The fetal thymus has a unique genomic copy number profile resulting from physiological T cell receptor gene rearrangement.

Somatic mosaicism, the presence of genetically distinct cells within an organism, has been increasingly associated with human morbidity, ranging from being a cause of rare syndromes to a risk factor for common disorders such as malignancy and cardiovascular disease. Previous studies interrogating the normal prevalence of somatic mosaicism have focused on adults. We here present an estimate of the baseline frequency of somatic mosaic copy number variation (CNV) at the time around birth, by sampling eight different organs from a total of five fetuses and newborns. Overall we find a significantly lower frequency of organ specific (i.e. mosaic) CNVs as compared to adults (p = 0.003; Mann-Whitney U-test). The rate of somatic CNV in adults has been estimated to around 2.2 CNV per organ assayed. In contrast, after stringent filtering, we found no organ-private CNVs in fetuses or newborns with exception of the thymus. This organ exhibited a specific genome profile in the form of deletions resulting from polyclonal T-cell receptor rearrangements. This implies that somatic non-immune related CNVs, if present at birth, are typically confined to very small cell populations within organs.

Consistency of blood pressure and impact on cardiovascular structure over 20 years in young men.

OBJECTIVE: To identify, in a prospective study, how blood pressure levels at the age of twenty predict hypertension and cardiovascular remodelling 20 years later. METHODS: Twenty-year-old men with blood pressure (BP) elevation [systolic blood pressure (SBP) 140-160 and/or diastolic blood pressure (DBP) 85-95 mmHg; blood pressure elevation (BPE) group] or normal BP [SBP 110-130 and DBP 60-80 mmHg; normal controls (NC) group] entered the study in 1987. In 2007, follow-up was conducted including ambulatory BP, echocardiography, anthropometric and intima media thickness (IMT) measurements. RESULTS: Assessed with 24-h ambulatory BP, the prevalence of hypertension was 35/47 (74.5%) and 1/17 (5.9%) in the BPE and NC group at follow-up respectively. Twenty-four hour mean arterial pressure (MAP) increased from 86.6 (0.8) to 97.2 (1.2) (P < 0.0001), and from 83.1 (1.5) to 88.1 (1.2) mmHg (P < 0.01) from baseline to follow-up in the BPE and NC group respectively. At follow-up, left ventricular mass index (LVMI) was 122 (4) and 106 (4) g m(-1) in the BPE and NC group (unpaired t-test; P < 0.01) respectively, whilst IMT was 0.61 (0.01) and 0.57 (0.01) mm in the BPE and NC group (P < 0.05) respectively. In a logistic regression model, prevalence of hypertension was best explained by office MAP and 24-h DBP at baseline (R(2) 0.333; P < 0.05). A combined model of office MAP, body mass index and insulin levels at baseline explained 56% of LVMI at follow-up. CONCLUSIONS: BP elevation in young age predicts hypertension and adverse cardiovascular remodelling at the age of 40 years. Baseline office MAP is the best predictor of hypertension, 24-h MAP and LVMI.

The influence of patent foramen ovale on oxygen desaturation in obstructive sleep apnoea.

Obstructive sleep apnoea (OSA) is associated with oxygen desaturation to a varying degree. A patent foramen ovale (PFO) may allow interatrial right-to-left shunting. The hypothesis of the current study was that oxygen desaturation will occur more often, in proportion to the frequency of respiratory disturbances, in OSA subjects with PFO than in those without. In a group of 209 subjects diagnosed with OSA, the proportion of desaturation to respiratory events was calculated as the ratio of oxygen desaturation index (ODI)/apnoea-hypopnoea index (AHI). A total of 15 cases with high proportional desaturation (ODI/AHI >or=0.66) were individually matched with 15 controls with low proportional desaturation (ODI/AHI or=20 bubbles passed over from the right to the left atrium after a single injection. The prevalence of large PFO was nine out of 15 (60%) in the high proportional desaturation group versus two out of 15 (13%) in the low proportional desaturation group. The median number of passing bubbles was positively correlated to minimum oxygen saturation among those with PFO. In conclusion, oxygen desaturation occurs more often, in proportion to the frequency of respiratory disturbances, in obstructive sleep apnoea subjects with a patent foramen ovale than in those without.

Energy transfer between fluorescein isothiocyanate and propidium iodide--a problem in the estimation of Tpot with the bromodeoxyuridine-DNA flow cytometry technique?

Energy transfer in flow cytometry can occur when two fluorochromes are bound in close proximity (generally within 100 A) and the emission spectrum of one fluorochrome overlaps significantly with the excitation spectrum of the other. The latter criterium is fulfilled for the fluorochromes fluorescein isothiocyanate and propidium iodide and also the former when they, e.g., are used in bromodeoxyuridine - DNA flow cytometry methods. In the present growth kinetic study using this method, we show that energy transfer does take place between fluorescein isothiocyanate and propidium iodide which results in a detected increase in DNA content with 2-3%. Despite the erroneous increase in the obtained DNA content values, this does not seem to have any influence on the calculation of DNA synthesis time and potential doubling time where the DNA content, based on the relative movement principle of the labelled cells, is used.

Comparison of different labelling index formulae used on bromodeoxyuridine-flow cytometry data.

The essence of the bromodeoxyuridine (BrdUrd)-flow cytometry (FCM) technique is that cells are labelled with the thymidine analogue BrdUrd. They are then allowed to progress through the cell cycle in a BrdUrd-free environment during the postlabelling time period. At a postlabelling time shorter than the length of the S phase (Ts), cells are fixed and prepared for FCM-mediated analysis of BrdUrd and DNA contents. From FCM-derived data, cell cycle kinetic parameters such as labelling index (LI), Ts, and potential doubling time (Tpot) can be calculated. Tpot is believed to be important in the evaluation of tumor aggressiveness and therapy response. Since LI is most commonly used together with Ts to calculate Tpot, it is important that both LI and Ts are independent of the time when cells are sampled. Several formulae to calculate LI and Ts have been presented. In the present paper, we deal with various formulae to calculate LI. These formulae differ in how they take into account unlabelled and BrdUrd-labelled cells in various fractions of the cell cycle. We present a new formula, which takes into consideration cells in the different fractions and thus makes LI theoretically independent of postlabelling time. Our results show that different LI values are obtained when different formulae are used to calculate LI. In addition, we show that the BrdUrd labelling period should be kept as short as possible.

Comparison of mathematical formulas used for estimation of DNA synthesis time of bromodeoxyuridine-labelled cell populations with different proliferative characteristics.

Growth kinetic data of human tumours, obtained by flow cytometric analysis of cells labelled with bromodeoxyuridine (BrdUrd) might provide prognostic information and allow prediction of response to radio- and chemotherapy. However, the theoretical models applied for calculation of growth kinetic data are not fully evaluated. The purpose of this study was to investigate the dependence of the estimation of DNA synthesis time (Ts) on sampling time after BrdUrd labelling, using four different mathematical formulas (Begg et al., White & Meistrich, White et al. and Johansson et al.) which have been developed for the evaluation of flow cytometry-derived data of BrdUrd-labelled cells. In addition, we have investigated the influence of the growth kinetic properties of the cell populations using two cultured cell lines (one slow and one fast growing), and two hetero-transplanted human tumours. The dependence of the estimation of Ts on sampling time was more or less pronounced, depending on the cell population examined and on the formula used. In the fast growing cell line, the estimates of Ts did not vary significantly with sampling time when using the formulas by White et al., whereas in the slow growing cell line, the estimates of Ts did not show any significant dependence on sampling time when using the formula by Johansson et al. In the tumours, the estimation of Ts depended on sampling time with all formulas used, although to different degrees. In one of the tumours, this was mainly caused by the influence of mouse cells, as we demonstrate. Our results indicate that the proliferative characteristics of a cell population should be taken into consideration when choosing a mathematical formula in order to attain Ts values that are independent of sampling time.

Radiation effects on S-phase duration, labelling index, potential doubling time and DNA distribution in head and neck cancer xenografts.

The effect of irradiation on S-phase duration (Ts), labelling index (LI), potential doubling time (Tpot), and cell cycle phase distributions was determined by DNA flow cytometry in xenografted human squamous cell carcinoma of the head and neck (SCCHN). Tumours were treated with a single dose of 3 Gy, and excised at intervals over a 90-h period. Six hours before each excision the tumours were labelled in vivo with bromodeoxyuridine (BrdUrd). Although the growth rate of irradiated tumours was comparable with that of untreated controls, analysis of BrdUrd uptake revealed a transient reduction of LI and a prolongation of Ts in irradiated tumours. Maximum mean Tpot was 931 days in irradiated tumours as compared to 13 days in untreated controls. The variations in Ts, LI and Tpot all occurred within the first hours after irradiation; during the remainder of the observation time, the values of the variables did not differ from those of untreated controls. In irradiated tumours the distribution of cells according to DNA content changed significantly on three occasions during the observation period: 1) Parallel to the initial lowering of LI and prolongation of Ts there was a transient increase in the proportion of cells in G0/G1 and a decrease in the proportion of cells in S and G2; 2) At 18 h, the most pronounced cell cycle phase redistribution occurred when the G0/G1 fraction decreased and the S and G2 phase fractions increased; 3) At 66 h (i.e., approximately one cell cycle later), the pattern was the same as that after 18 h. The findings suggest that the transient prolongation of DNA replication seen in SCCHN cells immediately after a single radiation dose is a symptom of DNA damage inflicted during late G1 or early S-phase, and that this disturbance in DNA synthesis is associated with the subsequent accumulation of cells in G2 phase.

Ornithine decarboxylase and S-adenosylmethionine decarboxylase expression during the cell cycle of Chinese hamster ovary cells.

Cells in mitosis were harvested from exponentially growing Chinese hamster ovary cells by the mitotic detachment technique. Immediately after harvesting, the mitotic cells were seeded in tissue culture flasks and incubated at 37 degrees C in a CO2 incubator. Care was taken not to perturb the progression of cells through the cell cycle. At every hour after seeding for 14 h, cells were collected for analysis of cell cycle distribution, cellular polyamine content, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) activities, and relative mRNA contents. The progression through the cell cycle was monitored by DNA flow cytometry. The putrescine, spermidine, and spermine levels were approximately doubled during the cell cycle: putrescine mainly during late S and G2, spermidine continuously during the entire cell cycle, and spermine mainly during G1 and S. The ODC activity was low in seeded mitotic cells and the enzyme was activated in late G1 and reached a plateau in S phase. A second burst in activity was observed during late S phase and maximal ODC activity was found at the S/G2 transition. The relative ODC mRNA level approximately doubled during the cell cycle and the increase in the relative level mainly took part during mid and late S phase. AdoMetDC activity increased in late G1 and a first maximum was observed during the G1/S transition. A second burst in activity was found in mid S phase. Maximal AdoMetDC activity was found in G2. The relative AdoMetDC mRNA approximately doubled during the cell cycle and the increase in the relative level mainly took place during late G1 and early S phase. Our results indicate that polyamine synthesis was regulated at transcriptional and translational/post-translational levels during the cell cycle of Chinese hamster ovary cells.

Influence of angiotensin II, alpha- and beta-adrenoceptors on peripheral noradrenergic neurotransmission in canine gracilis muscle in vivo.

Interactions between angiotensin II and adrenoceptor-mediated effects on peripheral sympathetic neurotransmission were investigated in constant flow blood-perfused canine gracilis muscle in situ, without and with pretreatment by non-competitive alpha-adrenoceptor blockade. Angiotensin converting enzyme (ACE)-inhibition by benazeprilat increased nerve stimulation (2 Hz, 4 min)-evoked noradrenaline (NA) overflow (+ 21 +/- 5%) with alpha-adrenoceptors intact, but reduced NA overflow (- 18 +/- 6%) when alpha-adrenoceptors were blocked. Vasoconstrictor responses were slightly reduced by benazeprilat. Subsequent infusion of angiotensin II (Ang II, 20 and 500 ng kg-1 min-1 i.v., raising arterial concentrations from 0.6 +/- 0.2 pM to 1390 +/- 240 and 25,110 +/- 3980 pM, respectively) failed to increase NA overflow or to enhance stimulation-evoked vasoconstriction. Adrenaline (0.4 nmol kg-1 min-1 i.v.) did not change evoked NA overflow before or after benazeprilat, either with or without alpha-adrenoceptor blockade, despite high concentrations (approximately 10 nM) in arterial plasma. Following benazeprilat, propranolol reduced NA overflow (- 24 +/- 3%) only if the alpha-adrenoceptors were blocked. In conclusion, benazeprilat reduced evoked NA overflow in the presence of alpha-adrenoceptor blockade to a similar degree as previously shown in the presence of neuronal uptake inhibition in this model. However, contrasting to our previous findings, benazeprilat enhanced NA overflow and reduced the post-junctional response to nerve stimulation in the absence of alpha-adrenoceptor blockade. This could be related to bradykinin accumulation during ACE-inhibition, in addition to the reduction of Ang II generation. Our data are not compatible with facilitation of NA release by circulating Ang II even at pharmacological dose levels. Although activation of prejunctional beta-adrenoceptors may facilitate evoked NA overflow in this model, circulating adrenaline is ineffective under physiological conditions even after alpha-adrenoceptor blockade. Also, beta-adrenoceptor-mediated prejunctional effects do not seem to involve Ang II in canine skeletal muscle in vivo.

Beta-adrenoceptors in human alveolar macrophages isolated by elutriation.

1. beta-adrenoceptors on human alveolar macrophages obtained by bronchoalveolar lavage (BAL) from healthy smoking volunteers (n = 26) were characterized by studying cyclic AMP (cAMP) accumulation in intact macrophages evoked by adrenaline or isoprenaline, with or without appropriate antagonists and by radioligand binding to macrophage membranes, using [125I]-iodopindolol (125IPIN) as beta-adrenoceptor ligand. 2. In a second study, cAMP responses of alveolar macrophages to isoprenaline and PGE1 and of peripheral blood lymphocytes to isoprenaline were compared in smoking and non-smoking healthy volunteers (n = 9 + 9), as our initial studies were performed in smokers, due to their higher cell yield. 3. BAL yielded 47 +/- 23 x 10(6) cells in smokers and 12 +/- 6 x 10(6) cells in non-smokers with a recovery of 82 +/- 8% in the elutriation step (means +/- s.d.). The cell preparation consisted of 99.2 +/- 0.8% macrophages and their viability (trypan blue exclusion) was 97.5 +/- 5.2%. 4. Isoprenaline or adrenaline increased cAMP accumulation approximately 40-fold with or without the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 10(-4) M), which enhanced basal and stimulated cAMP accumulation approximately five-fold. Peak responses were seen after 2 min. EC50s for isoprenaline and adrenaline were 3-5 x 10(-7) M. Phentolamine did not alter responses to adrenaline, indicating absence of inhibitory alpha 2-adrenoceptors. Propranolol inhibited isoprenaline induced cAMP accumulation stereoselectively; pD2-values were 8.2 for (-)-propranolol, 5.6 for atenolol and 7.5 for ICI 118,551, suggesting a predominance of beta 2-adrenoceptors. 5. Specific 125IPIN binding to macrophage membranes was rapid and saturable. Non-specific binding was determined in the presence of 1 microM (-)-propranolol. KD values were 71 +/- 7 pM and the density of specific binding sites was 36 +/- 3 fmol mg-1 protein (three experiments on a membrane pool from 10 subjects; r values for Scatchard analyses = 0.98 +/- 0.01). Similar values were obtained when 200 microM isoprenaline (+ GTP) was used to assess non-specific binding. Competition experiments again showed stereoselectivity for propranolol and a predominance of beta 2-adrenoceptors, as judged by the displacement of specific 125IPIN binding by atenolol and ICI 118,551. 6. Macrophages from smokers responded with less marked cAMP accumulation upon stimulation with isoprenaline or PGE1 than did cells from non-smokers (difference approximately 30%; P less than 0.05 for both agonists) in the presence of IBMX. Thus macrophages from smokers may produce less cAMP due to post-receptor changes in responsiveness.(ABSTRACT TRUNCATED AT 400 WORDS)