Bosiljka Plećaš-Solarović

Premature centromere division of the X chromosome in neurons in Alzheimer's disease.

Premature centromere division (PCD) represents a loss of control over the sequential separation and segregation of chromosome centromeres. Although first described in aging women, PCD on the X chromosome (PCD,X) is markedly elevated in peripheral blood lymphocytes of individuals suffering from Alzheimer disease (AD). The present study evaluated PCD,X, using a fluorescent in situ hybridization method, in interphase nuclei of frontal cerebral cortex neurons from sporadic AD patients and age‐matched controls. The average frequency of PCD,X in AD patients (8.60 ± 1.20%) was almost three times higher (p < 0.01) than in the control group (2.96 ± 1.20). However, consistent with previous studies, no mitotic cells were found in neurons in either AD or control brain, suggesting an intrinsic inability of post‐mitotic neurons to divide. In view of the fact that it has been well‐documented that neurons in AD can re‐enter into the cell division cycle, the findings presented here of increased PCD advance the hypothesis that deregulation of the cell cycle may contribute to neuronal degeneration and subsequent cognitive deficits in AD.

Age-associated changes in CD90 expression on thymocytes and in TCR-dependent stages of thymocyte maturation in male rats.

To elucidate the effects of ageing on T-cell-maturation, in 3- and 18-month-old rats, we analysed the expression of: (i) CD4/CD8/TCRalphabeta and (ii) Thy-1, which is supposed to be a regulator of TCRalphabeta signalling, and thereby the thymocyte selection thresholds. Since an essential role for TCRalphabeta signalling in the development of CD4+25+T(reg)-cells was suggested, the frequency of these cells was also quantified. We demonstrated that, as for mice, early thymocyte differentiational steps within the CD4-8- double negative (DN) developmental stage are age-sensitive. Furthermore, we revealed that TCRalphabeta-dependent stages of T-cell development are affected by ageing, most likely due to an impaired expression of Thy-1 on TCRalphabeta(low) thymocytes entering selection processes. The diminished frequency of the post-selection CD4+8+ double positive (DP) cells in aged rats, together with an overrepresentation of mature single positive (SP) cells, most probably suggests more efficient differentiational transition from the DP TCRalphabeta(high) to the SP TCRalphabeta(high) developmental stage, which is followed by an increase in pre-migration proliferation of the mature SP cells. Moreover, the study indicated impaired intrathymic generation of CD4+25+T(reg)-cells in aged rats, thus providing a possible explanation for the increased frequency of autoimmune diseases in ageing.

Age-dependent morphometrical changes in the thymus of male propranolol-treated rats.

In order to elucidate a putative role of neurally derived noradrenaline in the thymus development, and in maintenance of adult thymus structure, sexually immature male rats (21-day-old at the beginning of treatment) and young adult animals (75-day-old on the beginning of treatment) were treated with the non-selective beta-adrenoceptor antagonist propranolol (0.40 mg/100 g BW/day, s.c.) for 15 consecutive days, and their thymuses were analyzed stereologically. The effects of beta-adrenoceptor blockade were much more pronounced in sexually immature than in adult rats. In immature propranolol-treated rats the thymus size and volumes of both the main compartments (cortex and medulla) were significantly decreased reflecting, at least partly, a reduction in the overall number of thymocytes. Furthermore, in both the cortical subcompartments (outer and deep cortex) the mean diameter of thymocytes was increased. However, in adult rats exposed to propranolol treatment, only the volume of interlobular connective tissue was enlarged, whereas in the outer part of the cortex the mean thymocyte diameter was increased. These results indicate that the lack of sympathetic input (via beta-adrenoceptors) during the prepubertal period of development diminishes the normal thymus growth and/or accelerates the thymic involution that starts at puberty, immediately after its maximum size is reached, while it is less significant for the maintenance of the thymus size and structure in adults. Additionally, they suggest that distinct cell types, as well as thymocyte subsets, are sensitive to lack of beta-adrenoceptor-mediated influences in sexually immature and adult rats.

Thymopoiesis following chronic blockade of β-adrenoceptors

Abstract The present study was undertaken in order to further clarify putative role of the adrenergic innervation in the regulation of the intrathymic T‐cell maturation. For this purpose adult male DA rats were subjected to either 4‐day‐ or 16‐day‐long propranolol treatment (0.40 mg propranolol/100 g/day, s.c.) and the expression of CD4/8/TCRαβ on thymocytes, as well as thymocyte proliferative and apoptotic index, was assessed in these animals by flow cytometric analysis. Propranolol treatment, in spite of duration, increased both the thymocyte proliferative and apoptotic index (vs. respective vehicle‐treated controls). In 4‐day‐treated animals the thymus cellularity and thymus weight remained unaltered, while in 16‐day‐treated rats the values of both of these parameters were reduced (since increase in the thymocyte apoptotic index overcame that in the proliferative index). The treatments of both durations affected the thymocyte phenotypic profile in a similar pattern, but the changes were more pronounced in rats exposed to the treatment of longer duration. The relative proportion of the least mature CD4−8− double negative (DN) TCRαβ− cells was increased, those of thymocytes at distinct differentiational stages on the transitional route to the CD4+8+ double positive (DP) TCRαβlow stage decreased (all subsets of TCRαβ− in both groups of rats, and those with low expression of TCRαβ in rats subjected to 16‐day‐long treatment) or unaltered (all subsets of TCRαβlow cells in 4‐day‐treated rats). Furthermore, the percentage of CD4+8+ DP TCRαβlow cells was significantly elevated, as well as those of the most mature CD4+8− TCRαβhigh and CD4−8+TCRαβhigh cells (the increase in the percentage of former was much more conspicuous than that of the latter), while the relative proportion of their direct detectable precursors (CD4+8+ DP TCRαβhigh) was reduced. Thus, the present study: i) further supports notion of pharmacological manipulation of adrenergic action as an efficient means in modulation of the T‐cell development, and hence T‐cell‐dependent immune response, and ii) provides more specific insight into T‐cell maturation sequence point/s particularly sensitive to β‐adrenoceptor ligand action.

Morphometrical Characteristics of Age-Associated Changes in the Thymus of Old Male Wistar Rats

In order to provide a morphometrical description of the changes in the aged rat thymus and to relate them to apoptotic and proliferative activity of thymocytes, the thymuses from 3‐ and 18‐month‐old male Wistar rats and the percentages of bromodeoxyuridine‐incorporating and apoptotic cells in cultures of thymocytes were assessed by stereological analysis and flow cytometry, respectively. In old rats the volume of lymphoepithelial thymic tissue is markedly reduced, reflecting a sharp decrease in the total number of thymocytes. A reduction in the proliferative capacity of thymocytes and increase in their susceptibility to apoptosis are, most likely, primarily responsible for a 7‐fold reduction in thymic cellularity in old animals. Furthermore, only the volume of cortical compartment was affected by aging, while that of medulla, despite of reduced cellularity, was not significantly altered. The loss of functional tissue in aged thymus is compensated by a substantial increase in the volume of inter‐lobular connective and adipose tissue, so the thymic weight remained unaltered in old rats. These results suggest that thymus of aged Wistar rats exhibits morphological characteristics similar to those found in aged human thymus and thus may serve as an animal model for further investigations of thymus‐related changes in immunological aging.

DNA damage in Alzheimer disease lymphocytes and its relation to premature centromere division.

While Alzheimer disease (AD) is considered a neurodegenerative disorder, the importance of chromosome instability in non-neuronal cells is equally important, not only for shedding light on the etiology of the disease, but also for possible diagnostic purposes and monitoring the progress of the disease. Here, we evaluated the frequency of DNA damage and expression of premature centromere division (PCD) in peripheral blood lymphocytes of sporadic AD patients, age-matched and young controls. The results show that in male patients with AD, the frequencies of PCD and DNA damage were significantly greater (88%, p < 0.01 and 38%, p < 0.05, respectively) than in age-matched control group. AD females had significantly increased frequency of PCD (134%, p < 0.01) as well as a higher frequency of DNA damage (37%, p < 0.05). Ageing per se, both in males and females, shows significant increase of percentages of PCD (2.3 times, p < 0.01 and 2.8 times, p < 0.01, respectively) and DNA damage (63%, p < 0.01 and 50%, p < 0.01, respectively) comparing with young controls. In addition, a strong (R2 = 0.873, n = 6) and significant (p < 0.01) correlation between the frequencies of PCD and DNA damage was found in all examined groups. We may conclude that the increases in both parameters evaluated in this study are not only associated with normal ageing processes, but are markedly and significantly intensified in AD pathogenesis. Thus, our data support the view that AD is a generalized systemic disease, at least as for the increased DNA damage and PCD incidence in peripheral blood cells.

EFFECTS OF BETA-ADRENOCEPTOR BLOCKADE ON THE PHENOTYPIC CHARACTERISTICS OF THYMOCYTES AND PERIPHERAL BLOOD LYMPHOCYTES

The study revealed that β-adrenoceptor blockade with propranolol (0.40 mg/100 g/day, s.c.) in adult male DA rats: (i) increased the thymocyte proliferation and apoptosis, (ii) caused disturbances in kinetics of T cell differentiation leading to distinguishable changes in relative proportion of thymocytes at distinct maturational steps and to an expansion of the most mature single positive (CD4+, CD8+) thymocyte pool, (iii) affected the relative proportion of neither CD4+ nor CD8+ peripheral blood lymphocytes (PBL), and (iv) augmented the relative number of CD8+CD25+ cells. Thus, the results suggest the role of β-adrenoceptors in fine-tuning of T cell maturation, and, possibly, distribution and activation of distinct PBL subsets

Premature Centromere Division of Metaphase Chromosomes in Peripheral Blood Lymphocytes of Alzheimer's Disease Patients: Relation to Gender and Age

Chromosomal alterations are a feature of both aging and Alzheimers disease (AD). This study examined if premature centromere division (PCD), a chromosomal instability indicator increased in AD, is correlated with aging or, instead, represents a de novo chromosomal alteration due to accelerating aging in AD. PCD in peripheral blood lymphocytes was determined in sporadic AD patients and gender and age-matched unaffected controls. Metaphase nuclei were analyzed for chromosomes showing PCD, X chromosomes with PCD (PCD,X), and acrocentric chromosomes showing PCD. AD patients, regardless of age, demonstrated increased PCD on any chromosome and PCD on acrocentric chromosomes in both genders, whereas an increase in frequency of PCD,X was expressed only in women. This cytogenetic analysis suggests that PCD is a feature of AD, rather than an epiphenomenon of chronological aging, and may be useful as a physiological biomarker that can be used for disease diagnosis.

Oxytocin in corticosterone-induced chronic stress model: Focus on adrenal gland function

Chronic stress conditions can lead to considerable and extensible changes in physiological and psychological performances, and in emergence of risk for various somatic diseases. On the other hand, the neuropeptide oxytocin is reported to increase the resistance of the organism to stress and modulate activity of autonomic nervous system. Chronic corticosterone administration is used as a rat model for a state observed in terms of chronic stress exposure, when negative feedback mechanism of hypothalamus-pituitary-adrenal axis activity is disrupted. In our study, we aimed to investigate whether chronic administration of oxytocin (10 IU/400μL/day for 14days, s.c.) influenced adrenal gland morphology and activity in adult male Wistar rats during long-term corticosterone administration via drinking water (100mg/L for 21days). We examined the influence of treatments on the levels of adrenal gland hormones, corticosterone, adrenaline and noradrenaline, as well as their response to an acute stress challenge evoked by 15-min forced swimming. In addition, the expression of two main monoamine transporters, the noradrenaline transporter (NAT) and vesicular monoamine transporter 2 (VMAT2) in adrenal medulla was measured in the rats exposed to acute stress. Our results showed that oxytocin treatment prevented corticosterone-induced decrease in body weight gain, attenuated adrenal gland atrophy by increasing glandular weight, and the area of the zona fasciculate and reticularis. Chronic corticosterone intake blunted the response of all measured hormones to acute stress, whereas concomitant oxytocin treatment reversed adrenaline and noradrenaline response to acute stress. Furthermore, in adrenal medulla, oxytocin produced significant vasodilatation and stimulated expression of both catecholamine transporters detected both on mRNA and protein level. Our data suggest that oxytocin, by reducing atrophy of adrenal gland, and by increasing catecholamine storage capacity, may be beneficial in conditions accompanied with high glucocorticoid levels, such as chronic stress exposure.

The interface of hypothalamic–pituitary–adrenocortical axis and circulating brain natriuretic peptide in prediction of cardiopulmonary performance during physical stress

Brain natriuretic peptide (NT-pro-BNP) was implicated in the regulation of hypothalamic-pituitary-adrenocortical (HPA) responses to psychological stressors. However, HPA axis activation in different physical stress models and its interface with NT-pro-BNP in the prediction of cardiopulmonary performance is unclear. Cardiopulmonary test on a treadmill was used to assess cardiopulmonary parameters in 16 elite male wrestlers (W), 21 water polo player (WP) and 20 sedentary age-matched subjects (C). Plasma levels of NT-pro-BNP, cortisol and adrenocorticotropic hormone (ACTH) were measured using immunoassay sandwich technique, radioimmunoassay and radioimmunometric techniques, respectively, 10min before test (1), at beginning (2), at maximal effort (3), at 3rdmin of recovery (4). In all groups, NT-pro-BNP decreased between 1 and 2; increased from 2 to 3; and remained unchanged until 4. ACTH increased from 1 to 4, whereas cortisol increased from 1 to 3 and stayed elevated at 4. In all groups together, ΔNT-pro-BNP2/1 predicted peak oxygen consumption (B=37.40, r=0.38, p=0.007); cortisol at 3 predicted heart rate increase between 2 and 3 (r=-0.38,B=-0.06, p=0.005); cortisol at 2 predicted peak carbon-dioxide output (B=2.27, r=0.35, p<0.001); ΔACTH3/2 predicted peak ventilatory equivalent for carbon-dioxide (B=0.03, r=0.33, p=0.003). The relation of cortisol at 1 with NT-pro-BNP at 1 and 3 was demonstrated using logistic function in all the participants together (for 1/cortisol at 1 B=63.40, 58.52; r=0.41, 0.34; p=0.003, 0.013, respectively). ΔNT-pro-BNP2/1 linearly correlated with ΔACTH4/3 in WP and W (r=-0.45, -0.48; p=0.04, 0.04, respectively). These results demonstrate for the first time that HPA axis and NT-pro-BNP interface in physical stress probably contribute to integrative regulation of cardiopulmonary performance.