Edith Cortés

Malnutrition alters the rates of apoptosis in splenocytes and thymocyte subpopulations of rats

Malnutrition continues to be a major public health problem throughout the developing world. Nutritional deficiencies may be the most common cause of secondary immunodeficiency states in humans. It has been suggested that nutritional imbalances can induce apoptosis in a variety of cell types. The purpose of this study was to examine the effect of severe malnutrition on cell subsets and the frequency of spontaneous and/or dexamethasone‐induced cell death in vivo in the thymus and spleen from severely malnourished, lactating rats. Apoptosis frequency was estimated by flow cytometry using annexin‐V and terminal transferase‐mediated dUTP nick‐end labelling assay assays. The results obtained in the present study indicate that malnutrition is associated with a significant increase of spontaneously apoptotic cells in the thymus (9·8‐fold) and spleen (2·4‐fold). Increase in apoptosis was associated largely with CD4+CD8+ double‐positive thymocytes. Unexpectedly, similar frequencies of spontaneous apoptosis of these cells were found in both well‐nourished and malnourished rats. In contrast, consistent increases in the apoptosis of CD4‐CD8‐ double‐negative thymocytes were observed in malnourished rats. In addition, single‐positive CD8+ and single‐positive CD4+ thymocytes had higher frequencies of apoptosis in malnourished rats. The frequency of total dexamethasone‐induced apoptosis was found to be similar in both groups of animals. Nevertheless, in malnourished dexamethasone‐treated animals, the percentage of apoptotic double‐negative thymocytes was significantly higher than in well‐nourished animals, while the rate of apoptosis was lower among double‐positive cells. In general, the thymus appears more sensitive to the effects of malnutrition and dexamethasone than the spleen. Furthermore, double‐negative thymocytes appear to be the most affected.

Flow cytometric analysis of spontaneous and dexamethasone-induced apoptosis in thymocytes from severely malnourished rats

Severe malnutrition is widely distributed throughout the world, showing a high prevalence in developing countries. Experimental animal models have been useful to study the effects of malnutrition at different levels and ages. Apoptosis is a well recognised process of cell death occurring under several physiological and pathological conditions. It represents the principal mechanism involved in cell selection in the thymus. Thymocyte apoptosis induction by dexamethasone is one of the best characterised experimental models of programmed cell death. The aim of the present study was to determine whether severe malnutrition increased spontaneous and/or dexamethasone-induced apoptosis in vivo in thymocytes of experimentally malnourished rats during lactation. Thymocytes were obtained from malnourished rats at weaning (21d of age). Apoptosis frequency was estimated by the terminal transferase-mediated dUTP nick end labelling assay. Spontaneous apoptosis was 1.9 (sd 1.0) % in well nourished rats in contrast to 13.3 (sd 3.8) % in malnourished animals; this is seven times greater (P<0.001). Interestingly, the frequency of dexamethasone-induced apoptosis was similar in both groups of animals (47.9 (sd 10.1) % in well nourished rats and 53.8 (sd 8.0) % in malnourished rats). The results obtained in the present study indicate that malnutrition is associated with a significant increase of spontaneously apoptotic cells. In addition, the data showed that the fraction of thymocytes susceptible to dexamethasone-induced apoptosis was similar in well nourished and malnourished animals. The greater levels of spontaneously apoptotic cells associated with malnutrition could be related to alterations of the microenvironment of the thymus and/or to an obstruction of early thymocyte maturation.

Effector T lymphocytes in well-nourished and malnourished infected children

The mechanisms involved in impaired immunity in malnourished children are not well understood. CD4+ CD62L– and CD8+ CD28– do not express the naive cell markers CD62L and CD28, suggesting that they function as effector T cells. Using a flow cytometry‐based analysis we examined the proportions of CD4+ CD62L– and CD8+ CD28– T cell subsets in well‐nourished infected (WNI) and malnourished infected (MNI) children. Here we report that WNI children had a higher percentage of CD4+ CD62L– (11·1 ± 1·0) and CD8+ D28– (40·2 ± 5·0) T cell subsets than healthy (6·5 ± 1·0 and 23·9 ± 4·8) and MNI children (7·4 ± 1·1 and 23·1 ± 6·2, respectively) (P < 0·5). Data suggest that WNI children respond efficiently against pathogenic microbes. In contrast, relatively low numbers of circulating of CD4+ CD62L– and CD8+ CD28– T cells in MNI children may represent an ineffective response to infection. Levels of effector T cells in children with gastrointestinal infections versus those suffering from respiratory infections were also significantly different within the WNI group. While WNI children with gastrointestinal infections had higher absolute and relative values of CD8+, and CD8+ CD28– T subsets, by those with respiratory infections had higher values of CD4+ lymphocytes. However, due to the small number of subjects examined, our results in WNI children should be interpreted with caution and confirmed using a larger sample size. Our data suggest that altered expression of CD62L and CD28 receptors may contribute to impaired T cell function observed in MNI children.

Analysis of Mitomycin C-induced micronuclei in lymphocytes from malnourished infected children

The purpose of this study was to determine if peripheral blood lymphocytes from malnourished children with gastrointestinal or respiratory bacterial infection show increased frequencies of Mitomycin C (MMC)‐induced micronuclei as compared to well‐nourished, infected children. The results indicate that cells from malnourished, infected children had greater chromosome damage. This may indicate that such children would be more susceptible to environmental damage and malignant transformation.

Trimethoprim‐sulfamethoxazole increase micronuclei formation in peripheral blood from weanling well‐nourished and malnourished rats

The combination of trimethoprim and sulfamethoxazole (TMP‐SMX) is a widely used drug. In spite of this, there are few reports on its genotoxicity, and the results are controversial. Severe malnutrition is a complex condition that increases the susceptibility to infections. Consequently, drugs are extensively used in malnutrition cases. Experimental animal models have been widely used to study the effects of malnutrition. Neonatal rats were experimentally malnourished (UN) during lactation. The UN rats weighed 51.1% less than the well‐nourished (WN) controls and had lower concentrations of serum protein and blood lipids. The micronucleus (MN) assay is useful for detecting chromosome damage induced by nutritional deficiencies. In vivo rodent MN assays have been widely used to screen genotoxic agents. In this study, we have evaluated the frequency of spontaneous and TMP‐SMX‐induced micronuclei in the peripheral blood of weanling (21 days of age) rats using a flow cytometric analysis technique. The spontaneous frequency of micronucleated reticulocytes (MN‐RETs) was 2.7 times greater in the UN rats than in the WN rats. In rats that were not treated with TMP‐SMX, the percentage of reticulocytes was significantly lower (41.1%) in the UN rats than the WN controls. A therapeutic dose of TMP‐SMX (80 mg/kg (TMP), 400 mg/kg (SMX) for 48 hr) increased MN‐RETs in the WN and in the UN rats. The data demonstrate the genotoxic effect of this drug. The results indicate that severe protein‐calorie restriction and drug treatment enhance DNA damage in rat peripheral blood reticulocytes, potentially increasing the risk of negative effects on health. Environ. Mol. Mutagen., 2011. ©2011 Wiley‐Liss, Inc.

Fanconi anemia cells with unrepaired DNA damage activate components of the checkpoint recovery process

BackgroundThe FA/BRCA pathway repairs DNA interstrand crosslinks. Mutations in this pathway cause Fanconi anemia (FA), a chromosome instability syndrome with bone marrow failure and cancer predisposition. Upon DNA damage, normal and FA cells inhibit the cell cycle progression, until the G2/M checkpoint is turned off by the checkpoint recovery, which becomes activated when the DNA damage has been repaired. Interestingly, highly damaged FA cells seem to override the G2/M checkpoint. In this study we explored with a Boolean network model and key experiments whether checkpoint recovery activation occurs in FA cells with extensive unrepaired DNA damage.MethodsWe performed synchronous/asynchronous simulations of the FA/BRCA pathway Boolean network model. FA-A and normal lymphoblastoid cell lines were used to study checkpoint and checkpoint recovery activation after DNA damage induction. The experimental approach included flow cytometry cell cycle analysis, cell division tracking, chromosome aberration analysis and gene expression analysis through qRT-PCR and western blot.ResultsComputational simulations suggested that in FA mutants checkpoint recovery activity inhibits the checkpoint components despite unrepaired DNA damage, a behavior that we did not observed in wild-type simulations. This result implies that FA cells would eventually reenter the cell cycle after a DNA damage induced G2/M checkpoint arrest, but before the damage has been fixed. We observed that FA-A cells activate the G2/M checkpoint and arrest in G2 phase, but eventually reach mitosis and divide with unrepaired DNA damage, thus resolving the initial checkpoint arrest. Based on our model result we look for ectopic activity of checkpoint recovery components. We found that checkpoint recovery components, such as PLK1, are expressed to a similar extent as normal undamaged cells do, even though FA-A cells harbor highly damaged DNA.ConclusionsOur results show that FA cells, despite extensive DNA damage, do not loss the capacity to express the transcriptional and protein components of checkpoint recovery that might eventually allow their division with unrepaired DNA damage. This might allow cell survival but increases the genomic instability inherent to FA individuals and promotes cancer.