Blood Lymphocytes Of Healthy Individuals Research Articles

Haloperidol's Cytogenetic Effect on T Lymphocytes of Systemic Lupus Erythematosus and Rheumatoid Arthritis Patients: An In Vitro Study.

Investigating haloperidol's cytogenetic behavior in cultured human T lymphocytes of patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Four haloperidol solutions were added in cultures of peripheral blood lymphocytes of healthy individuals, SLE, and RA patients. After 72 hours of incubation, the cultured lymphocytes were plated on glass slides, and stained with the fluorescence plus Giemsa method, and sister chromatid exchanges (SCEs), proliferation rate index (PRI), and mitotic index (MI) were measured with the optical microscope. Result analysis revealed: (a) a statistically significant (p=0.001) dose-dependent increase of SCEs in SLE patients compared to healthy individuals; (b) a statistically significant (p=0.001) dose-dependent decrease of SCEs in RA patients for haloperidol concentrations 5, 10μg/mL; (c) a statistically significant (p=0.001) dose-dependent increase of SCEs in RA patients for haloperidol concentrations 20, 100μg/mL; and (d) a statistically significant (p=0.001) dose-dependent reduction of PRI and MI in both patient groups compared to healthy individuals. Furthermore, a correlation was observed between (a) SCE and PRI index variations, (b) MI and SCE index variations, and (c) PRI and MI index variations. Haloperidol affects T lymphocytes from SLE and RA patients by modifying DNA replication procedures, DNA damage response, and ferroptosis. Considering the wide use of haloperidol in neuropsychiatric symptoms of SLE and RA patients, further studies with more immune cell subsets are needed to evaluate its effects on human genetic material.

Olanzapine’s Cytogenetic Effect on T Lymphocytes in Systemic Lupus Erythematosus and Rheumatoid Arthritis Patients: In Vitro Study

This study will investigate olanzapine's cytogenetic behavior in cultured human T lymphocytes in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Three olanzapine solutions were added in cultures of peripheral blood lymphocytes of healthy individuals, SLE, and RA patients. After 72 hours of incubation, the cultured lymphocytes were plated on glass slides and stained with the fluorescence plus Giemsa method. Sister chromatid exchanges (SCEs), proliferation rate index (PRI), and mitotic index (MI) were measured with the optical microscope. There was a statistically significant (p=0.001) dose-dependent increase of SCEs in SLE and RA patients compared to healthy individuals and a statistically significant (p=0.001) reduction of PRI and MI in the highest concentration in the SLE group. Moreover, Spearman's rank correlation coefficient was applied to calculate the correlation between SCEs, PRI, and MI. Negative significant correlations were noticed for both patient groups concerning SCEs-PRI alterations and SCEs-MI alterations. Conversely, positive correlations were noticed for both patient groups for PRI-MI alterations. Conclusions: Olanzapine affects T lymphocytes from SLE and RA patients by modifying DNA replication procedures and DNA damage response. Considering the use of olanzapine in neuropsychiatric symptoms of SLE, further in vivo studies are necessary to evaluate its effect on human DNA.

Cytogenetic effects in mixed culture of blood cells from patients with chronic lymphocytic leukemia with blood lymphocytes of healthy individuals

Cytogenetic effects in mixed culture of blood cells from patients with chronic lymphocytic leukemia with blood lymphocytes of healthy individuals

Granulysin species segregate to different lysosome-related effector vesicles (LREV) and get mobilized by either classical or non-classical degranulation

Granulysin (GNLY) is a cationic antimicrobial, proinflammatory, and cytotoxic effector protein primarily expressed in human cytotoxic T and NK cells. Its two variants, the 15 kDa precursor and the mature 9 kDa protein processed by proteolysis, act on different microbes or infected and transformed target cells and utilize mechanistically different effector activities. In human peripheral blood lymphocytes of healthy individuals, both forms of GNLY are detected in TCR αβ+ (CD4+ and CD8+) T cells, TCR γδ+ T cells, and CD3−CD56+ NK cells. In general, classical cytotoxic cells (i.e. CD8+ TCR αβ+ T cells, TCR γδ+ T cells, and NK cells) contain effector proteins in higher abundance in more cells of the subset as compared to TCR αβ+ CD4+ T cells. Imaging flow cytometry analyses demonstrate that the subcellular localization and internal pools of 9 kDa and 15 kDa GNLY are virtually non-overlapping. The 9 kDa form is enriched in dense granules that also contain granzymes (Grz) and carry CD107a, whereas 15 kDa GNLY is associated with CD107a-negative lysosome-related effector vesicles. We further demonstrate that 15 kDa GNLY serves as an additional indicator for non-classical, PKC-dependent degranulation while the liberation of granules containing 9 kDa GNLY requires calcium mobilization. Our studies provide a deeper insight into the subcellular localization and release mechanisms of the individual GNLY species. This information will not only be useful for the interpretation of GNLY-related pathophysiologies, but also for the development of therapeutic interventions employing distinct GNLY effector functions for microbial targeting or immunoregulation.

Effect of concurrent chemoradiotherapy and radiotherapy alone on peripheral myeloid-derived suppressor and T regulatory cells in patients with nasopharyngeal cancer

Objective: To investigate the percentage of myeloid-derived suppressor cells (MDSC) and T regulatory cells (Treg) in peripheral blood of nasopharyngeal cancer (NPC) patients undergoing concurrent chemoradiotherapy or radiotherapy alone. Methods: Sixty NPC patients who received radiotherapy or concurrent chemoradiotherapy from September 2012 to November 2015 and 20 healthy individuals were included in this study. For the patients, the blood samples were collected at four time points: pre-radiation (Pre-RT), reaching a dose of 40 Gy (RT-40 Gy), finishing radiation (RT-finish) and three months after finishing radiation (3m-post-RT). Flow cytometry was used to evaluate the percentage of Treg (CD4(+) CD25(+) CD127(low/-)) and MDSC (HLA-DR(-)CD11b(+) CD33(+) ) cells in peripheral blood. Results: Treg and MDSC cells were present in peripheral blood lymphocytes of healthy individuals as a percentage of (7.50±1.62)% and (1.08±0.48)%, respectively. The proportions of peripheral Treg cells in patients at Pre-RT, RT-40 Gy, RT-finish and 3m-post-RT time points were (8.42± 1.52)%, (9.10±1.57)%, (8.87±1.56)% and (7.31±1.43)%, respectively, showing a statistically significant difference between Pre-RT and the other groups (P<0.05). At Pre-RT point, the percentage of Treg cells in Stage Ⅲ-Ⅳ patients [(8.63±1.39)%] was higher than that in Stage Ⅰ-Ⅱ [(7.65±1.94)%, P=0.042]. Moreover, the proportions of peripheral MDSC cells in patients at Pre-RT, RT-40 Gy, RT-finish and 3m-post-RT time points were (2.14±1.21)%, (4.08±1.90)%, (3.76±1.31)% and (1.52±0.88)%, respectively. The percentages of MDSC cells at RT-40 Gy and RT-finish points were significantly higher than those at Pre-RT, while the percentage of MDSC cells at 3m-post-RT was significantly lower than those at Pre-RT (P<0.05). At Pre-RT point, the percentage of MDSC cells in Stage Ⅲ-Ⅳ patients [(2.25±1.26)%] was higher than that in Stage Ⅰ-Ⅱ [(1.35±0.66)%, P=0.007]. At RT-finish point, the proportions of MDSC and Treg cells in patients with Ⅲ-Ⅳ grade of radiation induced oral mucositis [(4.41±1.27)% and (9.91±1.23)%] were significantly higher than those in Ⅰ-Ⅱ grade patients [(3.15±1.04)% and (8.41±1.52)%, both of P<0.05]. Conclusions: The proportions of MDSC and Treg cells in initial treated NPC patients are higher than healthy individuals, and they are also associated with the tumor stages. During the concurrent chemoradiotherapy and radiation, the percentage of MDSC and Treg cells is elevated, suggesting a decreased immune activity. The increase of MDSC and Treg cells is related to radiation induced oral mucositis.

CHORNOBYL CATASTROPHE: CYTOGENETIC EFFECTS OF LOW DOSE IONIZING RADIATION AND THEIR MODIFICATION

Among the long-term effects of the Chornobyl disaster the greatest concern of international medical and scientific community is given to the established fact of excess of the spontaneous level of cancer incidence in the exposed population. According to mo-dern concepts, the accumulation of chromosome aberrations, especially in radiosensitive cells, could be potentially oncogenic, and low doses of ionizing radiation could be promoters of the radiation-induced carcinogenesis. The results of our studies have shown that such substances as thymalin, inosine, ascorbic acid, caffeine could modify radiation-induced cytogenetic effects in peripheral blood lymphocytes of healthy individuals and exert protective or sensitizing action dependent on their concentrations, cell radiosensitivity, dose of irradiation, and relative biologic effectiveness of ionizing radiation. This article is part of a Special Issue entitled "The Chornobyl Nuclear Accident: Thirty Years After".

Abstract 1551: High-resolution parallel analysis of genome and transcriptome of single disseminated prostate cancer cells

Abstract Introduction: Manifestation of bone metastases is the most frequent cause of death among prostate cancer patients. Since the progression from non-metastatic (M0) to metastatic (M1) is highly unpredictable, molecular analysis of disseminated cancer cells (DCCs) detected before overt metastasis may provide an opportunity to dissect the early stages of systemic cancer and enable detection of critical therapy targets. However, we previously showed that early stage prostate DCCs acquire a bone marrow-associated transcriptomic phenotype (Guzvic et al. Cancer Res. 2014, 15;74(24):7383-94) impeding the definite identification of DCCs. To address this problem we developed a workflow of combined single-cell RNA-Seq and single-cell aCGH allowing high-resolution analysis of genomes and transcriptomes of individual cells. Here, we present a proof-of-concept study using DCCs isolated from bone marrow of prostate cancer patients. Materials and methods: We isolated 24 EPCAM-positive DCCs from bone marrow of prostate cancer patients. In addition, we analyzed two VCaP prostate cancer cells and two peripheral blood lymphocytes of healthy individuals. All isolated cells were subjected to combined whole genome and whole transcriptome amplification using Ampli1TM WGA Kit and a WTA approach (Klein CA et al., Nat Biotechnol. 2002, 20(4):387-92), respectively. The suitability of WTA and WGA products for downstream analyses were assessed using PCR-based QC-assays. Subsequently, WGA products were hybridized on high-resolution SurePrint aCGH arrays and analyzed with Agilent Genomic Workbench Software. WTA products were sequenced using Roche 454 GS FLX+ or Illumina HiSeq 2000 systems. Single-cell RNA-Seq data were evaluated using a bioinformatic pipeline adjusted to the needs of the single cell WTA method. Results: We provide two experimental protocols for single-cell RNA-Seq: (i) allowing detection of low-abundant transcripts and (ii) enabling comprehensive gene-expression analysis. The protocols were validated using VCaP cells, in which we could successfully detect low expression levels of TMPRSS2-ERG fusion transcripts. The established analysis allowed also detection of novel fusion transcripts in a M1-stage prostate cancer patient. Comprehensive gene expression analysis revealed presence of 4.000 to 13.000 transcripts expressed in bone marrow-derived cells. The comparison of RNA-Seq and aCGH data allowed to simultaneously detecting copy number alterations and corresponding changes in gene expression dosage. Thereby, the combined genome and transcriptome analysis of single cells enables to uncover the impact of genomic gains and losses on cellular transcriptomes. Conclusions: Combined genome and transcriptome analysis of patient-derived single DCCs is feasible and enables unambiguous identification and genotypic and phenotypic characterization of DCCs. Citation Format: Stefan Kirsch, Urs Lahrmann, Miodrag Guzvic, Zbigniew T. Czyz, Giancarlo Feliciello, Bernhard Polzer, Christoph A. Klein. High-resolution parallel analysis of genome and transcriptome of single disseminated prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1551.

Comparison of Individual Radiosensitivity to γ-Rays and Carbon Ions.

Carbon ions are an up-and-coming ion species, currently being used in charged particle radiotherapy. As it is well established that there are considerable interindividual differences in radiosensitivity in the general population that can significantly influence clinical outcomes of radiotherapy, we evaluate the degree of these differences in the context of carbon ion therapy compared with conventional radiotherapy. In this study, we evaluate individual radiosensitivity following exposure to carbon-13 ions or γ-rays in peripheral blood lymphocytes of healthy individuals based on the frequency of ionizing radiation (IR)-induced DNA double strand breaks (DSBs) that was either misrepaired or left unrepaired to form chromosomal aberrations (CAs) (simply referred to here as DSBs for brevity). Levels of DSBs were estimated from the scoring of CAs visualized with telomere/centromere-fluorescence in situ hybridization (TC-FISH). We examine radiosensitivity at the dose of 2 Gy, a routinely administered dose during fractionated radiotherapy, and we determined that a wide range of DSBs were induced by the given dose among healthy individuals, with highly radiosensitive individuals harboring more IR-induced breaks in the genome than radioresistant individuals following exposure to the same dose. Furthermore, we determined the relative effectiveness of carbon irradiation in comparison to γ-irradiation in the induction of DSBs at each studied dose (isodose effect), a quality we term “relative dose effect” (RDE). This ratio is advantageous, as it allows for simple comparison of dose–response curves. At 2 Gy, carbon irradiation was three times more effective in inducing DSBs compared with γ-irradiation (RDE of 3); these results were confirmed using a second cytogenetic technique, multicolor-FISH. We also analyze radiosensitivity at other doses (0.2–15 Gy), to represent hypo- and hyperfractionation doses and determined that RDE is dose dependent: high ratios at low doses, and approaching 1 at high doses. These results could have clinical implications as IR-induced DNA damage and the ensuing CAs and genomic instability can have significant cellular consequences that could potentially have profound implications for long-term human health after IR exposure, such as the emergence of secondary cancers and other pathobiological conditions after radiotherapy.

Telomere Length of Individual Chromosomes in Patients with Rheumatoid Arthritis.

We analyzed telomere length of individual chromosomes in peripheral blood lymphocytes of healthy individuals and patients with rheumatoid arthritis. Quantitative fluorescent in situ hybridization and subsequent computer analysis of metaphase chromosomes showed that distribution of telomere length on individual chromosomes is different under normal and pathological conditions. Patients with rheumatoid arthritis had significantly shorter chromosome 4p telomeres, which can be essential for pathogenesis of this multifactorial disease. Additionally, disease activity inversely correlated with telomere length on chromosome 10p carrying genes involved in T cell differentiation and proliferation.

Baseline frequency of chromosomal aberrations and sister chromatid exchanges in peripheral blood lymphocytes of healthy individuals living in Turin (North-Western Italy): assessment of the effects of age, sex and GSTs gene polymorphisms on the levels of genomic damage

Background: The increased exposure to environmental pollutants has led to the awareness of the necessity for constant monitoring of human populations, especially those living in urban areas.Aim: This study evaluated the background levels of genomic damage in a sample of healthy subjects living in the urban area of Turin (Italy). The association between DNA damage with age, sex and GSTs polymorphisms was assessed.Subjects and methods: One hundred and one individuals were randomly sampled. Sister Chromatid Exchanges (SCEs) and Chromosomal Aberrations (CAs) assays, as well as genotyping of GSTT1 and GSTM1 genes, were performed.Results: Mean values of SCEs and CAs were 5.137 ± 0.166 and 0.018 ± 0.002, respectively. Results showed age and gender associated with higher frequencies of these two cytogenetic markers. The eldest subjects (51–65 years) showed significantly higher levels of genomic damage than younger individuals. GSTs polymorphisms did not appear to significantly influence the frequencies of either markers.Conclusion: The CAs background frequency observed in this study is one of the highest reported among European populations. Turin is one of the most polluted cities in Europe in terms of air fine PM10 and ozone and the clastogenic potential of these pollutants may explain the high frequencies of chromosomal rearrangements reported here.

Властивості Na+ , K+ -активованої, Mg2+ -залежної АТФ-гідролази лімфоцитів крові у хворих на реактивний артрит

A significant role in the development and course of reactive arthritis (ReA) is played by T-lymphocytes as their development and systemic manifestations are based on immunological mechanisms. Additionally, the pathogenesis of many diseases is linked to changes in the structure and function of ion-transporting systems. Therefore, the aim of the study was to find out the kinetic properties of ATP-hydrolysis reaction involving Na+,K+-ATPase of peripheral blood lymphocytes of healthy individuals and patients with ReA. We used the current methodological approaches to the study of ATPase activity in saponin permeabilized cells. We conducted an analysis of the kinetic properties of ouabainsensitive Na+,K+-ATPase activity of saponin-perforated peripheral blood lymphocytes of healthy donors and patients with rheumatoid arthritis (RеA). We found out that in peripheral blood lymphocytes of patients with RеA primary active transport of Na+, K+ ions is slower and less intensive, though characterised by the same capacity, as in healthy donors. The affinity constant for ATP in peripheral blood lymphocytes in patients with RеA is greater by 2.9 times than its value in comparison with healthy donors. We established that in conditions of rheumatic pathology in immunocompetent cells, inhibition of Na+,K+-ATPase activity is not caused by reduction of speed of enzyme work, but by increase of affinity of ouabainsensitive Na+,K+-ATPase to ATP. At the same time, the Mg2+-binding center of Na+,K+-ATPase in patients with RеA is endogenous. We also found that affinity Na+,K+-ATPase to the ions K+ in peripheral blood lymphocytes of healthy donors is 2.4 times higher than in patients with RеA. We observed that Na+,K+-ATPase of peripheral blood lymphocytes of patients with RеA retains its endogenous receptor properties – sensitivity to ouabain does not change. It is assumed that under conditions of rheumatic pathology the impact on the Na+,K+-ATPase structure occurs both externally and on the cytoplasmic membrane surface. The above experimental data can be used for further clarification of the membrane mechanisms of ion exchange in immunocompetent cells of patients suffering from autoimmune diseases.

Evaluation of the genotoxicity and cytotoxicity in the general population in Turkey by use of the cytokinesis-block micronucleus cytome assay

The cytokinesis-block micronucleus cytome (CBMN-Cyt) assay was originally developed as an ideal system for measuring DNA damage, cytostasis and cytotoxicity. The objective of the present study is to simultaneously evaluate the background levels of micronuclei (MN), nucleoplasmic bridges (NPBs), nuclear buds (NBUDs), cell death (necrosis or apoptosis) and nuclear division index (NDI) in the peripheral blood lymphocytes of non-occupationally exposed, healthy subjects living in the city of Kayseri in Turkey. We used the CBMN-Cyt assay, taking into account factors – age, gender, and smoking habits – that might affect MN frequency and also other CBMN-Cyt assay parameters. Ninety-six healthy subjects (48 female and 48 male) were selected with ages varying between 21 and 60 years. The parameters, except for the number of binucleated (BN) cells with NPBs, showed no statistically significant difference between smokers and non-smokers. There were significant differences between female and male groups in MN frequency (higher in females) and in the number of NPBs (lower in females), while the other parameters were not significantly different between genders. The correlations between years of age and MN frequency, number of NPBs and the frequency of necrotic cells were statistically significant, while the correlations between the years of age and the other parameters were not. The results of the correlation analysis between years of smoking and MN frequency were positive, although no statistically significant correlation was found between the years of smoking and the other parameters. Among the smokers, no correlation was found either between the pack-years of smoking and the parameters assessed in this group. The results of the present study provide evidence of increasing MN frequency, number of NPBs and frequency of necrotic cells with increasing age in the peripheral blood lymphocytes of healthy individuals and influencing MN frequency and number of NPBs by gender.

Expression of RAN, ZHX-2, and CHC1L genes in multiple myeloma patients and in myeloma cell lines treated with HDAC and Dnmts inhibitors

Real time PCR is a powerful tool for studying the expression of genes involved in the pathophysiology of human diseases. Recent studies of the RAN (6p21), ZHX-2 (8q24.3), CHC1L (13q14.3) loci highlight the importance of these genes in multiple myeloma (MM) prognosis and therapeutic applications. Here, we described a detailed Real-Time PCR method for the detection of RAN, ZHX-2, and CHC1L expression, which could be applied in clinical situations. The expression profiles of these genes were studied in peripheral blood lymphocytes of healthy individuals, patients suffering from MM, and in the myeloma cell line, MOLP-8. Low expression levels of RAN, ZHX-2, and CHC1L were observed in myeloma patients, compared with peripheral blood lymphocytes and MOLP-8 cells. An inhibitor of histone deacetylases (TSA) had the ability to decrease expression of CHC1L and ZHX-2 in MOLP-8 cells, while expression of RAN was relatively stable in peripheral blood lymphocytes, control MOLP-8, and TSA- or 5-azacytidine treated MOLP-8 cells. In myeloma patients, we observed significant decreases in the expression of selected genes, but it was patient-specific. Our experiments illustrate new methodological approaches and troubleshooting for conducting gene expression studies in clinical laboratories.

Do GST Polymorphisms Modulate the Frequency of Chromosomal Aberrations in Healthy Subjects?

Rossi et al. (2009) described an association between chromosomal aberration (CA) frequency and cancer risk in a case–control study on 107 cancer cases and 291 controls, whereby they observed no modifying effect of polymorphisms in glutathione S-transferase M1 (GSTM1) and GSTT1. In our studies of 488 healthy individuals who shared the same environmental exposure in Slovakia and the Czech Republic, we observed a CA frequency of 2.35 ± 1.73 (mean ± SD) (Halasova et al. 2007; Musak et al. 2008; Naccarati et al. 2006; Slyskova et al. 2007; Vodicka et al. 2001, 2004a, 2004b). The frequencies (mean ± SD) for chromatid-type abberations (CTA) and chromosome-type aberrations (CSA) were 1.22 ± 1.21 and 1.15 ± 1.35, respectively. By analyzing modulating effects of genetic polymorphisms in GSTT1, GSTM1, and GSTP1 on CAs, CTAs, and CSAs (Table 1), we found no significant association between chromosomal damage and any of the studied polymorphisms. The results were further confirmed by logistic regression: for the GSTT1 null genotype, odds ratio (OR) = 1.35 [95% confidence interval (CI), 0.79–2.32; p = 0.27]; for the GSTM1 genotype, OR = 1.09 [95% CI, 0.74–1.62; p = 0.65]; and for a variant GSTP1 Val105Val genotype, OR = 0.83 [95% CI, 0.55–1.24; p = 0.36]. These data on a larger healthy population [previously published separately by Halasova et al. (2007), Musak et al. (2008), Naccarati et al. (2006), Slyskova et al. (2007), and Vodicka et al. (2001, 2004a, 2004b)] confirm the findings of Rossi et al. (2009) regarding GSTM1 and GSTT1 polymorphisms. Additionally, in our reanalysis, we did not observe any modulating effect of GSTP1 polymorphism on CA frequency. However, the modulating role of GST polymorphisms may not be excluded, particularly in interaction with heavy occupational exposure. In our study exploring chromosomal damage in tire-plant workers (Musak et al. 2008), CAs were significantly higher in subjects with GSTT1-null than in those with GSTT1-plus genotypes, particularly in association with smoking. Table 1 Distribution of analyzed genotypes and CA frequencies. In the past decade, CAs have been accepted as a predictive marker of cancer (Hagmar et al. 2004), particularly for colo-rectal and lung cancers (Boffetta et al. 2007; Norppa et al. 2006). Nevertheless, these studies, as well as the study of Rossi et al. (2009) may have limitations: For example, cohorts were recruited in various regions with different lifestyle and environmental backgrounds, and different laboratories were involved in processing and scoring the samples over many years. In earlier studies, virtually no data on individual susceptibility were available because of the lack of DNA for molecular analysis. The data on CAs presented here were obtained on healthy subjects from a homogeneous region with fairly similar socioeconomic background. The analysis of CAs reported in these studies (Halasova et al. 2007; Musak et al. 2008; Naccarati et al. 2006; Slyskova et al. 2007; Vodicka et al. 2001, 2004a, 2004b) were performed in two laboratories, using the same protocol and the same scoring criteria with regular slide exchanges to minimize interlaboratory and inter scorer differences. Also, native DNA from whole-blood samples for molecular genetic studies was collected simultaneously with the samples for cytogenetic investigations. Future prospective studies regarding CAs and cancer should be designed by taking into account the lifestyle and occupational/environmental exposures, along with factors of individual susceptibility. Some GST polymorphisms may modulate CA frequency through interaction with environmental factors. The next logical step for a confirmation of predictive values of CA frequencies in relation to cancer will be their determination in lymphocytes of cancer patients in association with clinical– pathological characteristics.

Association between frequency of chromosomal aberrations and cancer risk is not influenced by genetic polymorphisms in GSTM1 and GSTT1.

BackgroundThe frequency of chromosomal aberrations (CA) in peripheral blood lymphocytes of healthy individuals has been associated with cancer risk. It is presently unclear whether this association is influenced by individual susceptibility factors such as genetic polymorphisms of xenobiotic-metabolizing enzymes.ObjectivesTo evaluate the role of polymorphisms in glutathione S-transferase (GST) M1 (GSTM1) and theta 1 (GSTT1) as effect modifiers of the association between CA and cancer risk.MethodsA case–control study was performed pooling data from cytogenetic studies carried out in 1974–1995 in three laboratories in Italy, Norway, and Denmark. A total of 107 cancer cases were retrieved from national registries and matched to 291 controls. The subjects were classified as low, medium, and high by tertile of CA frequency. The data were analyzed by setting up a Bayesian model that included prior information about cancer risk by CA frequency.ResultsThe association between CA frequency and cancer risk was confirmed [ORmedium (odds ratio)medium = 1.5, 95% credibility interval (CrI), 0.9–2.5; ORhigh = 2.8, 95% CrI, 1.6–4.6], whereas no effect of the genetic polymorphism was observed. A much stronger association was seen in the Italian subset (ORhigh= 9.4, 95% CrI, 2.6–28.0), which was characterized by a lower technical variability of the cytogenetic analysis. CA level was particularly associated with cancer of the respiratory tract (ORhigh= 6.2, 95% CrI, 1.5–20.0), the genitourinary tract (ORhigh = 4.0, 95% CrI, 1.4–10.0), and the digestive tract (ORhigh = 2.8, 95% CrI, 1.2–5.8).ConclusionsDespite the small size of the study groups, our results substantiate the cancer risk predictivity of CA frequency, ruling against a strong modifying effect of GSTM1 and GSTT1 polymorphisms.

A study on the genotoxic effects of 8-Cl-cAMP on human lymphocytes in vitro

8-chloro-cyclic adenosine 3',5'-monophosphate (8-Cl-cAMP) is the most potent cAMP analogue that selectively inhibits a variety of cancer cell lines in vitro and tumors in vivo. Its action toward a variety of tumors, especially when coupled with other antitumor agents, have lead to phase I clinical investigations and recently phase II clinical investigations. Until today very little was done to evaluate its genotoxic potential. In order to evaluate its genotoxic potential we used the cytogenetic and cytokinesis block micronucleus assay in vitro on peripheral blood lymphocytes of healthy individuals. Using three concentrations (1 microM, 5 microM and 15 microM), 8-Cl-cAMP in normal human peripheral blood lymphocytes did not induce any cytogenetic aberrations of the structural type [chromatid breakage, isochromatid breakage and gaps], but did induce premature centromere separation (PCS) in all respective doses and increased the frequency of micronuclei (p <0.05) only in the highest dose (15 microM). Antiproliferative action of 8-Cl-cAMP was estimated by using the cytokinesis block nuclear division index (NDI). The results showed a decrease in the NDI of cells exposed to all doses of 8-Cl-cAMP when compared to control. Therefore, the overall results show a genotoxic potential of 8-Cl-cAMP in peripheral blood lymphocytes in vitro.

Automated detection of residual leukemic cells by consecutive immunolabeling for CD10 and fluorescence in situ hybridization for ETV6/RUNX1 rearrangement in childhood acute lymphoblastic leukemia

Among the various methods available for analyzing minimal residual disease, a new procedure for the cell-based approaches using consecutive phenotypic and genotypic analysis as revealed by immunofluorescent labeling and subsequent fluorescent in situ hybridization (FISH) has been developed. We are introducing a fluorescent microscopy-based technique by which not only cellular targets and immunological marker positivity, but also the FISH pattern was identified by automated scanning. For the latter one translocation-specific FISH pattern recognition was accomplished by using an automated scanning mode for the 3D determination of valid distances between FISH signals, to define the cutoff value for the shortest green–red spot distance differentiating positive cells from negative ones. The procedure was tested with CD10 + acute lymphoblastic leukemia cell line harboring the t(12;21)(p13;q22) resulting in the ETV6/RUNX1 rearrangement (formerly TEL/AML1), as well as peripheral blood lymphocytes of healthy individuals. Using the combined, automated method, a sensitivity of 98.67% and a specificity of 99.97% were obtained. The mean false positivity + 2 standard deviations cutoff level (0.09%) allows detection of leukemic cells with high accuracy, even a bit below the tumor load dilution of 10 −3, a value reported to be critical in clinical decision making.

CD8+ cytotoxic T lymphocytes isolated from allogeneic healthy donors recognize HLA class Ia/Ib–associated renal carcinoma antigens with ubiquitous or restricted tissue expression

AbstractAllogeneic hematopoietic stem cell transplantation can induce considerable tumor remissions in metastatic renal-cell carcinoma (RCC) patients. The precise effector mechanisms mediating these graft-versus-tumor reactions are unknown. We studied RCC-directed CD8+ T-cell responses in blood lymphocytes of healthy individuals matched with established RCC cell lines for HLA-class I. In 21 of 22 allogeneic mixed lymphocyte/tumor-cell cultures (MLTCs), RCC-reactive cytotoxic T-lymphocytes (CTLs) were readily obtained. From MLTCs, 121 CD8+ CTL clones with memory phenotype were isolated. Their anti–RCC reactivity was restricted by multiple classical HLA-Ia molecules, in particular by HLA-A2, -A3, -B7, -B44, -Cw7, and by a nonclassical HLA-Ib determinant. Extensive cross-reactivity analyses on a broad target panel identified CTLs that recognize antigens with expression restricted to renal tissue or to renal and colon tumors. Other CTLs were directed against antigens with broader tissue distribution being expressed in various epithelial and nonepithelial tumors or, additionally, in hematopoietic cells. With microcapillary liquid chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)/TOF mass spectrometry, we identified the HLA-A*0301-associated nonpolymorphic peptide KLPNSVLGR encoded by the ubiquitously expressed Eps15 homology domain–containing 2 gene as a CTL target. Defining human RCC antigens recognized by alloreactive CTLs may allow to improve the specificity and efficiency of allogeneic cell therapy (eg, specific donor-lymphocyte infusions or vaccination) in metastatic RCC patients.

Induction of apoptosis in human lymphocytes by the herbicide 2,4-dichlorophenoxyacetic acid

Dimethylammonium salt of 2,4-dichlorophenoxyacetic acid (DMA-2,4-D) is a widely used herbicide that is considered moderately toxic. In the present study we found that DMA-2,4-D is able to cause apoptosis in peripheral blood lymphocytes of healthy individuals and Jurkat T cells. Apoptosis induced by DMA-2,4-D was dose and time dependent, independent of Fas, TNF receptor 1 or the aromatic hydrocarbon receptor, and involved disruption of the mitochondrial transmembrane potential and activation of caspase-9. ZVAD-FMK, a broad-spectrum inhibitor of caspases, blocked DMA-2,4-D-induced apoptosis completely. While an inhibitor of caspase-9, as well as caspase-9 and caspase-3 inhibitors in combination, strongly blocked DMA-2,4-D-induced apoptosis, an inhibitor of caspase-3 had a moderate inhibitory effect. Unlike Fas-mediated apoptosis, the initiator caspase, caspase-8, was not involved in DMA-2,4-D-induced apoptosis. Transfection of Jurkat cells with Bcl-2 prevented DMA-2,4-D-induced disruption of the mitochondrial transmembrane potential and led to a complete blockage of apoptosis. Our data indicate that DMA-2,4-D kills human lymphocytes by initiating apoptosis via a direct effect on mitochondria. The activation of caspases occurs downstream of mitochondrial damage, and the dysfunction of mitochondria appears to be sufficient for triggering all downstream events leading to apoptosis.

Autoreactive human T cell lines recognizing ribosomal protein L7

Sera of patients suffering from systemic lupus erythematosus (SLE) frequently contain oligoclonal IgG autoantibodies with high affinity for the ribosomal protein L7 (rpL7). The humoral autoimmune response to rpL7 apparently is driven by antigen and T cell dependent. In order to analyze the T cell response to rpL7 we cultured peripheral blood lymphocytes of healthy individuals and SLE patients in the presence of recombinant rpL7. After 10 days, the cytokine response to re-stimulation with rpL7 was examined using a spot-ELISA. Measuring IFN-gamma secretion, the T cells of two patients and four healthy donors showed a significant increase in the number of spots as compared to control cells. Secretion of IL-4 or IL-10 was not detected. From the antigen-stimulated primary cultures we established by limiting dilution cloning six rpL7-reactive, IFN-gamma-secreting T cell lines which show a CD3+CD4+CD8- phenotype. One line additionally was shown to be positive for HLA-DR and CD45R0, but negative for CD27 and CD31. The cell lines carry alphabeta TCR chains which differ from each other in sequence and specificity. rpL7 fragments rich in basic amino acids could be identified as epitopes recognized by the TCR of three cell lines. Recognition of rpL7 is HLA-DR6 restricted or respectively HLA-DP restricted in the two cell lines analyzed.