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Ageing is associated with a progressive decline in thymic cytoarchitecture followed by a less efficient T cell development and decreased emigration of naive T cells to the periphery. These thymic changes are linked to increased morbidity and mortality from infectious, malignant and autoimmune diseases in old age. Therefore, it is of paramount importance to understand the thymic homeostatic processes across the life span, as well as to identify factors and elucidate mechanisms driving or contributing to the thymic involution. Catecholamines (CAs) derived from sympathetic nerves and produced locally by thymic cells represent an important component of the thymic microenvironment. In young rats, they provide a subtle tonic suppressive influence on T cell development acting via beta(2)- and alpha(1)-adrenoceptors (ARs) expressed on thymic nonlymphoid cells and thymocytes. In the face of thymic involution, a progressive increase in the thymic noradrenaline level, reflecting a rise in the density of noradrenergic nerve fibers and CA-synthesizing cells, occurs. In addition, the density of beta(2)- and alpha(1)-AR-expressing thymic nonlymphoid cells and the alpha(1)-AR thymocyte surface density also exhibit a pronounced increase with age. The data obtained from studies investigating effects of AR blockade on T cell development indicated that age-related changes in CA-mediated thymic communications, certainly those involving alpha(1)-ARs, may contribute to diminished thymopoietic efficiency in the elderly. Having in mind thymic plasticity in the course of ageing, and broadening possibilities for pharmacological modulation of CA signaling, we here present and discuss the progress in research related to a role of CAs in thymic homeostasis and age-related decay in the thymic naive T cell output. Copyright

The study was designed to explore the expression of different neuropeptides, viz. vasoactive intestinal peptide (VIP), calcitonin gene related peptide (CGRP), substance P (SP), bombesin and motilin in the cells of fetal and adult human thymus. Immunohistochemical staining revealed that cortical and medullary thymocytes were labeled by all antibodies, except those specific for motilin. Immunoreactive VIP and SP were observed in the solitary epithelial cells located in the subcapsular/subtrabecular cortex, at the corticomedullary junction and in the medulla. The cells within the subcapsular/subtrabecular monolayer, rare solitary cells in the deep cortex and epithelial cell network in the medulla, were labeled with antibodies to CGRP and bombesin. Hassall's corpuscles were labeled with all antibodies except that specific for SP. The obtained data obtained testify to the expression of different neuropeptides in human thymic lymphoid and non-lymphoid cells and suggest a role for neuroendocrine hormone-mediated mechanisms in the regulation of thymic homeostasis in humans.

Alpha(1)-adrenoceptors (alpha(1)-ARs) are involved in neuro-thymic and thymic intercellular communications, and consequently modulation of T-cell development. Ageing is associated with a number of changes in noradrenergic neuro-effector transmission, and possibly intercellular noradrenaline (NA)-mediated communication resulting in altered responses of target cells to NA. Thus, in old animals an altered NA modulation of thymopoiesis via alpha(1)-ARs may be expected. To test this hypothesis, in old and young adult Wistar rats we examined: 1) thymic NA levels, density of noradrenergic innervation and NA synthesizing cells, as well as alpha(1)-AR expression, and 2) then the effects of 14-day-long treatment with the alpha(1)-AR blocker, urapidil, on thymocyte development. Overall, the first part of study suggested augmented NA signalling to thymic cells via alpha(1)-ARs due to increased NA availability and alpha(1)-AR thymocyte surface density in old rats. The second part of study supported this assumption. Namely, although in rats of both ages urapidil affected the same thymocyte developmental steps ultimately leading to changes in the relative number of the most mature single positive TCR alpha beta(high) thymocytes, its effects were generally more prominent in old animals. Following urapidil treatment, the percentages of CD4+CD8- cells, including those showing a regulatory CD4+CD25+RT6.1- phenotype, were increased, while CD4-CD8+ cells decreased. In old rats, an augmented thymic escape of immature CD4+CD8+ cells was also registered. In rats of both ages the thymic changes were accompanied by alterations in the proportions of major cell populations in the T-lymphocyte compartment of both peripheral blood and spleen, leading to an increase in the CD4+/CD8+ T-cell ratio. These alterations were also more pronounced in old rats. Moreover, in old rats following urapidil treatment the proportion of TCR alpha beta + cells in the periphery was slightly greater reflecting, most

Multipla skleroza (MS) se, kao i većina drugih autoimunskih bolesti, češćejavlja kod žena nego kod muškaraca. Polni dimorfizam je zapažen i u kliničkomispoljavanju MS-e. Kod muškaraca se bolest javlja kasnije, motorni ispadi su teži iprimarno progresivni tok češći nego kod žena. Eksperimentalni autoimunskiencefalomijelitis (EAE) je animalni model MS-e, koji se kod osetljivih oglednihživotinja izaziva različitim indukcionim protokolima. Podaci vezani za polnidimorfizam u kliničkoj prezentaciji EAE-a su relativno oskudni, a postojećiinkonzistentni, pre svega zbog genetskih razlika između korišćenih životinjskihvrsta i sojeva, ali i varijacija u indukcionim protokolima. Hronobiološko starenjeorganizma uključuje i promene u imunskom sistemu koje karakteriše značajanporast autoimunskih fenomena. Uprkos tome, incidenca mnogih autoimunskihbolesti, uključujući i MS-u, se smanjuje kod starih. Podaci o osetljivosti starihoglednih životinja na indukciju EAE-a su heterogeni, u zavisnosti od vrste i sojaoglednih životinja, kao i od modela EAE-a. Mehanizmi koji stoje u osnovi manjeincidence autoimunskih bolesti kod starih jedinki nisu u potpunosti razjašnjeni. Jošsu manje poznati mehanizmi polnog dimorfizma u razvoju ovih bolesti kod starihživotinja, a posebno u čemu se oni razlikuju u odnosu na mehanizme odgovorne zaovaj fenomen kod mladih životinja.Ciljevi ove doktorske disertacije su bili da se: 1) ispitaju polne razlike ukliničkim parametrima indukovane autoimunske neuroinflamacije, kao važnepatogenetske komponente MS-e, na modelu aktivnog EAE-a, kod mladih adultnih(uzrast 3 meseca) i starih (uzrast 22-26 meseci) pacova Dark Agouti soja i 2)identifikuju ćelijski i molekularni mehanizmi odgovorni za uočene polne razlike.Dobijeni rezultati su pokazali postojanje polnog dimorfizma u incidenci itežini EAE-a i kod mladih i kod starih pacova. Incidenca EAE-a je kod pacova obauzrasta bila manja kod mužjaka nego kod ženki, s tim što su mladi mužjaci, zarazliku od starih, imali teži n, Multiple sclerosis (MS) is one of the most common organ-specificautoimmune diseases of the central nervous system. As in other autoimmunediseases, the prevalence of MS is higher in women than in men. The clinicalmanifestations of MS are also sexually dimorphic. Men exhibit later onset of thedisease, more severe motor symptoms and primary progressive course more oftenthan women. Experimental autoimmune encephalomyelitis (EAE) is an animalmodel induced in susceptible strains of animals. Data on sexual dimorphism in theclinical presentation of EAE are limited and inconsistent, reflecting, most likely, thedifferences in the genetic background of the experimental animals and theinduction protocols. Chronobiological ageing of the organism is accompanied byageing of the immune system. Immunosenescence is characterized by an increasein autoimmune phenomena. However, despite this phenomenon, the incidence ofmany autoimmune diseases, including MS, declines with ageing. Data on theinfluence of aging on the incidence and severity EAE are inconsistent. Additionally,data on sex differences in the clinical presentation of EAE in aged animals areextremely limited.The aim of the study was to 1) investigate sex differences in the incidenceand severity of autoimmune neuroinflammation, an important pathogeneticcomponent of MS, on an active EAE model in 3-month-old (young adult) and 22-26-month-old (aged) Dark Agouti rats and 2) identify the cellular and molecularmechanisms behind the observed sex differences. Irrespective of age, the incidenceof EAE was lower in male than in age-matched female rats. However, contrary toaged male rats, young male rats, which developed clinically manifested disease,exhibited more severe motor deficit than the age-matched female rats.Irrespective of age, at the peak of EAE, the greater mean maximal score wasassociated with: (i) greater number of overall and reactivated CD4+ T cells isolatedfrom spinal cord (SC); (ii) upregulated expression of mRNA for CD4

Multipla skleroza (MS) se, kao i većina drugih autoimunskih bolesti, češće javlja kod žena nego kod muškaraca. Polni dimorfizam je zapažen i u kliničkom ispoljavanju MS-e. Kod muškaraca se bolest javlja kasnije, motorni ispadi su teži i primarno progresivni tok češći nego kod žena. Eksperimentalni autoimunski encefalomijelitis (EAE) je animalni model MS-e, koji se kod osetljivih oglednih životinja izaziva različitim indukcionim protokolima. Podaci vezani za polni dimorfizam u kliničkoj prezentaciji EAE-a su relativno oskudni, a postojeći inkonzistentni, pre svega zbog genetskih razlika između korišćenih životinjskih vrsta i sojeva, ali i varijacija u indukcionim protokolima. Hronobiološko starenje organizma uključuje i promene u imunskom sistemu koje karakteriše značajan porast autoimunskih fenomena. Uprkos tome, incidenca mnogih autoimunskih bolesti, uključujući i MS-u, se smanjuje kod starih. Podaci o osetljivosti starih oglednih životinja na indukciju EAE-a su heterogeni, u zavisnosti od vrste i soja oglednih životinja, kao i od modela EAE-a. Mehanizmi koji stoje u osnovi manje incidence autoimunskih bolesti kod starih jedinki nisu u potpunosti razjašnjeni. Još su manje poznati mehanizmi polnog dimorfizma u razvoju ovih bolesti kod starih životinja, a posebno u čemu se oni razlikuju u odnosu na mehanizme odgovorne za ovaj fenomen kod mladih životinja. Ciljevi ove doktorske disertacije su bili da se: 1) ispitaju polne razlike u kliničkim parametrima indukovane autoimunske neuroinflamacije, kao važne patogenetske komponente MS-e, na modelu aktivnog EAE-a, kod mladih adultnih (uzrast 3 meseca) i starih (uzrast 22-26 meseci) pacova Dark Agouti soja i 2) identifikuju ćelijski i molekularni mehanizmi odgovorni za uočene polne razlike. Dobijeni rezultati su pokazali postojanje polnog dimorfizma u incidenci i težini EAE-a i kod mladih i kod starih pacova. Incidenca EAE-a je kod pacova oba uzrasta bila manja kod mužjaka nego kod ženki, s tim što su mladi mužjaci, za razli, Multiple sclerosis (MS) is one of the most common organ-specific autoimmune diseases of the central nervous system. As in other autoimmune diseases, the prevalence of MS is higher in women than in men. The clinical manifestations of MS are also sexually dimorphic. Men exhibit later onset of the disease, more severe motor symptoms and primary progressive course more often than women. Experimental autoimmune encephalomyelitis (EAE) is an animal model induced in susceptible strains of animals. Data on sexual dimorphism in the clinical presentation of EAE are limited and inconsistent, reflecting, most likely, the differences in the genetic background of the experimental animals and the induction protocols. Chronobiological ageing of the organism is accompanied by ageing of the immune system. Immunosenescence is characterized by an increase in autoimmune phenomena. However, despite this phenomenon, the incidence of many autoimmune diseases, including MS, declines with ageing. Data on the influence of aging on the incidence and severity EAE are inconsistent. Additionally, data on sex differences in the clinical presentation of EAE in aged animals are extremely limited. The aim of the study was to 1) investigate sex differences in the incidence and severity of autoimmune neuroinflammation, an important pathogenetic component of MS, on an active EAE model in 3-month-old (young adult) and 22- 26-month-old (aged) Dark Agouti rats and 2) identify the cellular and molecular mechanisms behind the observed sex differences. Irrespective of age, the incidence of EAE was lower in male than in age-matched female rats. However, contrary to aged male rats, young male rats, which developed clinically manifested disease, exhibited more severe motor deficit than the age-matched female rats. Irrespective of age, at the peak of EAE, the greater mean maximal score was associated with: (i) greater number of overall and reactivated CD4+ T cells isolated from spinal cord (SC); (ii) upregulated

Given that granulocyte macrophage colony-stimulating factor (GM-CSF) is identified as the key factor to endow auto-reactive Th cells with the potential to induce neuroinflammation in experimental autoimmune encephalomyelitis (EAE) models, the frequency and phenotype of GM-CSF-producing (GM-CSF+) Th cells in draining lymph nodes (dLNs) and spinal cord (SC) of Albino Oxford (AO) and Dark Agouti (DA) rats immunized for EAE were examined. The generation of neuroantigen-specific GM-CSF+ Th lymphocytes was impaired in dLNs of AO rats (relatively resistant to EAE induction) compared with their DA counterparts (susceptible to EAE) reflecting impaired CD4+ lymphocyte proliferation and less supportive of GM-CSF+ Th cell differentiation dLN cytokine microenvironment. Immunophenotyping of GM-CSF+ Th cells showed their phenotypic heterogeneity in both strains and revealed lower frequency of IL-17+ IFN-gamma+, IL-17+ IFN-gamma-, and IL-17-IFN-gamma+ cells accompanied by higher frequency of IL-17-IFN-gamma- cells among them in AO than in DA rats. Compared with DA, in AO rats was also found (i) slightly lower surface density of CCR2 (drives accumulation of highly pathogenic GM-CSF+ IFN-gamma+ Th17 cells in SC) on GM-CSF+ IFN-gamma+ Th17 lymphocytes from dLNs, and (ii) diminished CCL2 mRNA expression in SC tissue, suggesting their impaired migration into the SC. Moreover, dLN and SC cytokine environments in AO rats were shown to be less supportive of GM-CSF+ IFN-gamma+ Th17 cell differentiation (judging by lower expression of mRNAs for IL-1 beta, IL-6 and IL-23/p19). In accordance with the (i) lower frequency of GM-CSF+ Th cells in dLNs and SC of AO rats and their lower GM-CSF production, and (ii) impaired CCL2 expression in the SC tissue, the proportion of proinflammatory monocytes among peripheral blood cells and their progeny (CD45(hi) cells) among the SC CD11b+ cells were reduced in AO compared with DA rats. Collectively, the results indicate that the strain specificities in eff

There are little data on modulatory effects of estrogens on rat dendritic cell (DC) responses to inflammatory stimuli, and consequently their ability to activate and polarize CD4+ T lymphocyte-mediated immune responses. Splenic conventional DCs from young female Albino Oxford rats were activated in vitro with LPS (TLR4 agonist) or R848 (TLR7/8 agonist) in the presence and absence of 17 beta-estradiol (E2), and their allostimulatory and CD4+ lymphocyte polarizing ability in mixed leukocyte culture (MLC) were studied. Irrespective of the E2 presence, LPS and R848 up-regulated the expression of MHC II on DCs, so they exhibited enhanced allostimulatory capacity in co-culture with CD4+ lymphocytes. On the other hand, E2 promoted stimulatory action of both TLRs on OX62+ DC IL-23 production, augmented their stimulatory effects on IL-6 and IL-1 beta production, but diminished their enhancing effects on the expression IL-10 and IL-27 by DCs. Consequently, in MLC, OX62+ DCs activated/matured in the co-presence of E2 and either LPS or R848 increased the levels of IL-17, the signature Th17 cell cytoldne, when compared with those activated/matured in the absence of E2. GM-CSF levels were also increased in these MLC. Given that the expression of IL-7 mRNA was diminished in DCs activated/matured in the co presence of E2 and TLR, this increase most likely did not reflect enhanced differentiation of Th cells producing GM-CSF only (Th-GM). Conclusions: E2 augments capacity of LPS- and R848-activated/matured DCs from young rat spleen to induce differentiation of IL-17- and GM-CSF-producing cells. (C) 2016 Elsevier B.V. All rights reserved.

smanjena imunološka funkcija kod žena u menopauzi je uglavnom posledica nedostatka hormona ovarijuma u cirkulaciji. Supstituciona hormonska terapija najčešće podrazumeva nadoknadu estrogena, ali ne i progesterona. U ovom radu kao eksperimantalni model izolovane deficijencije progesterona korišćeni su pacovi stari 20 meseci ovarijektomisani na kraju reproduktivnog preioda (starosti 10 meseci) kod kojih je koncentracija estradiola (usled ekstragonadne sinteze) u nivou estradiola kod lažno ovarijektomisanih pacova iste starosti. Ispitivane je intraćelijska ekspresija receptora za estrogene (ER) i progesteron (PR) i sekrecija pro- i anti-inflamatornih citokina i krajnjih produkata metabolizma arginina u slezinskim i peritonealnim makrofagama, u bazalnim uslovima i nakon in vitro stimulacije sa lipopolisaharidom (LPS). Pokazano je da i peritonealne i slezinske makrofage ispoljavaju ER alfa i ER beta, kao i da ovarijektomija ne utiče na ekspresiju ER-a. Većina peritonealnih i slezinskih makrofaga je ispoljavala PR, a ovarijektomija je dovela do povećanja ekspresije PR samo u slezinskim makrofagama. Ovrijektomija je smanjila i sekreciju citokina iz slezinskih (IL-1beta) i peritonealnih makrofaga (TNF-alfa, IL-1beta, IL-10) i povećala sekreciju IL-10 iz slezinskih i TGF-beta iz peritonealnih makrofaga u bazalnim uslovima. Nakon stimulacije LPS-om, slezinske makrofage ovarijektomisanih pacova su sekretovale manje TNF-alfa i više IL-10, dok su peritonealne makrofage sekretovale manje IL-1beta i TGF-beta nego ćelije istog porekla iz lažno-ovarijektomisanih pacova. Ovarijektomija je smanjila sintezu uree i u slezinskim i u peritonealnim makrofagama stimulisanim LPS-om. Dugotrajna izolovana deficijencija progesterona u post-reproduktivnom periodu narušava ravnotežu u produkciji pro-/anti-inflamatornih citokina slezinskih i peritonealnih makrofaga.

Ischemic heart disease (IHD) is a condition in which there is an inadequate supply of blood and oxygen to a portion of the myocardium; it typically occurs when there is an imbalance between myocardial oxygen supply and demand. The most common cause of myocardial ischemia is atherosclerotic disease of an epicardial coronary artery (or arteries) sufficient to cause a regional reduction in myocardial blood flow and inadequate perfusion of the myocardium supplied by the involved coronary artery. Although risk factors for atherosclerosis development are largely known, the pathogenesis of disease is still matter of discussion. The investigation during the last decades have provided new date related to the role of endothelial dysfunction, and particularly role of immune cell-mediated mechanisms in pathogenesis of the disease. The data elucidating role of immunopathogenetic mechanisms in the development of atherosclerotic lesions and their complications provided a strong basis for designing a new generation of drugs to combat aterosclerosis, and thereby to prevent ischemic heart disease., Ishemijsku bolest srca karakteriše nedovoljno snabdevenje krvlju, odnosno kiseonikom, dela miokarda. Nastaje onda kada postoji nesklad između količine kiseonika koja se doprema cirkulacijom u miokard i njegovih potreba u kiseoniku. Najčešći uzrok smanjenog snabdevanja krvlju miokarda je aterosklerotska bolest jedne ili više epikardnih koronarnih arterija, koja sužava lumen i, sledstveno smanjuje. Iako su faktori rizika za razvoj ateroskleroze uglavnom poznati mehanizmi razvoja aterosklerotskih promena još uvek nisu u potpunosti razjašnjeni. Poslednje decenije donele su niz novih saznanja, vezanih za ulogu disfunkcije endotela u inicijaciji i progresiji ateroskerotskih promena i posebno za ulogu ćelija imunskog sistema i imunskih mehanizama u patogenezi ovog oboljenja. Saznanja vezana za ulogu imunopatogenetskih mehanizana stvorila su značajnu osnovu za dizajniranje nove generacije lekova za terapiju aterosklerose i time prevencu ishemijske bolesti srca.

The present review summarizes recent data on age-related thymic changes termed thymic involution, and highlights the putative role of perturbances in extrathymical and, possibly, intrathymical production of gonadal steroids and catecholamines in this process. Thymic involution has been envisaged as an extremely complex process involving multifactorial mechanisms along the bone marrow-thymic axis that accounts for the major manifestations of immunosenescence. These mechanisms include basic cell aging processes (for example, cell replication and programmed cell death) and processes unique to the immune system (such as generation of the T cell receptor repertoire and control of potentially autoreactive cells). Given that the onset of age-associated thymic involution coincides with the rise in gonadal steroid levels at puberty, a causal link between these events has been suggested. It has been shown that: (1) peripubertal gonadectomy causes substantial decrease in the level of noradrenaline in adult male and female thymus and (2) catecholamines, acting via alpha- and beta-adrenoceptor, produce suppressive effects on the thymic cellularity and production of both effector and regulatory T cells. Furthermore, the possibility that gonadal steroids contribute to thymic involution is discussed in this paper. In light of recent data indicating that effects of gonadal hormone deprivation on the thymic cellularity and function are long lasting but transitory, a putative role for the intrathymic sex steroid/catecholamine production in assuring the organ involution, under conditions of their limited supply by extrathymic sources, is also considered. Copyright

The interactions among the nervous, endocrine and immune system were studied by examining: i) thymic and thymocyte catecholamine levels in adult rats castrated (Cx) at postnatal day 3 and ii) effects of 14-day-long propranolol (P) treatment on main thymocyte differentiational molecule expression in adult non-Cx and Cx rat. The results demonstrated that castration in early postnatal period lowers levels of both neurally- and thymocyte-derived noradrenaline in adult rats, and thereby diminishes beta-adrenoceptor-mediated fine tuning of the T-cell differentiation/maturation. In non-Cx rats P affected TCR alpha beta-dependent stages of thymocyte differentiation/maturation decreasing frequency of CD4+8+ double positive (DP) TCR alpha beta(low) low cells entering selection processes and increasing relative number of positively selected DP TCR alpha beta(high) (most likely due to an increased thymocyte surface density of Thy-1 that is involved in negative control of TCR alpha beta-mediated signaling/selection thresholds) and the most mature CD4+8- TCR alpha beta(high) cells (including CD4+25+ regulatory cells). However, in Cx rats P failed to produce any significant changes in thymocyte subset composition.

Adult male Wistar rats were subjected to propranolol (P, 0.40 mg/100 g/day) or saline (S) administration (controls) over 14 days. The expression of major differentiation molecules on thymocytes and Thy-1 (CD90) molecules, which are shown to adjust thymocyte sensitivity to TCR alpha beta signaling, was studied. In addition, the sensitivity of thymocytes to induction of apoptosis and concanavalin A (Con A) signaling was estimated. The thymocytes from P-treated (PT) rats exhibited an increased sensitivity to induction of apoptosis, as well as to Con A stimulation. Furthermore, P treatment produced changes in the distribution of thymocyte subsets suggesting that more cells passed positive selection and further differentiated into mature CD4+ or CD8+ single positive (SP) TCR alpha beta(high) cells. These changes may, at least partly, be related to the markedly increased density of Thy-1 surface expression on TCR alpha beta(low) thymocytes from these rats. The increased frequency of cells expressing the CD4+25+ phenotype, which has been shown to be characteristic for regulatory cells in the thymus, may also indicate alterations in thymocyte selection following P treatment. Inasmuch as positive and negative selections play an important role in continuously reshaping the T-cell repertoire and maintaining tolerance, the hereby presented study suggests that pharmacological manipulations with beta-AR signaling, or chemically evoked alterations in catecholamine release, may interfere with the regulation of thymocyte selection, and consequently with the immune response.

The thymus structure, expression of CD4/CD8/TCR alpha beta on thymocytes and thymocyte proliferative and apoptotic indexes were analyzed in sexually immature 30-day-old and in sexually mature 60-day-old female rats neonatally androgenized (NA) by subcutaneous injection of 500 mu g testosterone propionate/day on days 1-3 and in their vehicle-administered counterparts. The treatment affected normal thymus development. Thus, at 30 days of age, there was a reduction in the thymus weight, reflecting a decrease in the main thymic compartments. However, at 60 days of age, thymus weight did not significantly differ from that in age-matched controls, since the cortical volume enlargement was followed by a proportional decrease in the medullary volume. In rats of both ages, the changes in thymic compartments most likely reflected alterations in the size of both lymphoid and nonlymphoid components. Furthermore, in NA rats, substantial changes in thymocyte phenotypic characteristics were registered, in spite of their age. In both groups of NA rats, a decrease in the relative proportion of the least mature CD4-8-TCR alpha beta- cells and in that of CD4+8- TCR alpha beta-/TCR alpha beta(low) cells followed by an increase in the percentage of their successor CD4+8+TCR alpha beta-/TCR alpha beta(low) cells was detected. In addition, in 30-day-old NA rats, the relative proportions of CD4+8+TCR alpha beta(high) cells ( just positively selected) and that of mature single positive (CD4+8- and CD4-8+) and CD4-8- double negative TCR alpha beta(high) cells, were reduced, while in 60-day-old NA rats only the percentage of CD4+8+TCR alpha beta(high) thymocytes was decreased. Thus, the study showed that the changes in the development of the hypothalamo-pituitary-gonadal axis induced by neonatal androgenization may affect the thymus development and intrathymic T-cell maturation. Copyright

In adult rats 3, 6 and 9 months post-orchidectomy performed at the age of 30 days the thymus weight, thymocyte yield and relative proportions of thymocyte subsets (delineated by expression of CD4/CD8 molecules and TCRalphabeta) were analyzed in order to elucidate a putative role of male gonadal hormones in the shaping of thymus size and intrathymic T cell Maturation, In 4-month-old control rats the thymus size and cellularity returned to the corresponding levels in 1-month-old rats, These levels were sustained during the following 6 months. In spite of that, the distribution of the main thymocyte subsets in these rats as Subjected to significant changes, probably due to an age-associated diminishing thymus ability to provide efficient T cell differentiation. The results added further v.,eight to a potential feedback regulatory role of CD4+8- cells in thymopoiesis. Furthermore. they revealed that the orchidectomy-induced (i) enlargement of the thymus size and enrichment of the thymic lymphoid cell content are of a limited duration and (ii) alterations in the relative proportion of thymocytes become quantitatively more pronounced with duration of the gonadal deprivation. Thus, the study also indicates that the age-associated changes in gonadal hormones may be, at least partly. responsible For the age-related reshaping a of the T cell maturation sequence, and hence for remodeling T cell dependent immune functions.

This study examined the effects of the principal ovarian steroids, 17 beta -estradiol (E) and progesterone (P), on the thymic structure and on the intrathymic development of T-cells. Adult female rats were ovariectomized (OVX) and treated for 13 days with physiological doses of either E or P; controls received an equivalent volume of vehicle. Ovariectomy produced a marked increase (vs, sham-operated controls) in thymus weight, which was associated with an increase in the volume and cellularity of both the medulla and cortex. Treatment of OVX rats with E reduced the thymic weight to value, which was significantly lower than that of sham-operated controls decreasing the volume of cortex below level in sham-OVX rats, and reversing the effect of ovariectomy on the volume of medulla. P only prevented the increases in thymus weight and cortical volume induced by OVX. However, unlike E, it had no discernable effect on the medullary volume. E treatment reduced the cellularity of the cortex and medulla to values, which were lower than those of sham-OVX rats, while P only reversed the effects of OVX on the cellularity of both the compartments. Ovariectomy also had a profound effect on the thymocyte profile, increasing the proportion of CD4(+)8(+)TCR alpha beta (-) cells and producing a corresponding decrease in the relative proportions of all TCR alpha beta (high) cell subsets. The decrease in the latter was opposed by treatment with E or P. However, the sensitivity of the less mature cells (except CD4(-)8(-)TCR alpha beta (-), the percentage of which was reduced by both hormones) to the two hormones differed. E reduced the relative proportion of CD4(-)8(+)TCR alpha beta (-), CD4(-)8(+)TCR alpha beta (low) and CD4(+) 8(+)TCR alpha beta (-) cells, while P increased the percentage of CD4(-) 8 +TCR alpha beta (low) cells. The results suggest that E anti P affect both the lymphoid and nonlymphoid compartments of the thymus, and that while P increases the volume of the nonlymphoid

This study was undertaken considering that, despite the broad use of the unopposed estrogen replacement therapy in elderly women, data on estrogen influence on the functional capacity of dendritic cells (DCs), and consequently immune response are limited. We examined the influence of 17 beta-estradiol on phenotype, cytokine secretory profile, and allostimulatory and polarizing capacity of splenic (OX62+) conventional DCs from 26-month-old (aged) Albino Oxford rats matured in vitro in the presence of LPS, a TLR4 agonist, and R848, a TLR7/8 agonist In the presence of 17 beta-estradiol, DCs from aged rats exhibited an impaired ability to mature upon stimulation with LPS, as shown by the lower surface density of MHC II and costimulatory CD80 and CD86 molecules. 17 beta-Estradiol alone enhanced CD40 expression in OX62+ DCs without affecting the expression of other costimulatory molecules, thereby confirming that the expression of this molecule is regulated independently from the regulation of other costimulatory molecules. However, although R848 upregulated the expression of MHC II and CD80 and CD40 costimulatory molecules on DCs, 17 beta-estradiol diminished the effect of this TLR agonist only on MHC II expression. In conjunction, the previous findings suggest that LPS and R848 elicit changes in the expression of costimulatory molecules via triggering differential intracellular signaling pathways. Furthermore, 17 beta-estradiol diminished the stimulatory influence of both LPS- and R848-matured OX62+ DCs on allogeneic CD4+ T lymphocyte proliferation in a mixed lymphocyte reaction (MLR). Moreover, as shown in MLR, the exposure to 17 beta-estradiol during LPS- and R848-induced maturation diminished Th1- and enhanced Th17-driving capacity and reduced Th1-driving capacity of OX62+ DCs, respectively. This suggests that LPS and R848 affect not only the surface phenotype, but also functional characteristics of OX62+ DCs triggering distinct intracellular signaling pathways. Coll

The study examined the putative role of ovarian hormones in shaping of rat peripheral T-cell compartment during post-reproductive period. In 20-month-old rats ovariectomized (Ox) at the very end of reproductive period, thymic output, cellularity and composition of major TCR alpha beta+peripheral blood lymphocyte and splenocyte subsets were analyzed. Ovariectomy led to the enlargement of CD8 + peripheral blood lymphocyte and splenocyte subpopulations. This reflected: (i) a more efficient thymic generation of CD8 + cells as indicated by increased number of CD4+CD8+double positive and the most mature CD4CD8+TCR alpha beta(high) thymocytes and CD8 + recent thymic emigrants (RTEs) in peripheral blood, but not in the spleen of Ox rats, and (ii) the expansion of CD8 + memory/activated peripheral blood lymphocytes and splenocytes. The latter was consistent with a greater frequency of proliferating cells among freshly isolated memory/activated CD8 + peripheral blood lymphocytes and splenocytes and increased proliferative response of CD8 + splenocytes to stimulation with plate-bound anti-CD3 antibody. The former could be related to the rise in splenic IL-7 and IL-15 mRNA expression. Although ovariectomy affected the overall number of CD4 + T cells in none of the examined compartments, it increased CD4+FoxP3 + peripheral blood lymphocyte and splenocyte counts by enhancing their generation in periphery. Collectively, the results suggest that ovariectomy-induced long-lasting disturbances in ovarian hormone levels (mirrored in diminished progesterone serum level in 20-month-old rats) affects both thymic CD8 + cell generation and peripheral homeostasis and leads to the expansion of CD4+FoxP3 + cells in the periphery, thereby enhancing autoreactive cell control on account of immune system efficacy to combat infections and tumors.

The study was undertaken considering age-related changes in susceptibility to experimental autoimmune encephalomyelitis (EAE) and a putative role of spleen in pathogenesis of this disease. The phenotypic and functional characteristics of T splenocytes were examined in young (3-month-old), middle-aged (8-month-old) and aged (26-month-old) Dark Agouti rats immunized for EAE with rat spinal cord in complete Freund's adjuvant The rat susceptibility to EAE induction, as well as the number of activated CD4+CD134+ lymphocytes retrieved from their spinal cords progressively decreased with aging. To the contrary, in rats immunized for EAE the number of activated CD4+ splenocytes, i.e., CD4+CD134+, CD4+CD25+FoxP3 and CD4+CD40L+ cells, progressively increased with aging. This was associated with age-related increase in (i) CD4+ splenocyte surface expression of CD44, the molecule suggested to be involved in limiting emigration of encephalitogenic CD4+ cells from spleen into blood and (ii) frequency of regulatory T cells, including CD4+CD25+FoxP3 + cells, which are also shown to control encephalitogenic cell migration from spleen into the central nervous system. In favor of expansion of T-regulatory cell pool in aged rats was the greater concentration of IL-10 in unstimulated, Concanavalin A (ConA)- and myelin basic protein (MBP)-stimulated splenocyte cultures from aged rats compared with the corresponding cultures from young ones. Consistent with the age-related increase in the expression of CD44, which is shown to favor Th1 effector cell survival by interfering with CD95-mediated signaling, the frequency of apoptotic cells among CD4+ splenocytes, despite the greater frequency of CD95+ cells, was diminished in splenocyte cultures from aged compared with young rats. In addition, in control, as well as in ConA-and MBP-stimulated splenocyte cultures from aged rats, despite of impaired CD4+ cell proliferation, IFN-gamma concentrations were greater than in corresponding cultures fro

The study was undertaken considering: i) that relative proportion of distinct subsets of splenic dendritic cells (DCs) is strain-specific and predictive for the susceptibility to autoimmune diseases; ii) age-related changes in endocytic, allostimulatory and polarizing capacity of splenic OX62+ DCs from Albino Oxford rats (relatively resistant to Th1/Th17-mediated diseases) and iii) strain specificities in age-related changes of mouse DCs. To ascertain whether there are strain specificities in age-related rat DC changes, we examined the influence of aging on OX62+ DCs from Dark Agouti (DA) rats prone to Th1/Th17-mediated autoimmune diseases. The study provided additional evidence that the predominance of CD4-cells within OX62+ DCs from young adult rats correlates with their susceptibility to Th1/Th17-mediated diseases. Consistently, lipopolysaccharide (LPS)-matured DCs from 3-month-old (young) rats exhibited Th1 driving force when co-cultured with allogeneic CD4+ T cells. This most likely reflected enhanced TNF-alpha and iNOS expression. Comparing with young rats, OX62+ DCs from 26-month-old (aged) rats showed: i) diminished endocytic capacity; ii) impaired ability to mature in vitro upon LPS stimulation (as indicated by lower MHC II, CD86 and CD40 surface expression), which is consistent with the increase in their IL-10 production, and iii) diminished allostimulatory capacity and loss of Th1-driving capacity in the mixed lymphocyte reaction. The latter, probably, reflected greater IL-10 production by LPS-stimulated DC from aged rats, as well as lower CD40 density on their surface. Overall, our findings suggest that aging might affect DA rat capability to mount an efficient Th1 immune response, and consequently susceptibility to Th1/Th17-mediated pathology.

Recent clinical evidence suggests that the therapeutic effect of massage involves the immune system and that this can be exploited as an adjunct therapy together with standard drug-based approaches. In this study, we investigated the mechanisms behind these effects exploring the immunomodulatory function of stroking as a surrogate of massage-like therapy in mice. C57/BL6 mice were stroked daily for 8 days either with a soft brush or directly with a gloved hand and then analysed for differences in their immune repertoire compared to control non-stroked mice. Our results show that hand-but not brush-stroked mice demonstrated a significant increase in thymic and splenic T cell number (p lt 0.05; p lt 0.01). These effects were not associated with significant changes in CD4/CD8 lineage commitment or activation profile. The boosting effects on T cell repertoire of massage-like therapy were associated with a decreased noradrenergic innervation of lymphoid organs and counteracted the immunosuppressive effect of hydrocortisone in vivo. Together our results in mice support the hypothesis that massage-like therapies might be of therapeutic value in the treatment of immunodeficiencies and related disorders and suggest a reduction of the inhibitory noradrenergic tone in lymphoid organs as one of the possible explanations for their immunomodulatory function.

Background: Aging influences immune response and susceptibility to EAE in a strain specific manner. The study was designed to examine influence of aging on EAE induction in Albino Oxford (AO) rats. Results: Differently from 3-month-old (young) rats, which were resistant to EAE induction, the majority of aged (24-26-month-old) rats developed mild chronic form of EAE. On 16th day post-immunization, when in aged rats the neurological deficit reached plateau, more mononuclear cells, including CD4+ T lymphocytes was retrieved from spinal cord of aged than young rats. The frequencies of IL-17+ and GM-CSF+ cells within spinal cord infiltrating CD4+ lymphocytes were greater in aged rats. To their increased frequency contributed the expansion of GM-CSF + IL-17 + IFN-gamma+ cells, which are highly pathogenic in mice. The expression of the cytokines (IL-1 beta and IL-23/p19) driving GM-CSF + IL-17 + IFN-gamma + cell differentiation in mice was also augmented in aged rat spinal cord mononuclear cells. Additionally, in aged rat spinal cord the expansion of GM-CSF + IL-17-IFN-gamma- CD4+ T lymphocytes was found. Consistently, the expression of mRNAs for IL-3, the cytokine exhibiting the same expression pattern as GM-CSF, and IL-7, the cytokine driving differentiation of GM-CSF + IL-17-IFN-gamma- CD4 + lymphocytes in mice, was upregulated in aged rat spinal cord mononuclear cells, and the tissue, respectively. This was in accordance with the enhanced generation of the brain antigen-specific GM-CSF+ CD4+ lymphocytes in aged rat draining lymph nodes, as suggested by (i) the higher frequency of GM-CSF+ cells (reflecting the expansion of IL-17-IFN-gamma- cells) within their CD4+ lymphocytes and (ii) the upregulated GM-CSF and IL-3 mRNA expression in fresh CD4+ lymphocytes and MBP-stimulated draining lymph node cells and IL-7 mRNA in lymph node tissue from aged rats. In agreement with the upregulated GM-CSF expression in aged rats, strikingly more CD11b + CD45(int) (activated microglia

Considering the crucial pathogenic role of CD4+ T cells in experimental autoimmune encephalomyelitis (EAE) and the opposite direction of the sexual dimorphism in the severity of the disease in 22-24-and 3-month-old dark agouti rats, sex differences in CD4+ T-cell-mediated immune response in aged rats immunized for EAE were examined and compared with those in young animals. In the inductive phase of EAE, fewer activated CD4+ lymphocytes were-retrieved from draining lymph nodes of male (developing less severe disease) compared with female rats, due, at least partly, to their lesser expansion. The former reflected a greater suppressive capacity of CD4+CD25+Foxp3+ cells. Consequently, CD4+ lymphocyte infiltration into the spinal cord of aged male rats was diminished. At the peak of EAE, the frequency of reactivated cells was lower, whereas that of the regulatory CD4+ cells was higher in male rat spinal cord. Consistently, microglial activation and the expression of proinflammatory/damaging cytokines in male rat spinal cord mononuclear cells were diminished. Additionally, the frequency of the highly pathogenic IL-17+IFN-gamma+ T lymphocytes infiltrating their spinal cord was lower. Together, these results point to (i) an age-specificity in CD4+ cell-mediated immune response and (ii) mechanisms underlying the sex differences in this response in aged rats. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

The influence of aging on phagocytic, antigen presenting and secretory capacity of resident Albino Oxford rat peritoneal macrophages cultured for 24 hours in medium alone and in the presence of either LPS or IL-4 was examined. The frequencies of CD68+, CD169+ and CCR7+ cells were comparable among fresh peritoneal cells from young (3-months-old) and aged (i8-months-old) rats, whereas those of CD163+, MHCII+ and CD86+ cells were lower within the same cell population in aged relative to young rats. Congruent with the latter findings, aged rat macrophages cultured in medium alone exhibited impaired allostimulatory properties. Additionally, their zymosan phagocytizing ability was reduced. Differently from young rat macrophages, aged rat macrophages changed neither allostimulatory nor zymosan phagocytizing ability upon in vitro treatment with either LPS or IL-4. In the presence of LPS, NO production comparably increased in macrophages from young and aged rats. However, in the presence of either LPS or IL-4 urea production increased only in macrophages from aged rats. Differently from LPS, which increased the prototypic proinflamrnatory cytokine production in macrophages from rats of both ages, the diminishing effect of IL-4 on their production was evident only in macrophages from young rats. Collectively, our results suggest that aging, besides diminishing influence on macrophage allostimulatory and zymosan phagocytizing ability, may impair their responsiveness to IL-4 in vitro. Consequently, an altered efficacy of inflammatory/immune responses in aged rats may be expected.

It has been hypothesized that maturational processes within the hypothalamo-pituitary-gonadal (HPG) axis and thymus are reciprocally regulated via neural pathways. To test this hypothesis, in the thymi of adult rats orchidectomized (ORX) at age of 1 (ORX-1), 7 (ORX-7) and 30 days (ORX-30): (i) noradrenaline (NA), dopamine (DA) and serotonin (5-HT) contents and acetylcholinesterase (AChE) activity were measured and (ii) the distribution of monoamine- and AChE-containing nerves and cells was examined by a sucrose phosphate glyoxylic acid (SPG) method and enzyme histochemistry, respectively. In all groups of ORX rats, the thymus weight was significantly increased over that in sham-ORX control rats. In the ORX-1 rats, the increase in the thymus weight was accompanied by a proportional increase in the content of both catecholamines and 5-HT; consequently the concentration of each of them remained unaltered. In these animals, the density of both SPG-stained thymus nerve fibers and cells also remained unchanged. In the ORX-7 rats, the rise in the thymus weight was followed by a proportional increase in the content of all monoamines, except for NA which was reduced. Therefore, in these rats neither the thymus concentrations of DA nor that of 5-HT differed from controls, while the concentration of NA was significantly decreased. The reduction in both NA content and concentration reflected a diminished density of SPG-positive nerve profiles. In the ORX-30 rats, the increase in thymus weight was neither paralleled by a proportional increase in the DA content nor in 5-HT, while the content of NA was decreased. Thus, in their thymi the concentration of both NA and DA, as well as that of 5-HT, were significantly reduced. In parallel with these changes, a decreased density of thymic SPG-positive nerve fibers and cells was found. In all ORX rats, the pattern of intrathymic distribution of SPG-positive fibers and cells remained unchanged. Orchidectomy affected neither the activity o

To assess a putative role of the neural pathways in transfer of information from the gonads to the thymus, adult AO rats were orchidectomized (ORX) or sham ORX (controls); sacrificed 1, 3, 7, or 15 days later and their thymi were analyzed for: (a) the concentrations of noradrenaline (NA), dopamine (DA) and serotonin (5-HT) and distribution of monoamine-containing nerve profiles and (b) the acetylcholinesterase (AChE) activity and distribution of AChE-containing nerve profiles. Three days after the castration, an elevation in the level of both catecholamines, reflecting an increase in the overall intensity of nerve fibers autofluorescence, was found. Seven days post castration neither NA nor DA concentration differed from the appropriate control values, while 15 days after the surgery the concentration of NA was lower than that in the controls, most likely, due to diminished density of noradrenergic nerve profiles. In both the rats sacrificed 7 and 15 days after orchidectomy the concentration of 5-HT was reduced as result of a decrease in the density of 5-HT-containing autofluorescent cells. The activity of AChE was depressed one day after the surgery; then increased, so that 3 days post castration its value was higher than that in the sham ORX. After this increase, AChE activity decreased being, at postoperative day 7 and 15, lower than that in the controls. It seems that this decrease in AChE activity reflected, not only a reduction in the density of AChE-containing nerve fibers, but also a decrease in the density of AChE positive cells. Thus, the results indicate that orchidectomy can evoke changes in the T-cell maturation altering modulatory influences on this process coming via neural route, as well as those coming from the mast cells and AChE positive epithelial cells which constitute important component of the thymus microenvironment.

Dendritske ćelije (DĆ) su profesionalne antigen-prezentujuće ćelije koje činesponu između urođenog i stečenog imuniteta i imaju važnu ulogu u imunskomodgovoru. Pored toga, DĆ imaju ulogu u uspostavljanju i održavanju imunološketolerancije, kao i u patogenezi autoimunskih bolesti. Istraživanja na polju biologijestarenja ćelija imunskog sistem su pokazala da tokom starenja komponente i urođenog istečenog imuniteta podležu promenama. Podaci vezani za uticaj starenja na DĆ nisukonzistentni i uglavnom se odnose na ćelije humanog porekla, ćelije primata i miševa,dok nema podataka o uticaju starenja na DĆ pacova.Cilj ove doktorske disertacije je bio da se ispita uticaj starenja na fenotipske ifunkcijske karakteristike konvencionalnih DĆ (kDĆ) pacova, kao i mogući značajsojnih razlika za nastanak starenjem uslovljenih promena ovih ćelija. Takođe, cilj je bioi da se utvrdi da li starenjem uslovljene promene kDĆ pacova koreliraju sa promenomosetljivosti pacova na indukciju organ-specifičnih autoimunskih bolesti. Ispitivane susveže izolovane kDĆ i stimulisane in vitro lipopolisaharidom (LPS). KonvencionalneDĆ su izolovane iz slezina pacova uzrasta 3 meseca (mladi odrasli pacovi) i 26 meseci(stari pacovi) Albino Oxford (AO) i Dark Agouti (DA) soja, koji pokazuju različitstepen osetljivosti na indukciju organ-specifične autoimunske bolesti posredovaneTh1/Th17 limfocitima, kao što je eksperimentalni autoimunski encefalomijelitis (EAE).Starenje dovodi do veće zastupljenosti ćelija CD4- fenotipa u populaciji svežeizolovanih kDĆ starih AO pacova za razliku od mladih životinja, kod kojih je brojnijaCD4+ subpopulacija, a ne utiče na zastupljenost ovih subpopulacija kod DA pacova,tako da je kod njih u oba uzrasta dominantna CD4- subpopulacija. Osim toga, svežeizolovane kDĆ starih AO pacova pokazuju nezreliji, a starih DA pacova zreliji fenotip,u odnosu na kDĆ mladih pacova odgovarajućeg soja, a što je procenjeno na osnovunivoa ispoljenosti aktivacionih molekula (CD80, CD86 i CD40). U s, Dendritic cells (DCs) are the professional antigen-presenting cells that form aninterface between innate and adaptive immunity and play an important role in theimmune response. They are also central in the establishment and maintenance ofimmune tolerance, as well as in the pathogenesis of autoimmune diseases. Aging isassociated with the progressive immune system changes, which involve all types ofimmune cells. The data on age-associated DC alterations origin mainly from studies inmice and human, and are largely inconsistent, while there are no data on the influenceof aging on rat DCs.The aim of this thesis was to elucidate the influence of aging on the phenotypicand functional characteristics of conventional DCs (cDCs), and the possible significanceof strain differences in the development of age-related changes. In addition, this thesiswas aimed to investigate a putative correlation between age-related changes in cDCbiology and rat susceptibility to induction of organ-specific autoimmune diseases.Freshly isolated cDCs and cDCs stimulated in vitro with lipopolysaccharide (LPS) wereinvestigated. The cells were isolated from the spleens of 3- (young adult) and 26 monthold(aged) rats of Albino Oxford (AO) and Dark Agouti (DA) strain, which arerelatively resistant (AO) and susceptible (DA) to the induction of Th1/Th17-mediatedorgan-specific autoimmune disease, as it is experimental autoimmune encephalomyelitis(EAE).In AO rats aging increased relative proportion of cells exhibiting CD4-phenotype within population of freshly isolated cDCs, while in young animals CD4+cells represented the predominant subpopulation within cDCs. In DA rats aging did notaffect cDC subset composition, so predominance of CD4- subset within cDCs in rats ofboth ages was found. Aging affected surface phenotype and endocytic capacity offreshly isolated cDCs in a strain-specific manner. Namely, the lower surface density ofactivation markers (CD80, CD86 and CD40) on cDCs from aged AO rats and theirhig

Dendritske ćelije (DĆ) su profesionalne antigen-prezentujuće ćelije koje čine sponu između urođenog i stečenog imuniteta i imaju važnu ulogu u imunskom odgovoru. Pored toga, DĆ imaju ulogu u uspostavljanju i održavanju imunološke tolerancije, kao i u patogenezi autoimunskih bolesti. Istraživanja na polju biologije starenja ćelija imunskog sistem su pokazala da tokom starenja komponente i urođenog i stečenog imuniteta podležu promenama. Podaci vezani za uticaj starenja na DĆ nisu konzistentni i uglavnom se odnose na ćelije humanog porekla, ćelije primata i miševa, dok nema podataka o uticaju starenja na DĆ pacova. Cilj ove doktorske disertacije je bio da se ispita uticaj starenja na fenotipske i funkcijske karakteristike konvencionalnih DĆ (kDĆ) pacova, kao i mogući značaj sojnih razlika za nastanak starenjem uslovljenih promena ovih ćelija. Takođe, cilj je bio i da se utvrdi da li starenjem uslovljene promene kDĆ pacova koreliraju sa promenom osetljivosti pacova na indukciju organ-specifičnih autoimunskih bolesti. Ispitivane su sveže izolovane kDĆ i stimulisane in vitro lipopolisaharidom (LPS). Konvencionalne DĆ su izolovane iz slezina pacova uzrasta 3 meseca (mladi odrasli pacovi) i 26 meseci (stari pacovi) Albino Oxford (AO) i Dark Agouti (DA) soja, koji pokazuju različit stepen osetljivosti na indukciju organ-specifične autoimunske bolesti posredovane Th1/Th17 limfocitima, kao što je eksperimentalni autoimunski encefalomijelitis (EAE). Starenje dovodi do veće zastupljenosti ćelija CD4- fenotipa u populaciji sveže izolovanih kDĆ starih AO pacova za razliku od mladih životinja, kod kojih je brojnija CD4+ subpopulacija, a ne utiče na zastupljenost ovih subpopulacija kod DA pacova, tako da je kod njih u oba uzrasta dominantna CD4- subpopulacija. Osim toga, sveže izolovane kDĆ starih AO pacova pokazuju nezreliji, a starih DA pacova zreliji fenotip, u odnosu na kDĆ mladih pacova odgovarajućeg soja, a što je procenjeno na osnovu nivoa ispoljenosti aktivacionih molekula, Dendritic cells (DCs) are the professional antigen-presenting cells that form an interface between innate and adaptive immunity and play an important role in the immune response. They are also central in the establishment and maintenance of immune tolerance, as well as in the pathogenesis of autoimmune diseases. Aging is associated with the progressive immune system changes, which involve all types of immune cells. The data on age-associated DC alterations origin mainly from studies in mice and human, and are largely inconsistent, while there are no data on the influence of aging on rat DCs. The aim of this thesis was to elucidate the influence of aging on the phenotypic and functional characteristics of conventional DCs (cDCs), and the possible significance of strain differences in the development of age-related changes. In addition, this thesis was aimed to investigate a putative correlation between age-related changes in cDC biology and rat susceptibility to induction of organ-specific autoimmune diseases. Freshly isolated cDCs and cDCs stimulated in vitro with lipopolysaccharide (LPS) were investigated. The cells were isolated from the spleens of 3- (young adult) and 26 monthold (aged) rats of Albino Oxford (AO) and Dark Agouti (DA) strain, which are relatively resistant (AO) and susceptible (DA) to the induction of Th1/Th17-mediated organ-specific autoimmune disease, as it is experimental autoimmune encephalomyelitis (EAE). In AO rats aging increased relative proportion of cells exhibiting CD4- phenotype within population of freshly isolated cDCs, while in young animals CD4+ cells represented the predominant subpopulation within cDCs. In DA rats aging did not affect cDC subset composition, so predominance of CD4- subset within cDCs in rats of both ages was found. Aging affected surface phenotype and endocytic capacity of freshly isolated cDCs in a strain-specific manner. Namely, the lower surface density of activation markers (CD80, CD86 and CD40) on cDCs from

Objective: Considering a causal role of androgens in thymic involution, age-related remodeling of peripheral T-cell compartments in the absence of testicular hormones was evaluated. Methods: Rats were orchidectomized (ORX) at the age of 1 month, and T-peripheral blood lymphocytes (PBLs) and splenocytes from young (75-day-old) and aged (24-month-old) rats were examined for differentiation/activation and immunoregulatory marker expression. Results: In ORX rats, following the initial rise, the counts of CD4+ and CD8+ PBLs diminished with aging. This reflected the decline in thymic export as shown by recent thymic emigrant (RTE) enumeration. Orchidectomy increased the count of both of the major T-splenocyte subsets in young rats, and they (differently from controls) remained stable with aging. The CD4+:CD8+ T-splenocyte ratio in ORX rats shifted towards CD4+ cells compared to age-matched controls. Although in the major T-cell subsets in the blood and spleen from aged ORX rats the numbers of RTEs were comparable to the corresponding values in age-matched controls, the numbers of mature naive and memory/activated cells substantially differed. Compared with age-matched controls, in aged ORX rats the numbers of CD4+ mature naive PBLs and splenocytes were reduced, whereas those of CD4+ memory/activated cells (predictive of early mortality) were increased. Additionally, in spleens from aged ORX rats, despite unaltered thymic export, CD4+CD25+FoxP3+ and natural killer T cell counts were greater than in age-matched controls. Conclusion: (i) Age-related decline in thymopoietic efficacy is not dependent on androgen presence, and (ii) androgens are involved in the maintenance of peripheral T-cell (particularly CD4+ cell) homeostasis during aging. (C) 2014 S. Karger AG, Basel

Aging is associated with the decline in immune response to infectious agents and tumors and increasing risk of autoimmunity, but the incidence of autoimmune diseases does not increase in the elderly. To elucidate the cellular and molecular mechanisms influencing clinical expression of autoimmunity in aged animals, the phenotypic and functional characteristics of mononuclear cells isolated from the spinal cords of 3-month-old (young) and 26-month-old (aged) Dark Agouti rats immunized to induce experimental autoimmune encephalomyelitis (EAE) - the model of multiple sclerosis, the most common autoimmune disease of the central nervous system, were examined. Aged rats were less susceptible to EAE induction, and the neurological and histological picture was milder in those rats which developed the clinically manifested disease. At the peak of the disease, several times fewer mononuclear cells and T lymphocytes were isolated from the spinal cords of aged rats compared with the young ones. The frequency of CD4+ cells among TCR alpha beta+ lymphocytes, as well as that of reactivated CD134(OX40)+ cells within its CD4+ T-lymphocyte subpopulation, was less in spinal cords of aged compared with young rats. Additionally, CD134 surface density on CD4+ lymphocytes was decreased in the spinal cord of aged rats. The changes in CD134 expression most likely reflected in part age-related intrinsic changes in CD4+ lymphocytes as the expression of this molecule was also impaired on in vitro stimulated naive CD4+ splenocytes from aged rats compared with young animals. In addition, greater frequency of CD8+ lymphocytes with regulatory phenotypes could also contribute to impaired CD4+ cell reactivation in aged rats. The increased apoptosis of CD4+ cells from aged rats was consistent with their impaired reactivation and it was accompanied by the greater frequency of CD4+CD11b+CD45(int/high) cells, which are supposed to be actively engaged in apoptotic cell phagocytosis and to have immunoregul

In humans, usual aging, differently from successful aging, is associated with deregulation of proinflammatory/anti-inflammatory cytokine balance. The corresponding data from rat studies are limited. Therefore, we examined (i) cytokine messenger RNA (mRNA) profile of fresh peritoneal cells from 6-(adult), 24-(old), and 31-month-old (long-lived) AO rats and (ii) proinflammatory (IL-1 beta and IL-6) and antiinflammatory (IL-10) cytokine, NO, and urea production in their LPS-stimulated cultures. Comparing with adult rats, cells from old ones expressed lower amount of TNF-alpha and IL-6 mRNAs, but greater amount of IL-1 beta mRNA. On the other hand, cells fromlong-lived rats exhibited a dramatic increase in IL-10 mRNA expression followed by diminished TNF-alpha and IL-6 mRNA expression, and comparable expression of IL-1 beta mRNA relative to adult rats. Consequently, IL-10/IL-1 beta mRNA ratio was greater in cells from long-lived rats than in adult and old rats. In LPS-stimulated peritoneal cell cultures (contained = 95 % macrophages) from old rats, concentration of common proinflammatory cytokines was higher than in those from adult rats. Comparing with adult and old rats, in LPS-stimulated macrophage cultures from long-lived rats, TNF-alpha and IL-6 concentrations were lower; IL-1 beta concentration was comparable or greater (in respect to adult rats), whereas that of IL-10 was strikingly higher. Consistently, in macrophage cultures from long-lived rats, NO (iNOS activity marker)/urea (arginase activity marker) ratio was less and not different from that in old and adult rats, respectively. The study suggests that macrophages from longlived rats, differently from those of old ones, have substantial ability to limit proinflammatory mediator production, which may contribute to their longevity.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis, a prototype of Th1/Th17-mediated organ-specific autoimmune disease. In the rat, susceptibility to development of these diseases is shown to be strain-and age-dependent. In adult rats of distinct strains, it correlates with splenic dendritic cell (DC) subset composition, which also exhibit age-related changes. The aim of this study was to examine influence of aging on: i) Albino Oxford (relatively resistant to EAE) and Dark Agouti (susceptible to EAE) rat development of EAE and ii) their splenic conventional (OX62+) DC population in respect to its subset composition and expression of mRNAs for proinflammatory and immunosuppressive cytokines. We used 3-month-old (young) and 26-month-old (aged) rats of AO and DA strain. The rats were immunized for EAE with rat spinal cord homogenate in complete Freund's adjuvant and clinical course of the disease was followed. Fresh OX62+DCs were examined for the expression of CD4 (using flow cytometry) and genes encoding cytokines influencing DC activation/maturation (TNF-alpha and IL-6) using RT-PCR. Additionally, in vitro lipopolysaccharide (LPS) activated/matured DCs were examined for the expression of genes encoding cytokines controlling Th1/Th17 cell polarization using RT-PCR. With aging, AO rats became more susceptible, whereas DA rats largely lose their susceptibility to the induction of EAE. In AO rats aging shifted CD4+: CD4-DC ratio towards CD4- cells, producing large amount of proinflammatory cytokines, whereas in DA rats CD4+: CD4-DC ratio remained stable with aging. In fresh DCs from rats of both the strains the expression of TNF-alpha mRNA increased with aging, whereas that of IL-6 mRNA decreased and increased in DCs from AO and DA rats, respectively. Following in vitro LPS stimulation OX62+ DCs from aged AO rats up-regulated the expression of mRNA for IL-23p19 (specific subunit of IL-23; crucial for sustained IL-17 production) and

Altered functions of macrophages with aging contribute to impairment of both innate and adaptive immunity in the elderly. The present study aimed to examine strain specificity of age-related changes in the phenotypic and functional characteristics of macrophages from DA and AO rats, which differ in average life span. Resident peritoneal macrophages from young (10-12 weeks old) and aged (98-104 weeks old) rats were tested for: (a) the surface expression of TLR4 and CD14; (b) the basal and LPS-induced production of TNF-alpha and IL-10; and (c) the basal and LPS-induced activity of iNOS and arginase, by measuring the levels of NO and urea, respectively, in the culture supernatants. Aging elevated TLR4 macrophage surface density in rats of both strains. Conversely, the age-related decrease in the surface density of CD14 co-receptor was detected only on macrophages from aged DA rats. Accordingly, with aging in DA rats, contrary to AO rats, upon LPS-stimulation both TNF-alpha and IL-10 levels decreased in culture supernatants. However, in rats of both strains TNF-alpha stimulation index (LPS-induced over basal production) remained stable with aging, but it was significantly greater in AO rats. Furthermore, with aging, IL-10 stimulation index decreased and increased in DA and AO rats, respectively. Age-related shift in urea stimulation index complied with the changes of IL-10 stimulation index during aging. In conclusion, the study suggests that the preserved ability of macrophages from aged AO rats to synthesize not only proinflammatory TNF-alpha, but also immunoregulatory IL-10 cytokine most likely contributes to their longer average life compared with DA rats.