Variability of methylation profiles of CpG sites in microRNA genes in leukocytes and vascular tissues of patients with atherosclerosis

In this study, we for the first time described the variability of methylation levels of 71 CpG sites in microRNA genes in leukocytes and blood vessels (coronary artery atherosclerotic plaques, intact internal thoracic arteries, and great saphenous veins) in patients with atherosclerosis using the Infinium HumanMethylation27 BeadChip microarray. Most of the analyzed CpG sites were characterized by the low variability, and most of these low-variable sites were hypomethylated in all tissue samples. CpG sites in coronary artery atherosclerotic plaques and leukocytes were similar in their methylation status. The highest variability of CpG methylation levels between different tissues was found for the CpG sites of the MIR10B gene; the methylation levels of these sites in leukocytes and atherosclerotic arteries were lower than in intact blood vessels. We also found that several cardiovascular disease risk factors, as well as medications, might affect methylation levels of CpG sites in microRNAs.     

Somatic genome variations in health and disease

It is hard to imagine that all the cells of the human organism (about 10(14)) share identical genome. Moreover, the number of mitoses (about 10(16)) required for the organism's development and maturation during ontogeny suggests that at least a proportion of them could be abnormal leading, thereby, to large-scale genomic alterations in somatic cells. Experimental data do demonstrate such genomic variations to exist and to be involved in human development and interindividual genetic variability in health and disease. However, since current genomic technologies are mainly based on methods, which analyze genomes from a large pool of cells, intercellular or somatic genome variations are significantly less appreciated in modern bioscience. Here, a review of somatic genome variations occurring at all levels of genome organization (i.e. DNA sequence, subchromosomal and chromosomal) in health and disease is presented. Looking through the available literature, it was possible to show that the somatic cell genome is extremely variable. Additionally, being mainly associated with chromosome or genome instability (most commonly manifesting as aneuploidy), somatic genome variations are involved in pathogenesis of numerous human diseases. The latter mainly concerns diseases of the brain (i.e. autism, schizophrenia, Alzheimer's disease) and immune system (autoimmune diseases), chromosomal and some monogenic syndromes, cancers, infertility and prenatal mortality. Taking into account data on somatic genome variations and chromosome instability, it becomes possible to show that related processes can underlie non-malignant pathology such as (neuro)degeneration or other local tissue dysfunctions. Together, we suggest that detection and characterization of somatic genome behavior and variations can provide new opportunities for human genome research and genetics.     

Somatic genomic variations in extra-embryonic tissues

In the mature chorion, one of the membranes that exist during pregnancy between the developing fetus and mother, human placental cells form highly specialized tissues composed of mesenchyme and floating or anchoring villi. Using fluorescence in situ hybridization, we found that human invasive cytotrophoblasts isolated from anchoring villi or the uterine wall had gained individual chromosomes; however, chromosome losses were detected infrequently. With chromosomes gained in what appeared to be a chromosome-specific manner, more than half of the invasive cytotrophoblasts in normal pregnancies were found to be hyperdiploid. Interestingly, the rates of hyperdiploid cells depended not only on gestational age, but were strongly associated with the extraembryonic compartment at the fetal-maternal interface from which they were isolated. Since hyperdiploid cells showed drastically reduced DNA replication as measured by bromodeoxyuridine incorporation, we conclude that aneuploidy is a part of the normal process of placentation potentially limiting the proliferative capabilities of invasive cytotrophoblasts. Thus, under the special circumstances of human reproduction, somatic genomic variations may exert a beneficial, anti-neoplastic effect on the organism.     

Anti-interferon activity of peripheral blood leukocytes in patients infected with HIV-1

The study was aimed at anti-interferon activity of different components of the in vitro system cells MT-4--HIV-1 as well as the culture fluid of peripheral blood leukocytes from patients at different stages of HIV infection and from patients with mixed infection (HIV and chronic hepatitis C) in comparison with patients infected with hepatitis C virus alone and healthy persons. Anti-interferon activity was detected in all groups of patients, its detection rate varying within 33% and 68%. The tendency towards increased detection rate of anti-interferon activity in HIV-infected patients in parallel with decreased number of CD4+ lymphocytes was noted. These data made it possible to suggest that increased detection rate of anti-interferon activity in the culture fluid of peripheral blood leukocytes from HIV-infected patients in parallel with decreased number of CD4+ lymphocytes could result from pathogenetic processes in the body, leading to a decrease in therapy effectiveness of HIV-infected patients with the preparations of alpha-interferon, especially in patients with a low content of CD4+ cells.     

Phenotype of peripheral blood leukocytes and survival of patients with metastatic colorectal cancer

Immune dysfunction is prevalent in metastatic cancer. Few patients with colorectal cancer metastases are cured, and among the strategies aimed at improving the therapeutic results in patients with metastatic colorectal cancer, immunotherapy is being increasingly investigated. We evaluated retrospectively the prognostic significance of peripheral blood leukocytes in 59 patients with metastatic colorectal cancer. The relative numbers of CD3+, CD3+CD4+, CD3+CD8+, NK (CD3-CD16+CD56+), CD3+DR+, CD3+CD25+, CD3+CD69+, CD19+, CD19+CD23+, CD8+CD28+, CD8-CD28+, CD8+CD57+, CD14+DR+ and CD14+CD16+ leukocytes were analyzed by two-color flow cytometry. A three-step approach was adopted to identify predictors of prognosis using regression analysis. Based on the results of univariate survival analysis, the absolute number of white blood cells, NK/CD3+CD69+ and NK/white cell count ratios were significant indicators of prognosis. In the multivariate regression analysis a model was obtained using a single parameter, the NK/CD3+CD69+ ratio, predicting the survival with 10-15% power of regression. The present results indicate that the NK/CD3+CD69+ ratio in peripheral blood may be an independent variable in a regression model predicting the overall survival of patients with colorectal cancer metastases to be tested in prospective studies.     

ABCA1 gene expression in peripheral blood lymphocytes and macrophages in patients with atherosclerosis

ABCA1 transporter is known to play important role in the cholesterol transport from peripheral tissues. However its contribution in atherosclerosis development remains not completely understood. Using Real Time PCR, a significant reduction of ABCA1 mRNA level in leukocytes of patients with atherosclerosis was determined when compared with controls. Mean ABCA1 expression levels in leukocytes for the group of patients and for the control group are 0.57 +/- 0.28 and 0.93 +/- 0.14 (p = 0.02). At the same time we detected a significant increase of ABCA1 mRNA level in macrophages of patients when compared with controls. Mean ABCA1 expression levels in macrophages for the group of patients and for the control group are 1.32 +/- 0.10 and 0.90 +/- 0.14 (p = 0.014). In summary, we suggest that expression level of ABCA1 gene may contribute to the development of atherosclerosis.     

Evidence for acrolein-modified DNA in peripheral blood leukocytes of cancer patients treated with cyclophosphamide

Monitoring human populations for specific DNA modifications has been made possible by developing highly sensitive immunoassays employing antibodies specific for carcinogen-DNA adducts. While these techniques have been used to follow occupationally and environmentally exposed populations, results have been limited by the lack of exposure data with which to correlate adduct formation. Cancer patients treated with precisely known doses of anticancer drugs can be studied to examine the association between drug dose and adduct formation. This study examined acrolein-modified DNA in patients treated with the anticancer drug cyclophosphamide (CP) and in newly diagnosed patients prior to treatment. Employing 2 different detection methods, enzyme-linked immunosorbent assay (ELISA) and immuno-dot blot (IDB), acrolein-modified DNA was identified in a total of 6 of 12 (50%) treated patients and in 0 of 15 untreated patients. Formation of acrolein-modified DNA was examined as a function of lifetime CP dose, recent CP dose, time since last treatment, regime of treatment, and smoking history; however no clear trends were observed.     

Gene expression patterns in peripheral blood leukocytes in patients with recurrent ciguatera fish poisoning: Preliminary studies

Ciguatera fish poisoning (ciguatera) is a common clinical syndrome in areas where there is dependence on tropical reef fish for food. A subset of patients develops recurrent and, in some instances, chronic symptoms, which may result in substantial disability. To identify possible biomarkers for recurrent/chronic disease, and to explore correlations with immune gene expression, peripheral blood leukocyte gene expression in 10 ciguatera patients (7 recurrent and 3 acute) from the U.S. Virgin Islands, and 5 unexposed Florida controls were evaluated. Significant differences in gene expression were noted when comparing ciguatera patients and controls; however, it was not possible to differentiate between patients with acute and recurrent disease, possibly due to the small sample sizes involved.     

Cytochemistry of nucleoproteids and some cathionic proteins in the peripheral blood leukocytes of patients with lung cancer.

In 40 patients with untreated lung cancer cytochemical studies of the peripheral blood leukocytes were conducted by means of a cytological method for the simultaneous staining of nucleoproteids (RNP and DNP) and some cathionic proteins (after Zvetkova and Zvetkov [60]). Changes were detected in the RNP cytoplasmic contents of lymphocytes, of which the most outstanding were the reduction and uneven distribution of RNP granules, their frequent extracellular expulsion by means of microclasmatoses, as well as changes in the staining of cathionic proteins of RNP accompanied by an increased nuclear chromatin condensation in the small and medium-sized lymphocytes. Parellel to reducing of the percentage of these cells in the peripheral blood of patients with advanced neoplastic disease an increased number of lymphoblastoid and monoblastoid cells is established with RNP diffusely stained, but reduced in quantity and localized in the cytoplasmic periphery and projections (compared to Downey type II atypical cells). By means of one of the variants of the method (modified type of Feulgen's reaction) a characteristic distribution and structuring of the nuclear chromatin is established in mono- and polymorphonuclear cells, most clearly expressed in the nuclei of monocytes and monoblastoid cells, as well as in nuclei of neutrophil granulocytes. In these cellular types a more specific nuclear modelling (microhypersegmentation) is observed resulting in multiple irregular nuclear projections on the nuclear surface, probably caused by subkaryolemal distribution of uneven chromatin thickenings. The changes are also recorded in the cathionic protein containing secondary cytoplasmic granules in granulocytes-neutrophils and eosinophils, probably associated with changes in the lysosomal and phagocytic functions of these cells in neoplastic diseases. The authors discuss the importance of the obtained results in connection with data on the participation of lymphocytes and neutrophils in the immune response to tumour antigenic stimuli during the course of the neoplastic process, as well as with data on the suppressive effect of antigenic (serum, viral) factors, possibly affecting the synthesis and the transport of cellular nucleoproteids (RNP and DNP) in leukocytes of cancer patients.     

Distribution of peripheral blood leukocytes in acute heatstroke.

We examined 11 heatstroke patients (mean rectal temperature 41.4 +/- 0.3 degrees C) and 40 healthy subjects to determine the effects of hyperthermia on peripheral blood leukocyte distribution. Precooling samples were taken on admission. Whole blood was incubated with conjugated monoclonal antibodies, and erythrocytes were eliminated by FACS lysing solution. Lymphocyte subsets were detected by specific mouse monoclonal antibodies: Leu-4/CD3+ (T-cells), Leu-3a/CD4+ (T-helper cells), Leu-2a/CD8+ (T-suppressor-cytotoxic cells), Leu-11/19/CD16+/CD56+ (natural killer cells), and Leu-12/CD19+ (B-cells). Immunofluorescence was measured with a flow cytometer. The number of circulating leukocytes and lymphocytes was significantly increased in heatstroke patients. This lymphocytosis was mainly due to an increase in T-suppressor-cytotoxic cells and natural killer cells. The absolute number of lymphocytes and T-suppressor-cytotoxic cells significantly correlated with the degree of hyperthermia (r = 0.62, P = 0.04; r = 0.751, P = 0.007, respectively). There was a significant decrease in the percentages of T-, B-, and T-helper cells and increase in T-suppressor-cytotoxic and natural killer cells, giving a marked decrease in the ratio of T-helper to T-suppressor-cytotoxic cells. We conclude that heatstroke is associated with leukocytosis and significant alteration in absolute number and percentage of circulating lymphocyte subpopulations.     

Genotoxic effect of ozone in human peripheral blood leukocytes

The genotoxic effect of ozone was studied in human leukocytes in vitro, using the single cell gel electrophoresis (SCGE) assay. Cell treatment for 1 h at 37 degrees C with 0.9-5.3 mM O(3) resulted in a dose-dependent increase of DNA damage, comparable to that induced by 4-40 mM of H(2)O(2), used as a positive control. This effect of ozone was reversed by post-treatment incubation of the cells for 45-90 min at 37 degrees C, and prevented by pre-incubation of the cells with catalase (20 microg/ml). These results demonstrate that O(3) induces DNA-damage in primary human leukocytes. The damage is rapidly repaired, and probably mediated by the formation of H(2)O(2).