Peripheral Blood Lymphocyte Cultures Research Articles

Involvement of (IgG and IgM)-secreting B lymphocytes in severity of autoimmune hepatitis type 1

Autoimmune hepatitis type 1 (AIH1) is an autoimmune disease attacks the liver and characterized by periportal inflammation, elevated immunoglobulins, and autoantibodies. The central role of B lymphocyte in pathogenicity of AIH1 is unclear. Here, the effect of antibody-secreting cells activity in terms of number of plaque-forming cells (PFC) on severity of AIH1 was evaluated. The high number of PFC-(IgG and IgM) in peripheral blood of patients with AIH1 was observed and this was concomitant with increase in the number of CD4+ T lymphocyte, immunoglobulin (Ig) (IgG and IgM) concentrations, and levels of liver function tests (LFTs). However, the negative correlation (r < -0.5, P < 0.05) between the numbers of PFC-(IgG and IgM) and CD8+ T lymphocytes was reported here. The present study showed the positive relationship between the concentrations of Ig (IgG and IgM) and levels of LFTs (r > +0.5, P < 0.05). The high expression of IL-10 mRNA was found in the tissue culture of peripheral blood lymphocytes obtained from patients with AIH1 as compared with that isolated from control group (P < 0.05). The current study proved the direct role of (IgG and IgM)-secreting cells in severity of AIH1. This was associated with CD4+ cell numbers and IL-10 mRNA expression, and mediated by IgG and IgM.

Sister chromatid exchange as an index of chromosome instability in chondrodystrophic chickens (Gallus domesticus)

Commercially bred broiler chickens reared in inadequate breeder-created habitat conditions often suffer from a disease that afflicts their lower limbs, chondrodystrophy. The sister chromatid exchange (SCE) test makes it possible to detect chromosome instability that corresponds with elevated vulnerability of the organism to genotoxic factors of a mutagenic and carcinogenic nature. The frequency of SCE in chromosomes of chondrodystrophic and healthy broilers was analyzed. Chromosome preparations were obtained from our in vitro culture of peripheral blood lymphocytes stained using the fluorescence plus Giemsa technique. The SCE/cell mean of the population under analysis was 6.8 ± 1.6. The SCE/cell mean in the chromosomes of the chondrodystrophic chickens was 8.5 ± 1.0. The healthy chickens had an SCE/cell mean of 5.1 ± 1.3. Statistically significant differences were identified between both chicken groups. Moreover, a higher SCE/cell mean was observed in the males: 6.9 ± 1.7 compared with 6.7 ± 1.7 in the females. However, this difference was not statistically significant. Additionally, SCE incidence in the first 3 biggest autosome pairs (1, 2, 3) was analyzed in detail. The number of identified SCE was proportional to chromosome length. The most exchanges were observed in the proximal region of the chromosomes under analysis.

Analysis of Chromosome Aberrations Contained in Vitro Human Peripheral Blood Lymphocytes After Treatment with Ceftriaxone

The invention and use of antibiotics in treating infections is one of the greatest achievements of the twentieth century medicine. Antibiotics are one of the categories of pharmaceuticals with a broad and increasing application. The goal of this paper is to analyze the influence of different set of test concentrations of ceftriaxone antibiotics on the occurrence of chromosome aberrations after in vitro treatment with concentrations: 0.15 mg/ml, 0.25 mg/ml and 0.50 mg/ml. In the study was used the blood of healthy donors in vitro, treated with different concentrations of ceftriaxone. Ceftriaxone is a semisynthetic cephalosporine third generation antibiotic, with broad spectrum of activity and with specific characteristics (Nerlove et al. 1996). As a biomarker of genetic damage are used the methods of cultivation of peripheral blood lymphocytes and analysis of chromosome aberrations. Cytogenetic analysis of ceftriaxone genotoxicity was performed in 48-hour culture of human peripheral blood lymphocytes by test of standard chromosome aberration analysis according to Moorhead, with certain modifications. Insight into the frequency and type of chromosome aberrations is obtained by analyzing 100 metaphases per sample. By the analysis of 100 metaphases per sample was determined that the relative frequency of metaphases with chromosome aberrations is increased with increasing concentrations of ceftriaxone in lymphocyte culture. The increase in the frequency of structural aberrations was also positively correlated with the applied ceftriaxone concentrations. Metaphases with numerical and structural aberrations are registered in lymphocyte cultures treated with ceftriaxone concentration of 0.25 mg/ml and 0.50 mg/ml, but this increase was not significant compared to the control cells cultures. Significantly increased frequency of metaphases with structural chromosome aberrations in cultured human peripheral blood lymphocytes treated with concentrations of 0.25 mg/ml and 0.5 mg/ml compared to the control confirming clastogenic potential of this drug. Ceftriaxone also expressed aneugenic activity at the highest test concentration (0.50 mg/ml), confirming a statistically significant difference in the frequency of numerical aberrations in cultures treated with doses of 0.5 mg/ml.

Genotoxic effects of anesthetics in operating room personnel evaluated by micronucleus test

Exposure to certain chemical agents causes damage to the genetic material. There is controversy about the genotoxic and/or mutagenic effect caused by acute or chronic anesthetics exposure. The aim of this research was to assess and compare the frequency of micronuclei (MN) in professionals working in operating rooms and other hospital areas. The genotoxic and cytotoxic effects were evaluated in lymphocytes using the micronucleus test and the proliferative index, respectively. We examined peripheral blood lymphocyte cultures from 30 operating room professionals (exposed group) and 30 professionals non-exposed in other hospital areas of the same hospital (control group). There was no statistically significant difference in proliferative index between the groups. Nevertheless, there was an increase in MN frequency in binucleated (BN) cells in the exposed group (p=0.0003) compared to the control group. Moreover, there was a statistically significant difference between genders (p=0.0187), and the frequency of MN in BN cells from women was higher than in men. Therefore, gender influenced the frequency of MN. The age and period of working time in an operating room influenced the MN frequency only in women professionals. Thereby, there was a genotoxic effect in occupationally exposed professionals, and the micronucleus test could be used in the biomonitoring of human populations for evaluating the risk of developing cancer.

Сравнительный анализ результатов цитогенетических обследований контрольных групп лиц в различных отечественных лабораториях

The incidence of unstable chromosome aberrations in peripheral blood lymphocytes from unirradiated control subjects was analyzed using cytogenetic data obtained from 9 cytogenetic laboratories located in Moscow, St.-Petersburg, Obninsk, and Dubna (Russia). The objective of this study was to estimate the level and spectrum of spontaneous chromosome aberrations in human lymphocytes. 1140 blood samples were taken from 1112 subjects (594 men and 546 women) aged 1 to 72. The total metaphase number was 466795. The uniform Giemsa method for peripheral blood lymphocyte cultures was used. After counting 466795 metaphases, 4288 chromosomal aberrations of various types were classified. The most frequent types of aberrations were acentrics and chromatid deletions. They made up 90% of the total number of aberrations. The remaining 10% were exchange aberrations. The number of chromosome exchanges (dicentrics and centric rings) was twice the number of chromatid exchanges. Overall, the portion ofcells with chromosomal or (and) chromatid aberrations was 0.89 +/- 0.01%; the frequency of acentrics was 0.29 +/- 0.01; the frequency of dicentrics was 0.046 +/- 0.003; the frequency of unstable chromosome aberrations was 0.35 +/- 0.01; and the frequency of chromatid aberrations was 0.57 +/- 0.01 per 100 cells.

Genotoxic and Cytotoxic Evaluation of Pyrethroid Insecticides λ-Cyhalothrin and α-Cypermethrin on Human Blood Lymphocyte Culture

The present study aimed to investigate the genotoxic, cytotoxic and aneugenic effects of 1, 2, 3.75, 7.5, 15, 30 μM concentrations of the insecticides λ-cyhalothrin (LCT) and α-cypermethrin (CYP) on human peripheral blood lymphocyte culture using micronucleus (MN) and fluorescence in situ hybridisation (FISH) methods. All the concentrations were tested to assess the MN and apoptosis effects, and 1 and 2 μM LCT and 7.5 and 15 μM CYP concentrations were tested for FISH analysis. The cytotoxic effect was also observed using trypan blue and the acridine orange/ethidium bromide fluorescence staining method to measure the apoptotic effect. It was observed that both of the insecticides had a cytotoxic effect at all the concentrations (p ≤ 0.001) and apoptotic effect for LCT at 15-30 μM (p ≤ 0.05; p ≤ 0.01) for CYP between 2 and 30 μM concentrations (p ≤ 0.05; p ≤ 0.01). The micronuclei that developed after exposure were induced because of an aneugenic effect (p ≤ 0.001). LCT and CYP might be spindle poisons or caused damaged to centromere/kinetochore function.

Immunotherapy with LAK Cells and Anti-CD20 Monoclonal Antibodies for Follicular Lymphoma: Enhanced Antibody-Dependent Cell Cytotoxicity of LAK Cells in Association with GA101 Rather Than Rituximab

AbstractAbstract 4881 BackgroundThe current gold standard treatment for follicular lymphoma (FL) consists in the association of rituximab, that is an anti-CD20 monoclonal antibody (mAb), and one among many possible chemotherapy regimens, followed by maintenance with the same mAb. However, LF is still considered incurable. Hence, it is necessary to keep investigating new strategies aiming at improving the overall clinical results.As an anti-tumor mechanism, antibody-dependent cellular cytotoxicity (ADCC) depends on both the immune system effector arm and the antibody structure. The culture of peripheral blood lymphocytes with IL-2 activates killer cell subpopulations (LAK cells), mainly consisting of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), both with significant cytotoxic capacity. The combination of LAK cells with a mAb could achieve a synergistic effect by enhancing ADCC and potentially achieving clinical efficacy. As structural modifications in mABs may alter their biological activity, new anti-CD20 with structural changes in the Fc portion of the immunoglobulin have been synthesized in order to obtain a greater therapeutic effect. GA101 is a new anti-CD20 mAB that binds with higher affinity to the CD20 type II epitope, increasing the ADCC effect as compared to to rituximab. Currently, no data support the notion of a possible synergistic effect of GA101 and LAK cells. AimThe objective of this work was to assess the cytotoxic capacity of LAK cells generated from the peripheral blood of patients with FL. In addition, we aimed at ascertaining whether the combination of rituximab and LAK could improve the functional activity of LAK alone. Finally, we conducted a comparative study of two anti-CD20 antibodies structurally different, namely rituximab and GA101, used in combination with LAK cells. Material and methodsLAK cells were expanded in vitro from 39 peripheral blood samples of patients with FL. Cytotoxicity studies were performed using chromium release assays in which LAK cells targeted K562 (as NK target), Daudi (as LAK cells target) and CRL-1596 (CD20 positive) cells. Overall, mononuclear cells were isolated and basal cytotoxicity was measured in 39 peripheral blood samples from patients with FL. The ability of two anti-CD20 antibody (rituximab and GA101) to enhance LAK cell activity was evaluated against the CRL-1596 cell line. As a control, an irrelevant antibody (cetuximab) was used. To perform these studies, target cells were incubated with effector cells and antibody at 10 mg/ml concentration. ResultsResults are expressed as mean percentages. Basal cytotoxicity against K562, Daudi and CRL-1596 cells was 19.4%, 11.9% and 1.5% respectively. After culture with IL-2, the cytotoxicity activity against the same cell lines was reassessed and in all cases a statistically significant increase was observed (p<0,05). In particular, cytotoxicity against K562, Daudi and CRL-1596 was 39.5%, 39.6% and 21.1%, respectively.Furthermore, we studied the rituximab effect on the cytotoxic capacity against CRL-1596. The observed cytotoxicity of LAK cells with rituximab was 39.9% vs 23.6% for LAK cells alone (p<0,001). In parallel, the GA101 effect on the cytotoxic capacity against CRL-1596 was also evaluated. The cytotoxicity of LAK cells with GA101 was 52.3% vs 23.6% for LAK cells alone (p<0,001). Vice versa, no difference was observed in the cytotoxic activity of LAK cells against CRL1596 cell line when the effector cells were incubated with the irrelevant antibody cetuximab (26.0% vs 23.6%).Finally, when comparing the ability of both anti-CD20 antibodies to enhance ADCC, we observed a significant difference in favor of GA101 (GA101: 52.3%, rituximab: 39.9%; p<0,001). ConclusionsLAK cells generated from peripheral blood lymphocytes by culture with IL-2 in patients with LF show a higher cytotoxic activity than naive lymphocytes. The observed cytotoxic capacity of these LAK cells against a CD20 positive cell line (CRL-1596) is enhanced by the addition of anti-CD20 mAbs. Interestingly, GA101 was consistently more effective than rituximab in enhancing the cytotoxic capacity of LAK cells. Disclosures:No relevant conflicts of interest to declare.

In vivo synergistic cytogenetic effects of aminophylline on lymphocyte cultures from patients with lung cancer undergoing chemotherapy

BackgroundThe anti-cancer and cytogenetic effects of aminophylline (AM) have been demonstrated in several clinical trials. The aim of the present study was to investigate the in vivo cytogenetic effects of AM in newly diagnosed patients with small cell (SCLC) and non-small cell lung cancer (NSCLC), receiving chemotherapy for the first time. MethodsSister chromatid exchanges (SCEs) and proliferation rate index (PRI) were evaluated in peripheral blood lymphocyte cultures from six patients with SCLC and six patients with NSCLC after the in vitro addition of AM and after the in vivo administration of AM in patients receiving chemotherapy. ResultsThe in vitro addition of AM significantly increased SCEs only in SCLC patients (p<0.001). The in vivo administration of AM after chemotherapy increased SCEs in both cancer types (SCLC: p<0.001, NSCLC: p=0.003) and this increase was synergistic, the rates of SCEs in the presence of AM were higher than the expected SCE values if the increases above background for chemotherapy and AM were independent and additive (SCLC: p<0.001, NSCLC: p=0.008). Although in both groups of patients cell division delays were observed after the combined chemotherapy plus in vivo AM treatment, the correlation between the magnitude of the SCE response and the PRI depression was not statistically significant (p>0.05). ConclusionsThese observations suggest that AM enhances the results of concurrently administered chemotherapy by synergistically increasing its cytogenetic effects in patients with lung cancer.

Sister chromatid exchange in Greenleg Partridge and Polbar hens covered by the gene-pool protection program for farm animals in Poland

A basic assay that detects genotoxic DNA damage disrupting DNA replication and repair mechanisms is the sister chromatid exchange test. The frequency of sister chromatid exchanges was analyzed in chromosomes of the following hen breeds: Greenleg Partridge and Polbar. Chromosome preparations were obtained from our in vitro culture of peripheral blood lymphocytes stained using the fluorescence plus Giemsa (FPG) technique. The sister chromatid exchange (SCE)/cell mean of the hens under analysis was 7.83 ± 1.76 (7.22 ± 1.70 in the Greenleg Partridge and 8.43 ± 1.61 in the Polbar population). Statistically significant differences were identified between the hen breeds. A higher mean number of SCE/cell was observed in the group of hens producing fewer eggs (8.55 ± 1.51) compared with the group with a better egg yield (7.10 ± 1.65). The differences were statistically significant. Additionally, SCE frequency in the first, second, and third chromosome was analyzed in detail. The highest number of SCE was observed in the first and the lowest in the third chromosome. The SCE distribution in the particular regions of the analyzed chromosomes was also studied. The most numerous exchanges were observed in the proximal region, followed by the interstitial and distal areas.

Chromosomal abnormality in patients with secondary amenorrhea.

Secondary amenorrhea is a condition in which there is cessation of menses after at least one menstruation. It is a symptom of different diseases, such as hormonal disturbances which range from pituitary to ovarian origin, as well as chromosomal abnormalities. Knowledge of the distinct cause of secondary amenorrhea is of tremendous benefit for the management and monitoring of patients. In this study, we determine the chromosomal abnormalities in patients with secondary amenorrhea in Southwest Iran. We selected 94 patients with secondary amenorrhea who referred to our Cytogenetic Ward from 2004 until 2009. For karyotyping, peripheral blood lymphocyte cultures were set up by conventional technique. In this study, 5.3% (n=5) of patients with secondary amenorrhea presented with chromosomal abnormalities, of which all contained an X element. The chromosomal abnormalities were: i) 45, X (n=1); ii) 47, XXX (n=1); iii) 45, X [13]/ 45, Xi(X)q[17] (n=1); iv) 45, X[12]/46,X,+mar[12] (n=1); and v) 46,X,del(Xq)(q23q28) (n=1). Our study revealed that some causes of secondary amenorrhea could be due to chromosomal abnormalities. Therefore, cytogenetic studies should be important tests in the evaluation of patients with secondary amenorrhea.

Immune attributes of cardiac-derived adherent proliferating (CAP) cells in cardiac therapy

Cardiac-directed cell therapies show potential to reduce mortality and morbidity in heart disease. However, high functional efficacy should be complimented with low immunogenicity, in particular if allogeneic cell sources are applied. Therefore, we aimed to examine cardiac-derived adherent proliferating (CAP) cells with respect to their immunogenicity and immune modulatory features in vitro. Human CAP cells were isolated from cardiac biopsies and screened in a CFSE-based proliferation assay in co-cultures with phytohaemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBMCs) or mixed lymphocyte cultures (MLCs) to assess their potential to induce immune cell proliferation or activation by flow cytometry. Moreover, levels of pro- and anti-inflammatory cytokines in supernatants of co-cultures were analysed. The capacity of CAP cells to induce the generation of regulatory T cells (Tregs) was determined by flow cytometric measurement of FoxP3 expression. CAP cells of different donors (n = 5) showed low immunogenicity in co-cultures with human allogeneic PBMCs. In addition, they induced no change in the normal alloantigen-driven immune responsiveness in MLCs. However, CAP cells significantly reduced the induction of immune cell proliferation in PBMCs cultures stimulated with the polyclonal trigger PHA. Adding CAP cells into MLCs or PHA-stimulated cultures resulted in significantly reduced levels of TNFα or IFNγ, respectively, compared to controls without CAP cells. At early time points (day 2), interaction of CAP cells with PBMCs resulted in elevated proportions of FoxP3+ CD4+ CD25(high+) cells. The results indicate that CAP cells have low immunogenicity and could be advantageous in cardiac repair by reducing inflammatory cytokines and inducing regulatory T cells.

Aqueous Extracts of Lippia turbinata and Aloysia citriodora (Verbenaceae): Assessment of Antioxidant Capacity and DNA damage

The aim of the present work was to make a contribution to the knowledge of aqueous extracts of Lippia turbinata and Aloysia citriodora (Verbenaceae; infusion and decoction) in relation with the establishment of its antioxidant activity and lack of DNA damage, for its potential use in therapeutics. The cytogenotoxic profile was evaluated through genotoxic biomarkers such as mitotic index, cellular proliferation kinetics, sister chromatid exchanges, single-cell gel electrophoresis assay, and micronucleus test in human peripheral blood lymphocyte cultures. No statistical differences were found (P > .05) between control and exposed cultures, even between both aqueous extracts. The total antioxidant capacity was shown to be higher in the decoction than in the infusion and both aqueous extracts protected against protein carbonylation and lipid peroxidation, the decoction being more efficient than the infusion (P < .005). These results suggest the safe use of these medicinal plants as chemoecologic agents in therapeutics.

Genotoxic and cytotoxic effects of doxycycline in cultured human peripheral blood lymphocytes

Doxycycline (DOX) is a broad-spectrum tetracycline antibiotic used in the treatment of many infections. In this study, the genotoxic and cytotoxic effects of DOX in cultured human peripheral blood lymphocytes were investigated by measuring chromosome aberrations (CAs), cytokinesis-block micronucleus (CBMN) assay, mitotic index (MI), and nuclear division index (NDI). Cultures were treated with DOX at three concentrations (2, 4, and 6 µg/mL) for 48 hours. Mitomycin C (MMC) was used as a positive control. All the tested concentrations of DOX for MI and the higher concentrations (4 and 6 µg/mL) for NDI significantly decreased mitotic activity. However, there are no significant differences between negative control and all the tested concentrations of DOX for CA and MN frequencies. In conclusion, our results indicate that DOX has a cytotoxic effect, but not a genotoxic effect, on human peripheral blood lymphocyte cultures. Further detailed studies, especially about the cell-cycle kinetics of DOX, are required to elucidate the decreases in dividing cells and make a possible risk assessment on cells of patients receiving therapy with this drug. Further, if the specific cytostatic and cytotoxic potential of DOX to different types of cancer cells is investigated in detail, it may also have been used as an antitumoral drug.

Chromosomal Rearrangements In Lymphocytes Of Head and Neck Squamous Cell Carcinoma Treated With Chemotherapeutic Agents

Squamous cell carcinoma especially of oral cavity is one of the most prevalent diseases in the world. Chromosomal rearrangements are known to play important role in the pathogenesis of many diseases including cancer. In case of Head and Neck Squamous Cell Carcinoma, chromosomal changes are detectable at all stages of tumor development providing excellent opportunity for chromosomal prognosis and therapy. The present work aimed to study the frequency and pattern of chromosomal aberrations in human peripheral blood lymphocyte culture of freshly diagnosed Head and Neck squamous cell carcinoma patients. Further In vitro anticancer drugs (5-Fluorouracil {5-FU } and Cisplatin) effects were studied for clastogenicity. Results indicated significant impact of chemotherapeutic agents on the frequency of different types of chromosomal rearrangements.

Sister chromatid exchange in selected horse breeds (&amp;lt;i&amp;gt;Equus caballus&amp;lt;/i&amp;gt;)

Abstract. In studies of chromosome instability, the sister chromatid exchange (SCE) test is a particularly sensitive cytogenetic assay for detecting DNA damage. SCE tests of chromosome instability were performed in the group of 6 horse breeds (Pure-bred Arabian, Malapolski horse, Polish noble half-bred, Polish cold-blooded, Hucul and Polish Konik). The chromosome preparations were obtained from our in vitro culture of peripheral blood lymphocytes stained using the FPG technique. The mean number of SCEs/cell in the analysed population of horses was 5.14±1.44. The mean frequency of SCEs in the 6 analysed horse breeds varied depending on the breed. Statistically significant differences were observed between the horse breeds (P&lt;0.01). No statistically significant differences in the number of SCEs per cell were found between the males and females (5.10±1.34 and 5.20±1.52, respectively). The horses were also assessed for the number of SCEs/cell in relation to the age of the animals. The differences between the age groups were statistically significant (P&lt;0.01).

A Study of Anticancer Drug Treatment on Satellite Associations in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma is one of the most prevalent diseases worldwide. Satellite associations are known to play an important role in the pathogenesis of certain diseases including cancer . The present work aimed to study the frequency of satellite association in human peripheral blood lymphocyte culture of freshly diagnosed oral squamous cell carcinoma patients. In vitro anticancer drugs (5-Fluorouracil {5-FU } and Cisplatin) were studied for the frequency of satellite association. Results showed significant impact of chemotherapeutic agents on the frequency of satellite associations of dif ferent groups of acrocentric chromosomes.

Induction of micronuclei on Greek hairdressers occupationally exposed to chemical mixtures

Since the early 20th century, hairdressers (HD) have been exposed to a wide range of harmful chemical products. To determine the possible genetic damage to HD, as a result of their occupational exposure to combinations of different chemical factors, we applied the micronucleus assay on their peripheral blood lymphocytes cultures. The micronucleus assay was performed on blood samples from 20 Greek female HD and 20 control women, having no connection with the occupation, from the same area. In the results analysis, parameters included were age, smoking habits, and duration of occupational exposure. The results of our study showed a significant increase in HD micronuclei (MN) frequency, compared to the controls (13.4 ± 1.00 vs. 8.05 ± 0.65). The frequency of large-size MN was significantly higher in the HD and presented potential correlation with the phenomenon of aneuploidy. A statistically significant difference in the frequency of MN between HD and controls who smoked was observed, while this was not the case with the non smoker groups. However, multiple regression analysis showed no significant correlation between smoking habits and MN frequency. The observed increase of the frequency of MN in HD is attributed to the long-term occupational exposure of HD in combination with different chemical factors. Since in the literature there are very few similar studies, further combined studies are suggested on a larger number of HD from different countries, combining biological and molecular techniques, as well as chemical analytical methods of determining and tracing the chemical factors in both the occupational environment and their organisms.

338 Interferon alpha 21 levels are elevated in peripheral blood lymphocytes and aortic smooth muscle cells with the 9p21.3 coronary artery disease risk genotype

The 9p21.3 locus confers risk for coronary artery disease (CAD) by an unknown mechanism. A recent report identified several long-range enhancers, at CDKN2B, CDKN2A, MTAP and between the interferon omega-1 (IFNW1) and interferon alpha-21 (IFNA21) genes, whose chromatin structure is influenced by single nucleotide polymorphisms within the 9p21.3 risk locus as far as 1 million base pairs away. However, whether protein expression from the genes in the vicinity of these long-range enhancers is correlated with the 9p21.3 risk genotype was not known. Here, we applied immunoblot analysis using antibodies specific to the proteins encoded by the genes near the long-range enhancers to test for their association with the 9p21.3 risk genotype. Using protein extracts from primary cultures of human aortic smooth muscle cells (n = 6) and peripheral blood lymphocytes from patients with CAD (n = 18) genotyped for the 9p21.3 risk locus using SNP microarrays, we found a clear positive correlation (P < 10-4) between IFNA21 levels and 9p21.3 risk genotype in both cell types. No association between MTAP expression and 9p21.3 genotype was seen in either cell type. In contrast, CDKN2A protein expression in both cell types, and CDKN2B protein expression in aortic smooth muscle cells was negatively associated with 9p21.3 risk genotype. Thus, the presence of long-range enhancers influenced by the 9p21.3 risk locus does not predict the direction of associated gene expression. Elevated IFNA21 and reduced CDKN2A protein expression is linked to and likely accounts for the risk of CAD associated with the 9p21.3 locus.

The results of 25 years of cytogenetic investigation of survivors who were exposed to different doses of irradiation during the chernobyl accident

Using routine methods over 25 years, changes in the registered levels of chromosomal aberrations were studied in the peripheral blood lymphocyte cultures of 74 patients who were irradiated as a result of the Chernobyl accident. The initial dosage estimations by mean dicentric frequency varied from 0.2 to 9.8 Gy. Generally, a double exponential type model was most adequate for the quantitative description of the elimination of cytogenetic indexes associated with different types of unstable chromosomal aberrations. Great individual variability of the elimination rate of chromosomal aberrations and its dependency on the value of the originally estimated dosage were found during the first period. A computer method for retrospective dos-age estimation was developed based on this data. The method is based on analysis of cell distributions in accordance with the number of dicentrics and, as a whole, unstable chromosomal aberrations contained in them. In addition, the dynamics of the translocation frequencies in the peripheral blood lymphocyte cultures of a number of patients from this contingent were investigated, beginning at 10 years after the irradiation using the FISH method of chromosome staining.

Evaluation of the genotoxicity and cytotoxicity of fexofenadine in cultured human peripheral blood lymphocytes

Fexofenadine (FXF) is a new non-sedating antihistamine used in the treatment of seasonal allergic rhinitis and chronic idiopathic urticaria. Studies on FXF genotoxicity and cytotoxicity in cultured human peripheral blood lymphocytes have not been reported so far. Therefore, the present study is the first report investigating the genotoxic and cytotoxic effects of FXF in cultured human peripheral blood lymphocytes in vitro. Cultures were treated with FXF at three concentrations (50, 100 and 150μg/ml) for 24 and 48h. Endpoints analyzed included: mitotic index (MI), nuclear division index (NDI), chromosomal aberrations (CA) and micronucleus (MN) assay. Mitomycin C (MMC) was used as a positive control. The results of CA and MN assays showed that FXF was not genotoxic at all the concentrations tested, meanwhile MI and NDI results showed dose-dependent decrease and significant differences were found for at least one concentration. In conclusion, the results of this study suggest that FXF has a cytotoxic effect but not genotoxic effect on human peripheral blood lymphocyte cultures. Further cytogenetic studies, especially about the cell cycle kinetics of FXF are required to elucidate the decreases in dividing cells, and biomonitoring studies should also be conducted with patients receiving therapy with this drug.