Microsatellite DNA instability and COPD exacerbations.

Increased frequency of microsatellite DNA instability (MSI) has been detected in the sputum of chronic obstructive pulmonary disease (COPD) patients. The aim of the present study was to investigate the relationship between MSI in sputum cells and exacerbation frequency, which is an important parameter in the clinical course of the disease. Induced sputum samples and peripheral blood obtained from 36 patients with COPD at stable state were analysed. The control group consisted of 30 nonsmoking healthy subjects. DNA was extracted and analysed for MSI using the following microsatellite markers: RH70958, D5S207, D6S2223, D6S344, D6S263, G29802, D13S71, D14S588, D14S292 and D17S250. Following MSI analysis, exacerbations were recorded for 3 yrs in total. No MSI was detected in healthy nonsmokers. A total of 18 (50%) out of 36 patients exhibited MSI in their sputum cells. Patients who exhibited MSI showed significantly increased frequency of exacerbations compared with patients that did not. In addition, a significantly increased frequency of purulent and of severe type exacerbations was found in patients exhibiting MSI. Patients positive for marker G29802, D13S71 or D14S588 presented increased exacerbation frequency. The significant association between microsatellite DNA instability and chronic obstructive pulmonary disease exacerbations indicates that somatic mutations could be involved in the pathogenesis and natural history of the disease.

Risk factors for smoking among school adolescents in Greece.

OBJECTIVE: We conducted this study among school adolescents to identify factors, which influence schoolchildren to smoke. METHODS: We carried out a cross-sectional study in a sample of 924 students of all classes (ages 12-18 years old) in 15 public high schools in a semi-urban area in Crete, Greece, using a questionnaire. The questionnaire comprised of 46 questions covering children's lifestyle habits regarding daily activities and leisure time, frequency of risk-taking behaviour, knowledge about the hazards and long-term consequences of smoking. RESULTS: 23.9% of participants were experimental smokers and 18.6% were current smokers. 11.4% of the total population was daily smokers. There was a significant increase in the prevalence of experimental and current smokers with school grades, while peaks in last grades were observed. Boys started smoking earlier than girls, mean (standard error) age 13.4 (2.3) years vs. 14.1 (2.3) years, P = 0.01. Stepwise logistic regression analysis showed a positive relationship between current smoking and having brother or sister smoking [odds ratio (95% confidence interval) 2.7 (1.7-4.4) and 1.8 (1.1-3.3) respectively], having more than three friends who were smokers [2.6 (2-3.4)] and last school grade [1.4 (1.2-1.7)]. Students appeared to be informed about long-term smoking hazards and had negative views on children who smoke especially in the lower grades. CONCLUSIONS: Prevention programmes should be imposed early in elementary schools while cessation policies should target at all grades, in particular at critical grades depending on population-specific characteristics.

Exacerbations and lung function decline in COPD: new insights in current and ex-smokers.

AIM: To investigate whether there is a significant relationship between an increased frequency of exacerbations and the rate of forced expiratory volume in 1s (FEV(1)) decline in COPD patients. METHODS-MEASUREMENTS: About 102 COPD patients (44 smokers, 58 ex-smokers) participated in a 3-year prospective study. Exacerbations were identified as worsening of patient's respiratory symptoms as recorded on diary cards. Spirometry was performed every 6 months. The effect of frequent exacerbations on lung function was investigated using random effects models. RESULTS: The median (mean(95% CI)) annual exacerbation rate was 2.85 (3.1 (2.7-3.6)). Patients with an annual exacerbation rate over the median rate had significantly lower baseline post-bronchodilation FEV(1)(%pred), higher MRC dyspnoea score and chronic cough compared to patients who had an annual exacerbation rate less than the median. The average annual rate of FEV(1)(%pred), adjusted for smoking decline (DeltaFEV(1)), was found significantly increased in frequent compared to infrequent exacerbators (P=0.017). The highest DeltaFEV(1) was observed in smokers frequent exacerbators and a significant interaction between exacerbation frequency and DeltaFEV(1) was also observed in ex-smokers. CONCLUSIONS: Our findings suggest that an increased frequency of exacerbations is significantly associated with FEV(1) decline even in ex-smokers. Thus, smoking and frequent exacerbations may have both negative impact on lung function. Smoking cessation and prevention of exacerbations should be a major target in COPD.

Normal bone marrow hematopoietic stem cell reserves and normal stromal cell function support the use of autologous stem cell transplantation in patients with multiple sclerosis.

Bone marrow (BM) stem cell reserves and function and stromal cell hematopoiesis supporting capacity were evaluated in 15 patients with multiple sclerosis (MS) and 61 normal controls using flow cytometry, clonogenic assays, long-term BM cultures (LTBMCs) and enzyme-linked immunosorbent assays. MS patients displayed normal CD34+ cell numbers but a low frequency of colony-forming cells (CFCs) in both BM mononuclear and purified CD34+ cell fractions, compared to controls. Patients had increased proportions of activated BM CD3+/HLA-DR+ and CD3+/CD38+ T cells that correlated inversely with CFC numbers. Patient BM CD3+ T cells inhibited colony formation by normal CD34+ cells and patient CFC numbers increased significantly following immunomagnetic removal of T cells from BMMCs, suggesting that activated T cells may be involved in the defective clonogenic potential of hematopoietic progenitors. Patient BM stromal cells displayed normal hematopoiesis supporting capacity indicated by the CFC number in the nonadherent cell fraction of LTBMCs recharged with normal CD34+ cells. Culture supernatants displayed normal stromal derived factor-1 and stem cell factor/kit ligand but increased flt-3 ligand levels. These findings provide support for the use of autologous stem cell transplantation in MS patients. The low clonogenic potential of BM hematopoietic progenitors probably reflects the presence of activated T cells rather than an intrinsic defect.

Activated T-lymphocytes with myelosuppressive properties in patients with chronic idiopathic neutropenia.

To characterize the cellular components responsible for the impaired granulopoiesis in chronic idiopathic neutropenia (CIN), we investigated the origin of the proapoptotic cytokine producing cells in the bone marrow (BM) microenvironment of CIN patients. We found that the interferon gamma (IFN gamma) and/or Fas-ligand expressing cells in patient BM mononuclear cells and long-term BM culture stroma cells were the CD3(+) T-lymphocytes but not the CD14(+) monocytes/macrophages. The percentage of activated T-lymphocytes was increased in patients' BM as indicated by the proportions of human leucocyte antigen (HLA)-DR(+), CD25(+), CD38(+), CD69(+) and Fas(+) cells within the CD3(+) fraction. Intracellular IFN gamma expression was higher in the BM than peripheral blood of the patients and was associated with increased BM T-lymphocyte numbers. In crossover experiments, patient CD3(+) T-lymphocytes conferred autologous and allogeneic haemopoietic progenitor cell colony inhibition. Patients' T-cell receptor repertoire and polymerase chain reaction analysis did not reveal any clonal T-lymphocyte expansion, suggesting the absence of a direct, antigen-driven recognition of CD34(+) myeloid progenitor cells by patient T-lymphocytes. We conclude that CIN patients have increased number of activated T-lymphocytes in the BM, probably in the setting of a localized polyclonal immune reaction and that these cells confer an inhibitory effect on myelopoiesis through myelosuppressive cytokines including Fas-ligand and IFN gamma.

Acute myeloid/NK precursor cell leukemia with trisomy 4 and a novel point mutation in the extracellular domain of the G-CSF receptor in a patient with chronic idiopathic neutropenia.

Chronic idiopathic neutropenia (CIN) has been well recognized as a granulocytic disorder not associated with increased risk to malignant transformation. Four cases, however, of acute myeloid leukemia have been recently reported in patients with CIN. In the current paper, we report on a CIN patient who developed acute myeloid/natural killer (NK) precursor cell leukemia 11 years after diagnosis and 4 months after initiation of treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF). Leukemic cells had trisomy 4 as the sole cytogenetic abnormality and, also, a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSFR) leading to truncated protein with a loss of 36 amino acids. There was no evidence that this receptor transmitted signals even in the presence of high doses of rhG-CSF in the cultures. We consider that CIN may be a preleukemic condition, at least in a subset of patients, and that rhG-CSF administration is unlikely to be involved in the leukemic transformation in this patient, although such a possibility could not be completely ruled out.

Increased cell apoptosis in bone marrow trephine biopsies and immunomagnetically isolated myeloid progenitor cells in patients with chronic idiopathic neutropenia.

The frequency of apoptotic cells in bone marrow trephine biopsies and cytospins of immunomagnetically isolated myeloid progenitor cells was determined in 39 patients with chronic idiopathic neutropenia (CIN) and 12 hematologically normal individuals using the in situ end-labeling (ISEL) apoptosis detection method. We found that 66.7% of the patients but none of the normal controls displayed apoptotic cells equal to or higher than 5% of the total mononuclear cells in bone marrow biopsies (p<0.01). In the double stain, we also found that the proportion of apoptotic CD15(+) myeloid precursor cells did not differ significantly between patients and control subjects, while the proportion of apoptotic CD34(+) hemopoietic cells could not be estimated with accuracy because of the presence of CD34(+) endothelial cells. Significantly increased apoptosis was noted in cytospins of immunomagnetically isolated patient CD34(+) and CD34(+)/CD33(+) cells but not CD34(-)/CD33(+) cells, compared to the controls ( p<0.001, p<0.02 and p>0.05, respectively). These findings confirm and extend our previous observations in flow-cytometric studies of apoptosis in CIN, indicating that increased apoptosis in CIN bone marrow concerns mainly the CD34(+) and CD34(+)/CD33(+) progenitor cell compartments. We conclude that the accelerated apoptosis in these compartments may account for the impaired neutrophil production in CIN patients.

Potent inhibitory action of red wine polyphenols on human breast cancer cells.

Breast cancer (one of the most common malignancy in Western societies), as well as esophagus, stomach, lung, bladder, and prostate cancer, depend on environmental factors and diet for growth and evolution. Dietary micronutriments have been proposed as effective inhibitory agents for cancer initiation, progression, and incidence. Among them, polyphenols, present in different foods and beverages, have retained attention in recent years. Red wine is a rich source of polyphenols, and their antioxidant and tumor arresting effects have been demonstrated in different in vitro and in vivo systems. In the present study, we have measured the antiproliferative effect of red wine concentrate, its total polyphenolic pool, and purified catechin, epicatechin, quercetin, and resveratrol, which account for more than 70% of the total polyphenols in red wine, on the proliferation of hormone sensitive (MCF7, T47D) and resistant (MDA-MB-231) breast cancer cell lines. Our results indicate that polyphenols, at the picomolar or the nanomolar range, decrease cell proliferation in a dose- and a time-dependant manner. In hormone sensitive cell lines, a specific interaction of each polyphenol with steroid receptors was observed, with IC(50)s lower than previously described. Interaction of polyphenols with steroid receptors cannot fully explain their inhibitory effect on cell proliferation. In addition, discrete antioxidant action on each cell line was detected under the same concentrations, both by modifying the toxic effect of H(2)O(2), and the production of reactive oxygen species (ROS), after phorbol ester stimulation. Our results suggest that low concentrations of polyphenols, and consecutively, consumption of wine, or other polyphenol-rich foods and beverages, could have a beneficial antiproliferative effect on breast cancer cell growth.

Wine antioxidant polyphenols inhibit the proliferation of human prostate cancer cell lines.

The effect of different wine antioxidant polyphenols (catechin, epicatechin, quercetin, and resveratrol) on the growth of three prostate cancer cell lines (LNCaP, PC3, and DU145) was investigated. A dose- and time-dependent inhibition of cell growth by polyphenols was found at nanomolar concentrations. The proliferation of LNCaP and PC3 cells was preferentially inhibited by flavonoids (catechin, epicatechin, and quercetin), whereas resveratrol was the most potent inhibitor of DU145 cell growth. Possible mechanisms of action were investigated: 1) The competition of polyphenols for androgen binding in LNCaP cells revealed significant interaction only in the case of high concentrations of quercetin, at least at five orders of magnitude higher than the concentrations needed for cell growth inhibition. All other phenols showed low interactions. 2) Oxygen species production after mitogen stimulation and H2O2 sensitivity of these cell lines did not correlate with the observed antiproliferative effects, ruling out such a mode of action. 3) NO production revealed two different patterns: LNCaP and DU145 cells produced high concentrations of NO, whereas PC3 cells produced low concentrations. Phorbol ester stimulation of cells did not reveal any additional effect in LNCaP and DU145 cells, whereas it enhanced the secretion of NO in PC3 cells. Polyphenols decreased NO secretion. This effect correlates with their antiproliferative action and the inhibition of inducible NO synthase. It is therefore proposed that the antiproliferative effect of polyphenols is mediated through the modulation of NO production. In conclusion, our data show a direct inhibitory effect of low concentrations of antioxidant wine phenols on the proliferation of human prostate cancer cell lines mediated by the production of NO, further suggesting potential beneficial effects of wine and other phenol-containing foods or drinks for the control of prostate cancer cell growth.

Opioid alkaloids and casomorphin peptides decrease the proliferation of prostatic cancer cell lines (LNCaP, PC3 and DU145) through a partial interaction with opioid receptors.

Opioid agonists (ethylketocyclazocine, etorphine, [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Ala2, N-Me-Phe4-Gly-ol]enkephalin (DAGO), [D-Ser2,Leu5]enkephalin-Thr6 (DSLET) and morphine were found to inhibit the proliferation of human prostate cancer cell lines (LNCaP, DU145, and PC3), in a dose-dependent manner. The 50% inhibitory concentrations (IC50) were in the picomolar range. In many cases, this effect was antagonized by the general opioid antagonist, diprenorphine, indicating the existence of specific opioid binding sites. Saturation binding experiments with selective ligands and effectors showed no opioid sites on the LNCaP cell line, kappa1 and mu sites on the PC3 cell line, and kappa1, kappa3 and mu sites on the DU145 cell line. In other cases, the opioid effect was not antagonized by diprenorphine, indicating that the action of opioids might be mediated through other membrane receptors. Furthermore, casomorphin peptides, issued from bovine alpha- (alpha-casein-90-95 and alpha-casein-90-96) and beta-caseins (beta-casomorphin and beta-casomorphin-1-5), and human alphaS1-casein (alphas -casomorphin and alphaS1-casomorphin amide) inhibited cell proliferation of human prostate cell lines, also by a mechanism partly involving opioid receptors. As opioid neurons can be found in the prostate gland, and casomorphin peptides might reach the gland through the general circulation, the above findings indicate a putative role of opioids in prostate cancer cell growth.