycle (or have a lower/different turnover), and that there may be a higher immune stimulus making the NK cells take longer to activate the apoptosis process.
Keywords: pregnancy; systemic lupus erythematosus; NK cells.
NK cells are a subtype of mononuclear cells morphologically distinct of lymphocytes B and T due to its increased granular density. NK cells participate in immunoregulation, haematopoiesis, reproduction and neuroendocrine interactions. Also play significant role in host defense against certain microorganisms, in part through their ability to secrete cytokines, such as IFN-γ, TNF, and GM-CSF,1 among others.2
They are capable of producing cytokines, pro and anti-inflammatory, and lead to death of target cells by apoptosis. Thus, have implications in the pathogenesis of several human diseases. By the nature of cytokine production, NK cells takes part in the production regulation of antibodies dependent on T cells in autoimmune diseases. A reduction in NK cell activity is seen in SLE. Thus, the modulation of NK cell activity may be important in the pathogenesis and control of autoimmune diseases, infectious, and neoplastic.3
NK cells in pregnancy
The gestational period is a unique model in nature. It includes a highly complex immune status, with frequent exacerbation of illness or pre-existing changes. From the immunological standpoint, it has been a great challenge trying to understand what mechanisms are involved in no rejection of the placenta by the maternal immune system, since the placenta, being of embryonic origin, contains genetic material, both maternal and paternal.4 The innate immune system is active in pregnancy and, due to the relative suppression of adaptive immunity, may play an important role in maternal immune defense.5 Furthermore, the innate immune system has a dominant influence on reproduction. Analyses of cell types, which are present at the egg implantation site, show that T and B cells, typical of the adaptive immune system, are rare, while the predominant population consists of NK cells.6 The main types of immune cells in secretory endometrium are T cells, NK cells, and macrophages. T cells comprise approximately 45% of leukocytes in the proliferative phase endometrium, and this number remains constant throughout the secretory phase, and during pregnancy. However, their number apparently decreases due to the increase of NK cells during the secretory phase and early pregnancy.7 Several reports suggest that uterine NK cells have an important role in reproduction. They are hormonally regulated, increasing in number during the luteal phase of the menstrual cycle, when the implantation occurs; are present in early pregnancy when trophoblastic cells invade the spiral arterioles. They are particularly abundant around the fetal infiltration derived from extravillous trophoblastic cells. NK cells (CD56+) proliferate actively in the decidua during the luteal phase. In these phases, other mucosal elements (epithelial glands and stromal cells) cease proliferation and begin differentiation, except for endothelial cells, which continue to proliferate at this stage. However, the stimulus for the proliferation of NK cells in vivo is still unknown.8 NK cells in decidua are in close contact with the trophoblast fetal-maternal interface. However, decidual NK do not exert cytolytic activity against trophoblastic cells. Several studies have shown that the general cytotoxicity of decidual NK is reduced, when compared with peripheral blood NK cells.9
Pregnancy in SLE
For many years, SLE patients were warned not to become pregnant, but with a greater knowledge of the disease pathophysiology mechanisms and clinical manifestations during pregnancy, associated with medical care, pregnancy is a normal event in women with SLE. Normal but not monotonous. The pregnancy of SLE patients remains a high risk pregnancy, although most women do not present major complications.10 The autoimmune disease is primarily a disorder of immune reactivity, which changes tolerance against various substances that starts behaving as autoantigen. Similarly, the success of pregnancy depends on maternal tolerance or non-immune reactivity against paternal antigens. Maternal tolerance seems to be associated with the development of several specific mechanisms that protect the fetus from maternal immune cytotoxic attacks.11
NK cells in SLE
Since 1980, many evidences have been collected suggesting an association between the decrease in the number and activity of NK cells and autoimmune diseases. In a study of 71 patients with SLE, it was found that the number of NK cells (CD16+ CD56+) in the peripheral blood of these patients was only one third of the levels of controls - a highly significant difference. The proportion of NK cells in peripheral blood was significantly lower in patients with moderate or severe disease, compared with patients with inactive disease, and more depressed in patients with severe lupus nephritis.1 Green et al. found low levels of NK cell activity in first-degree relatives of patients with SLE, suggesting that the correlation of NK cell deficiency with patient activity is important in the pathogenesis of the disease and not secondary to the disease process or to treatment with drugs. This deficiency was both in cell number and function.12
SLE NK cells seem to change on quantity and cytotoxicity, which is related to its pathogenesis, and yet there is no investigation about these cells presentation during pregnancy in SLE. Perhaps, a better control of immune response is the key to prevent the majority of SLE patients to develop disease activity during pregnancy. This pilot study objective was to evaluate the quantity and the life cycle of NK cells in peripheral blood and try to correlate with disease activity.
MATERIAL AND METHODS
This study was reviewed and approved by the Research Ethics Committee of Pedro Ernesto University Hospital and the Research Ethics Committee of the Maternity School of UFRJ, and was registered in SISNEP.
We evaluated four study groups. Group 1) pregnant women with SLE (PLES), which was the target group; and other three control groups: Group 2) pregnant women without autoimmune diseases (Gcontrols), Group 3) women with SLE (PLES), Group 4) women without autoimmune diseases (Controls). The activity of SLE was assessed by SLEPDAI13 in pregnant women, and the SELENA-SLEDAI14,15 in non-pregnant women. Each group was composed by 10 child-bearing age women, except the Control group with 11 individuals. Inclusion criteria were: pregnant women with SLE who began monitoring the pregnancy latest by twenty weeks of pregnancy, pregnant control groups, patients with SLE and controls matched for age and skin color. Exclusion criteria were: 1) more than twenty weeks of pregnancy at the time of material collection (blood and urine) for the study; 2) non-pregnant women over 40 (not in child-bearing age); 3) SLE carrier associated with another autoimmune disease, except antiphospholipid antibody syndrome; 4) infectious processes that require treatment with systemic antibiotics up to seven days before study entry. In the PLES group, were accepted only patients treating SLE with prednisone (Pred), chloroquine diphosfate (CQN), hydroxychloroquine (HCQ) and azathioprine (AZA), as these are the most commonly used medications for SLE treatment during pregnancy. At the day of urine and blood sample collection, it was applied a questionnaire to patients to assess factors that could interfere with the immune response: fever, flu, allergies, use of medication, physical activity, exposure to sun and sea, smoking, use of alcohol, and mood state.
To determine the total number of NK cells, 50 μL of blood (containing up to 0.5 x 106 cells/mL per tube) were stained with 3 μL of anti-CD56-PE (BD 347747 California, USA) and incubated for twenty minutes at 4 ºC. Then the cells were lysed with Quicklise (Cytognos, Salamanca, Spain) for 15 minutes, and the remaining cells were washed twice with buffer saline pH 7.2 frozen. Soon after that, the cells were centrifuged for three minutes at 400 x g. To quantify the percentage of apoptosis in NK cells (CD56+), it was used the kit for detection of apoptosis from BD Pharmingen (California, USA), stained with annexin V and propidium iodide. In this test, the apoptosis is quantified by the detection of fosfatidilserina exposed on the surface of apoptotic cells using annexing V that has affinity for it,16 and PI detects cells alive or dead. The cells are resuspended in 300 μL of saline buffer, 2.5 μL of annexin-V-FITC and 2.5 μL of PI (FL-3) were added, followed by homogenization, and incubated for 15 minutes in a darkroom. After incubation, it was added 300 μL of buffered saline to cells, which were processed and acquired in the FACSCalibur cytometer (BD Biosciences, CA, USA) at the maximum of one hour. 10 thousand events were acquired in channels FL-1, FL-2 and FL-3. The analysis was performed with multiparametric approach, to visualize the movement of migration living cells to those in apoptosis (initial and late) and dead. It was used the Paint'A'Gate program for this analysis and quantification of these population.
Statistical evaluation was divided into descriptive and inferential analysis, the first aimed to characterize the groups investigated, while to obtain inferences, we tried to compare them to identify possible statistical differences, always considering α = 0.05.
The descriptive analysis led to the estimates measures of location (mean and median) and dispersion (standard deviation and coefficient of variation) for quantitative variables (age, CD56%+, CD56+ absolute/mm3, living cell, initial apoptosis, late apoptosis, dead cells, total apoptosis, gestational age), as advocated by Costa Neto.17 This reference was also the base for frequency tables development of qualitative variables (skin color, humor, questionnaire, Pred, CQN / HCQ, AZA, SLEPDAI and SLEDAI14,15).
Also in the description field, Pearson's linear correlation and covariance were estimated, both estimated by quantitative variables.18 Cramer's V coefficient correlation was estimated for the questionnaire variable against CD56+% CD56+ abs/mm3 and total apoptosis, since it involves qualitative variable.
Comparison among groups demanded that the proximity to the normal distribution were evaluated for quantitative variables, which was conducted by the Shapiro-Wilk test, given the low number of individuals/group.17
The normality enabled a comparison of four groups to be performed by two-way ANOVA.19 When proximity to the normal distribution was not observed, it was applied the Kruskal-Wallis test20 (CD56+%, CD56+ abs, initial apoptosis, late apoptosis, dead cell).
PLES and GLES groups were compared for some variables that were present only in those groups, then the statistical difference investigation was designed by Mann-Whitney test20 (gestational age).
For qualitative variables, the comparison of frequencies was done by the Chi-Square test19 (skin color, humor, questioning, Pred, CQN / HCQ, AZA). Statistical analysis were made based on the coefficient of variation (CV).17
CV < 20.00% - low variability, the individuals had uniform results, and the characterization of the variable was performed by mean and standard deviation.
CV > 20.00% - high variability, the individuals had discrepant results, and the characterization of the variable was performed by median and coefficient of variation.
GLES patients, at the time of evaluation, had gestational age ranging from 10 weeks and 3 days to 20 weeks, and Gcontrols from 9 weeks and 6 days to 18 weeks. Gestational age was estimated in days, and the GLES group was 105.00 days ± 23.55%, and Gcontrols group 100.45 days ± 16.73%. The age of women studied in the four groups ranged from 17 years and 11 months to 40 years old, the GLES group was 25 years old ± 24.66%, while in PLES, Gcontrols and Controls were respectively 32.40 ± 6.20 years, 25.80 ± 4.85 years and 30.91 ± 5.01 years, which a priori indicated that the groups were uniform with regard to age. Therefore, this variable should have no impact on other variables, except in GLES. Counting of CD56+ cells was obtained (percentage and absolute value). Percentage showed high variability, and the medians were: GLES group, 0.10 ± 91.01%; PLES, 0.12 ± 27.65%; Gcontrols, 0.15 ± 45.70% and Controls, 0.08 ± 47.47%. Individual values are shown in Table 1.
After determining the total amount of NK cells, these were analyzed for their viability, and differentiated in the stages of: live cells, cells at initial apoptosis, cells at late apoptosis and dead cells. Value of total apoptosis was considered as the sum of initial apoptosis, late apoptosis and dead cell stages. This determination was based on the consideration that, once the cells enter into apoptosis, its natural course is death. The results of each stage were calculated as percentages and analyzed in relation to its average, standard deviation, median and the coefficient of variation (Table 2). Living cells showed low variability, with means: GLES group, 0.91 ± 0.06%; PLES, 0.95 ± 0.03%; Gcontrols, 0.86 ± 0.11%; and Controls, 0.88 ± 0.08. The groups with SLE patients had a higher number of live NK, compared with patients without the disease; and the GLES group had lower value than PLES group, as well as Gcontrols group when compared to Control group (Table 2).
The initial apoptosis, late apoptosis and dead cell stages showed high variability, as well as total apoptosis stage. The median total apoptosis, GLES 0.06 ± 73.45%, 0.04 ± 54.86% PLES, Gcontrols 0.11 ± 75.72%, and Controls 0.11 ± 62.30%, was lower in groups of SLE patients when compared with the groups of patients without disease (Table 2) (Figure 1).
Some patients were receiving medications during the study period, except treatment with systemic antibiotics. We analyzed the frequency of the use of these medications (Table 3).
The Shapiro-Wilk test was applied for variables of age, CD56+, living cell, initial apoptosis, late apoptosis, dead cell and total apoptosis (Table 4). Then, the ANOVA test was applied for the variables of age (P = 0.02), living cell (P = 0.06), total apoptosis (P = 0.03), and we also applied the Fisher's test for age and total apoptosis (Table 5). There was significant difference in total apoptosis of Gcontrols and Controls groups in relation to PLES group, but it was not possible to identify differences in GLES group.
Results of the activity indices of SLE, SLEPDAI13 and SELENA/SLEDAI14,15 were analyzed in relation to CD56+%, CD56+ abs, living cell and total apoptosis (Table 6), and was observed a dependence with CD56+ abs, but reverse, i.e., when the variability of CD56+ increased, SLEPDAI13 and SELENA/SLEDAI14,15 decreased. There was a relationship, but no a correlation between CD56+% and CD56+ abs: the dependence only occurred in CD56+ abs. The correlations of live cells and total apoptosis were not significant (P > 0.05). There was no dependency between the variables. Due to the small number of individuals in each group and/or a variable that interfered in the results, it was not possible to check values statistically significant.
NK cells are the predominant population of leukocytes in the uterine mucosa, and much has been studied to examine their role at the time of implantation and maintenance of pregnancy. Changes in regulation of NK cells have been associated with reproductive changes, such as spontaneous abortion, implantation failure and infertility, and preeclampsia.21 Miscarriage is associated with an increase in NK CD56dimCD16+cells and decrease in CD56brightCD16- in the endometrium during the luteal phase.22 A study that assessed the levels of NK cells in peripheral blood in the pre-conception and post-conception found no significant changes of these levels when women with a history of recurrence miscarriages were studied and compared to normal controls. However, when subpopulations of NK cells were assessed, the CD56+CD16+ cells were moderately reduced in pregnant compared with non-pregnant women.21 It is important to remember that the values of NK cells count does not specifically reflect the immune response of pregnancy, and that the quantity and activity of these cells can fluctuate according to different variables, such as hormonal effects, physical activity, time of day, and sympathetic response to stress. Moreover, the number of NK cells of the peripheral blood is not necessarily correlated with their cytotoxicity.21 In our study, we used a questionnaire containing some of these variables, which could interfere in the values of NK cells, to try to minimize errors in the interpretation of the results. There was no correlation between the values of NK cells and the variables studied (fever, flu, allergies, use of medication, physical activity, exposure to sun and sea-bathing, smoking, use of alcohol and humor).
Although still controversial, some studies show an increase in levels of SLE activity adapted for pregnancy. The increased in SLE activity has profound implications on the fetal outcome, being associated with the lower number of live births and prematurity. However, the presence of lupus anticoagulant increases the risk of fetal loss.23 There are reports of reduction in the number of NK cells in the peripheral blood of patients with SLE, and this decrease was more pronounced in patients with active disease.1 This study found no reduction in the number of NK cells in pregnant patients and/or controls with or without SLE, but the small number of individuals in each group may have hindered the evaluation. The NK cell activity was not evaluated, only the cell cycle. There were no reports found on the evaluation of cell cycle of the NK cells in SLE patients.
Schepis et al. found an increase of NK cells in patients with SLE, regardless of the activity of the disease. This increase was related to the increase of INF type I (first-response cytokine produced mainly by dendritic cells; appears to be involved in the pathogenesis of SLE), an abundant cytokine in SLE that can lead to an increase of NK cells in vitro. Serum levels of INF-α were increased in patients with disease activity, but not in those without disease activity.24 In this study, we found no significant differences in NK cell count between the groups, there was a high variability in the results. However, when related to the indices of activity of SLE (SLEPDAI13 and SELENA / SLEDAI14,15), we verified an inverse relationship with the absolute number of CD56+, but this relationship was not maintained in the percentage of CD56+cells, so there was no correlation between the percentage and the absolute number. These results were not statistically significant.
There is a discussion whether the blood tests with increased levels of NK cells can be reproduced, or the values of patients with pregnancy loss are within the rate of normality, and if high levels in the blood may represent a mobilization in response to stress. The increase of the levels of NK cells may derive, in part, from a response to the stress of the venipuncture (elevation of NK count was found in the initial sample but not twenty minutes later). However, high counts of NK cells in the peripheral blood could be important for events in the maternal-fetal interface, the increase of stress in the activity of NK cells and their mobilization, suggesting an important role in the pathophysiology of fetal loss.25 These differences in literature on the values found for NK cells in SLE patients may be related to the difficulty of determining a relationship between the values of NK cells and its correlation with the disease activity, due to several factors that can interfere with the immune response.
In this study, it was evaluated in addition to the count of NK cells its viability and quantified the number of living cells in the initial and late stages of apoptosis, dead cells and total apoptosis. The results of living cells showed low variability and the groups of SLE patients (GLES and PLES) had a higher number of NK living cells, compared with patients without the disease.The group of pregnant women (GLES) showed a lower value group than the non-pregnant women group (PLES), as well as the group of women without disease (Gcontrols) when compared to the Control group. Perhaps, this shows that in the peripheral blood there are a smaller number of live NK cells in early pregnancy. In general, apoptotic cells are rapidly removed by phagocytosis, due to the induction of changes in the cell surface by the process of apoptosis. This prevents the release of physiological intracellular constituents, including nucleosomes, which are formed during the process of apoptosis through cleavage of chromatin by nucleases. Therefore, disorders in apoptosis or phagocytosis of apoptotic cells have been proposed in the development of autoimmune diseases, especially SLE.26 Several studies using mice with molecule deficiencies (e.g., C1q, and IgM) or receptors involved in phagocytosis of apoptotic cells have shown that the change in the removal of apoptotic cells leads to autoimmunity against nucleosomes and glomerulonephritis. Moreover, defects in the clearance of apoptotic cells have been described in mice and patients with lupus. Nucleosomes are not only important for the induction of the disease, but also may influence the clearance of apoptotic cells. Furthermore, autoantibodies formed in SLE may also modulate this process. Therefore, during disease progression when nucleosomes autoantibodies begin to move, they may amplify the disease by inhibiting the clearance of apoptotic cells.26 In this study, it was found that total apoptosis was decreased in the groups of patients with SLE, when compared with groups without disease.
It was not possible to determine the predominant profile of NK cells, but perhaps this increase in the amount of living cells and decrease in total apoptosis may suggest that NK cells in SLE patients, pregnant or not, remain alive and maybe active for a greater period.
In this pilot study, it was not possible to demonstrate differences in the NK cells values of patients with or without the disease. As for the cell cycle of NK cells, the living cells in the groups of SLE patients were in greater number than in the controls without the disease; pregnant women had lower values than non-pregnant women in groups with SLE and in groups of patients without the disease. The total apoptosis was decreased in all groups of SLE patients when compared with groups without the disease, but less in non-pregnant patients than in pregnant women.
These differences between living cells counts and total apoptosis may show that in SLE patients, pregnant or not, the NK cells have a long life (or a lower/different turnover), suggesting that there may be a greater immune stimulation, making NK cells take longer to activate the process of apoptosis. Due to the small number of patients in each group, the results may have been just a coincidence. We will continue collecting samples of new cases within the same protocol.
To the doctors and residents of Rheumatology and Obstetrics at the State University of Rio de Janeiro and the Obstetrics section of UFRJ, where we have performed part of our study with pregnant women; to the employees of Bronstein Laboratory, RJ (Diagnostics of America SA), where the determinations were processed.
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NK cells in pregnant patients with SLE: a preliminary study
Alessandra Cardoso PereiraI; Mônica Cristina Brandão dos Santos LimaII; Nilson Ramires de JesúsIII; Marcus Montello FrançaIV; Homero da Silva Nahum JuniorV; Roger Abramino LevyVI
Publication in this collection
03 Aug 2009
Date of issue
28 Apr 2009
26 June 2008