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Starenje utiče na incidenciju većine inflamatornih autoimunskih bolesti, što se povezuje sa starenjem imunskog sistema, tzv. imunološkim starenjem (engl. immunosenescence). Budući da je pokazano da postoje značajne individualne razlike u procesu starenja ispitivan je značaj genetskih faktora (sojnih razlika) za starenjem uslovljene promene u incidenciji inflamatornih autoimunskih bolesti kod pacova. Korišćen je model eksperimentalnog autoimunskog encefalomijelitisa (EAE) indukovanog inokulacijom homogenata singene kičmene moždine u kompletnom Frojndovom adjuvansu uz ko-administraciju inaktivisane Bordetella pertussis, koji mimikrira ranu inflamatornu fazu multiple skleroze, najčešće inflamatorne autoimunske bolesti centralnog nervnog sistema, čija incidencija pokazuje jasnu uzrasnu zavisnost (smanjuje se starenjem). U istraživanja su uključene adultne i stare ženke pacova Dark Agouti (DA) soja, koji pokazuju izrazitu osetljivost na indukciju EAE-a u adultnom uzrastu i pacova Albino Oxford (AO) soja, koji su u adultnom uzrastu relativno rezistentni na indukciju EAE-a. Cilj je bio i da se definišu ćelijski i molekularni mehanizmi, koji bi mogli da budu odgovorni za moguće sojno zavisne starenjem uzrokovane razlike u osetljivosti na indukciju ove bolesti.Rezultati dobijeni u okviru ove doktorske disertacije pokazali su da starenje smanjuje incidenciju EAE-a i težinu bolesti kod DA pacova, a da kod AO pacova dovodi do razvoja bolesti blagog protrahovanog toka, koja pokazuje značajnu incidenciju (62,5%). Razlike u kliničkom ispoljavanju bolesti kod ova dva soja pacova su se mogle povezati sa sojnim razlikama u mehanizmima koji su uključeni u kontrolu intenziteta primarnog (auto)imunskog odgovora u drenirajućim limfnim čvorovima, ali i migracije aktivisanih neuroantigen-specifičnih T pomoćničkih 17 (engl. T helper 17, Th17) ćelija, posebno zadržavanja u slezini (ekspresija CD44s molekula na površini Th ćelija i zastupljenost CD4+CD25+Foxp3+ regulatornih T-limfocita), kao, Aging affects the incidence of most inflammatory autoimmune diseases, which is associated with the aging of the immune system, the so-called immunosenescence. Since it has been shown that there are significant individual differences in the aging process, the significance of genetic factors (strain differences) for age-related changes in the incidence of inflammatory autoimmune diseases in rats was investigated. The model of experimental autoimmune encephalomyelitis (EAE) induced by inoculation of the syngenic spinal cord homogenate in a complete Freund's adjuvant with co-administration of inactivated Bordetella pertussis was used. This model mimics the early inflammatory phase of multiple sclerosis, the most common inflammatory autoimmune disease of the central nervous system, whose incidence shows a clear age dependency (decreases with aging). Young adult and aged female rats of Dark Agouti (DA) strain, which show high susceptibility to EAE induction at an young adult age and rats of Albino Oxford (AO) strain, which are relatively resistant to EAE induction at an young adult age were used in this research. The aim of this doctoral dissertation was to define cellular and molecular mechanisms that could be responsible for presumed strain specificities in age-related changes in the disease induction and development.The results obtained in this doctoral dissertation showed that aging decreases the incidence of EAE and the disease severity in DA rats, while in AO rats it leads to development of mild clinical disease of prolonged duration, exhibiting a significant incidence (62.5%). Differences in the clinical manifestations of the disease in these two rat strains could be associated with strain differences in the mechanisms involved in controlling the intensity of the primary (auto) immune response in the draining lymph nodes, but also the migration of activated neuroantigen-specific T helper 17 (Th17) cells, in particular their retention in the spleen (Th cell surface

Natural killer (NK) cells, influencing dendritic cell (DC)-mediated CD4+ lymphocyte priming in draining lymph nodes (dLNs) and controlling spinal cord (SC) infiltration with encephalitogenic CD4+T lymphocytes, modulate EAE (multiple sclerosis model). This study examined their putative contribution to age-related differences in EAE development in Dark Agouti (DA) (exhibiting age-related decrease in EAE susceptibility) and Albino Oxford (AO) (becoming susceptible to EAE with aging) rats. Aging increased NK cell number in dLNs from rats of both strains. In AO rats, but not in DA ones, it also increased the numbers of IFN-γ-producing NK cells (important for DC activation) and activated/matured DCs, thereby increasing activated/matured DC/conventional Foxp3-CD4+ cell ratio and activated CD25+Foxp3-CD4+ cell number. Aging in DA rats diminished activated/matured DC/conventional Foxp3-CD4+ cell ratio and activated Foxp3-CD4+ cell number. However, MBP-stimulated CD4+ cell proliferation did not differ in dLN cell cultures from young and aged AO rats (as more favorable activated/matured DC/Foxp3-CD4+ cell ratio was abrogated by lower intrinsic CD4+ cell proliferative capacity and a greater regulatory CD25+Foxp3+CD4+ lymphocyte frequency), but was lower in those from aged compared with young DA rats. At SC level, aging shifted Foxp3-CD4+/cytotoxic CX3CR1+ NK cell ratio towards the former in AO rats, so it was less favorable in aged AO rats exhibiting prolonged neurological deficit compared with their DA counterparts. The study showed strain and age differences in number of IFN-γ-producing NK cells in EAE rat dLNs, and suggested that their pathogenetic relevance depends on frequency and/or activity of other cells involved in CD4+ T cell (auto)immune response.

Starenje utiče na incidenciju većine inflamatornih autoimunskih bolesti, što se povezuje sa starenjem imunskog sistema, tzv. imunološkim starenjem (engl. immunosenescence). Budući da je pokazano da postoje značajne individualne razlike u procesu starenja ispitivan je značaj genetskih faktora (sojnih razlika) za starenjem uslovljene promene u incidenciji inflamatornih autoimunskih bolesti kod pacova. Korišćen je model eksperimentalnog autoimunskog encefalomijelitisa (EAE) indukovanog inokulacijom homogenata singene kičmene moždine u kompletnom Frojndovom adjuvansu uz ko-administraciju inaktivisane Bordetella pertussis, koji mimikrira ranu inflamatornu fazu multiple skleroze, najčešće inflamatorne autoimunske bolesti centralnog nervnog sistema, čija incidencija pokazuje jasnu uzrasnu zavisnost (smanjuje se starenjem). U istraživanja su uključene adultne i stare ženke pacova Dark Agouti (DA) soja, koji pokazuju izrazitu osetljivost na indukciju EAE-a u adultnom uzrastu i pacova Albino Oxford (AO) soja, koji su u adultnom uzrastu relativno rezistentni na indukciju EAE-a. Cilj je bio i da se definišu ćelijski i molekularni mehanizmi, koji bi mogli da budu odgovorni za moguće sojno zavisne starenjem uzrokovane razlike u osetljivosti na indukciju ove bolesti. Rezultati dobijeni u okviru ove doktorske disertacije pokazali su da starenje smanjuje incidenciju EAE-a i težinu bolesti kod DA pacova, a da kod AO pacova dovodi do razvoja bolesti blagog protrahovanog toka, koja pokazuje značajnu incidenciju (62,5%). Razlike u kliničkom ispoljavanju bolesti kod ova dva soja pacova su se mogle povezati sa sojnim razlikama u mehanizmima koji su uključeni u kontrolu intenziteta primarnog (auto)imunskog odgovora u drenirajućim limfnim čvorovima, ali i migracije aktivisanih neuroantigen-specifičnih T pomoćničkih 17 (engl. T helper 17, Th17) ćelija, posebno zadržavanja u slezini (ekspresija CD44s molekula na površini Th ćelija i zastupljenost CD4+CD25+Foxp3+ regulatornih T-limfocita), kao, Aging affects the incidence of most inflammatory autoimmune diseases, which is associated with the aging of the immune system, the so-called immunosenescence. Since it has been shown that there are significant individual differences in the aging process, the significance of genetic factors (strain differences) for age-related changes in the incidence of inflammatory autoimmune diseases in rats was investigated. The model of experimental autoimmune encephalomyelitis (EAE) induced by inoculation of the syngenic spinal cord homogenate in a complete Freund's adjuvant with co-administration of inactivated Bordetella pertussis was used. This model mimics the early inflammatory phase of multiple sclerosis, the most common inflammatory autoimmune disease of the central nervous system, whose incidence shows a clear age dependency (decreases with aging). Young adult and aged female rats of Dark Agouti (DA) strain, which show high susceptibility to EAE induction at an young adult age and rats of Albino Oxford (AO) strain, which are relatively resistant to EAE induction at an young adult age were used in this research. The aim of this doctoral dissertation was to define cellular and molecular mechanisms that could be responsible for presumed strain specificities in age-related changes in the disease induction and development. The results obtained in this doctoral dissertation showed that aging decreases the incidence of EAE and the disease severity in DA rats, while in AO rats it leads to development of mild clinical disease of prolonged duration, exhibiting a significant incidence (62.5%). Differences in the clinical manifestations of the disease in these two rat strains could be associated with strain differences in the mechanisms involved in controlling the intensity of the primary (auto) immune response in the draining lymph nodes, but also the migration of activated neuroantigen-specific T helper 17 (Th17) cells, in particular their retention in the spleen (Th cell surface

Males exhibit stronger sympathetic nervous system (SNS) activity, but weaker primary CD4 + T-cell (auto) immune responses. To test the role of catecholamines, major end-point SNS mediators, in this dimorphism, influence of propranolol (beta-adrenoceptor blocker) on mitogen/neuroantigen-stimulated CD4 + T cells from female and male EAE rat draining lymph node (dLN) cell cultures was examined. Male rat dLNs exhibited higher noradrenaline concentration and frequency of beta(2)-adrenoceptor-expressing CD4 + T lymphocytes and antigen presenting cells. Propranolol, irrespective of exogenous noradrenaline presence, more prominently augmented IL-2 production and proliferation of CD4 + lymphocytes in male than female rat dLN cell cultures. In neuroantigen-stimulated dLN cells of both sexes propranolol increased IL-1 beta and IL-23/p19 expression and IL-17 + CD4 + cell frequency, but enhanced IL-17 production only in male rat CD4 + lymphocytes, thereby abrogating sexual dimorphism in IL-17 concentration observed in propranolol-free cultures. Thus, beta-adrenoceptor-mediated signalling may contribute to sex bias in rat IL-17-producing cell secretory capacity.

Pharmacological blockade of alpha(1)-adrenoceptor is shown to influence development of experimental autoimmune encephalomyelitis (EAE), an IL-17-producing CD4+TCR+ (Th17) cell-mediated disease mimicking multiple sclerosis. Considering significance of CD4+ cell priming for the clinical outcome of EAE, the study examined alpha(1)-adrenoceptor-mediated influence of catecholamines, particularly those derived from draining lymph node (dLN) cells (as catecholamine supply from nerve fibers decreases with the initiation of autoimmune diseases) for CD4+ cell priming. The results confirmed diminishing effect of immunization on nerve fiber-derived noradrenaline supply and showed that antigen presenting and CD4+ cells synthesize catecholamines, while antigen presenting cells and only CD4+CD25+Foxp3+ regulatory T cells (Tregs) express alpha(1)-adrenoceptor. The analysis of influence of alpha(1)-adrenoceptor antagonist prazosin on the myelin basic protein (MBP)-stimulated CD4+ lymphocytes in dLN cell culture showed their diminished proliferation in the presence of prazosin. This was consistent with prazosin enhancing effect on Treg frequency and their Foxp3 expression in these cultures. The latter was associated with upregulation of TGF-beta expression. Additionally, prazosin decreased antigen presenting cell activation and affected their cytokine profile by diminishing the frequency of cells that produce Th17 polarizing cytokines (IL-1 beta and IL-23) and increasing that of IL-10-producing cells. Consistently, the frequency of all IL-17A+ cells and those co-expressing GM-CSF within CD4+ lymphocytes was decreased in prazosin-supplemented MBP-stimulated dLN cell cultures. Collectively, the results indicated that dLN cell-derived catecholamines may influence EAE development by modulating interactions between distinct subtypes of CD4+ T cells and antigen presenting cells through alpha(1)-adrenoceptor and consequently CD4+ T cell priming.

An accumulating body of evidence suggests that development of autoimmune pathologies leads to thymic dysfunction and changes in peripheral T-cell compartment, which, in turn, perpetuate their pathogenesis. To test this hypothesis, thymocyte differentiation/maturation in rats susceptible (Dark Agouti, DA) and relatively resistant (Albino Oxford, AO) to experimental autoimmune encephalomyelitis (EAE) induction was examined. Irrespective of strain, immunization for EAE (i) increased the circulating levels of IL-6, a cytokine causally linked with thymic atrophy, and (ii) led to thymic atrophy reflecting partly enhanced thymocyte apoptosis associated with downregulated thymic IL-7 expression. Additionally, immunization diminished the expression of Thy-1, a negative regulator of TCR alpha beta-mediated signaling and activation thresholds, on CD4+CD8+ TCR alpha beta(lo/hi) thymocytes undergoing selection and thereby impaired thymocyte selection/survival. This diminished the generation of mature CD4+ and CD8+ single positive TCR alpha beta(hi) thymocytes and, consequently, CD4+ and CD8+ recent thymic emigrants. In immunized rats, thymic differentiation of natural regulatory CD4+Foxp3+CD25+ T cells (nTregs) was particularly affected reflecting a diminished expression of IL-7, IL-2 and IL-15. The decline in the overall thymic T-cell output and nTreg generation was more pronounced in DA than AO rats. Additionally, differently from immunized AO rats, in DA ones the frequency of CD28- cells secreting cytolytic enzymes within peripheral blood CD4+ T lymphocytes increased, as a consequence of thymic atrophy-related replicative stress (mirrored in CD4+ cell memory pool expansion and p16(INK4a) accumulation). The higher circulating level of TNF-alpha in DA compared with AO rats could also contribute to this difference. Consistently, higher frequency of cytolytic CD4+ granzyme B+ cells (associated with greater tissue damage) was found in spinal cord of immunized DA rats compared with

The thymus is sexually differentiated organ providing microenvironment for T-cell precursor differentiation/maturation in the major histocompatibility complex-restricted self-tolerant T cells. With increasing age, the thymus undergoes involution leading to the decline in efficacy of thymopoiesis. Noradrenaline from thymic nerve fibers and "(nor) adrenergic" cells is involved in the regulation of thymopoiesis. In rodents, noradrenaline concentration in thymus and adrenoceptor (AR) expression on thymic cells depend on sex and age. These differences are suggested to be implicated in the development of sexual diergism and the age-related decline in thymopoiesis. The programming of both thymic sexual differentiation and its involution occurs during the critical early perinatal period and may be reprogrammed during peripubertal development. The thymic (re) programming is critically dependent on circulating levels of gonadal steroids. Although the underlying molecular mechanisms have not yet been elucidated fully, it is assumed that the gonadal steroid action during the critical perinatal/peripubertal developmental periods leads to long-lasting changes in the efficacy of thymopoiesis partly through (re) programming of "(nor) adrenergic" cell networks and AR expression on thymic cells.

Introduction: It has been suggested that: i) noradrenaline synthesis in ,,adrenergic“ immune cells changes during development of EAE and multiple sclerosis and ii) noradrenaline influences EAE development through a1−adrenoceptor. To elucidate mechanisms standing behind this phenomenon, a1−adrenoceptor−mediated influence of draining lymph node (dLN) cell−derived noradrenaline on CD4+ T−cell response in dLNs from Dark Agouti rats of both sexes immunized for EAE was examined. Methods: Cells recovered from dLNs on 7th day post−immunization were examined for noradrenaline synthesis/content and a1B−adrenoceptor expression using HPLC and/or flow cytometry. Additionally, effects of prazosin (a1- AR blocker) on CD4+ T−cell proliferation, the frequency of IL−17+ CD4+ T−cells and regulatory (Foxp3+CD25+) CD4+ T−cells (Tregs), activational/maturational molecule expression on antigen presenting cells (APCs) and their cytokine profile in dLN cell culture were examined using flow cytometry and/or qRT−PCR/ELISA. Results: Irrespective of sex, conventional CD4+ T−cells and Tregs, and APCs from rat dLNs synthesized noradrenaline, while only Tregs and APCs expressed a1B-adrenoceptor. In myelin basic protein−stimulated dLN cell cultures from rats of both sexes prazosin increased Treg frequency and Foxp3 expression, but diminished co−stimulatory CD80 and CD86 molecule expression on APCs, thereby reducing CD4+ cell proliferation.

To understand strain-specificities of immune system in aged rats and their immunopathological implications, CD4+T lymphocyte-mediated neuroinflammation in experimental autoimmune encephalomyelitis (EAE) was studied in two strains. Upon immunization for EAE, 22-24-month-old Albino Oxford (AO) rats developed milder neurological deficit of prolonged duration compared with their Dark Agouti (DA) counterparts. Consistently, they exhibited: (i) diminished neuroantigen-specific CD4+T lymphocyte generation in draining lymph nodes (reflecting lower density of high-affinity IL-2 receptor complex on their surface and higher CD4+FoxP3+CD25+regulatory cell frequency); (ii) less favorable spinal cord expression of CXCL12 and CCL2, and consequently diminished infiltration of neuroantigen-specific CD4+T lymphocytes, including highly pathogenic IL-17+IFN-gamma+ones, and inflammatory monocytes into the spinal cord and (iii) subsequently impaired CD4+T lymphocyte reactivation/survival and differentiation into highly pathogenic IL-17+cells (reflecting downregulated expression of IL-1 beta, IL-6 and IL-23/p19). On the other hand, when the neurological deficit reached maximum/plateau, in AO rat spinal cord was found lower CD4+FoxP3+CD25+ cell frequency followed by higher frequency of IL-10-producing CD8+T cells, which most likely also belong to regulatory T lymphocytes. Thus, the altered relation between regulatory T cell and effector CD4+T cell subsets was linked with persistence of mild neuroinflammation in AO rat EAE model. (C) 2017 Elsevier B.V. All rights reserved.

The study was undertaken considering that: i) androgens affect beta(2)-adrenoceptor (AR)-mediated catecholamine (CA) action in many tissues; and ii) peripubertal changes in both circulating androgen and thymic CA levels are implicated in rat thymic involution. Its aims were to: i) explore putative effects of the late prepubertal orchidectomy on thymic CA:beta(2)-AR complex in young adult rats, and ii) delineate the direct effects of testicular hormone withdrawal on the CA:beta(2)-AR complex from those elicited secondarily through altered influence of this complex components on each other's availability. Upon showing that prepubertal orchidectomy augmented the efficacy of thymopoiesis through increasing the thymocyte surface density of Thy-1, whose expression is negatively regulated by beta(2)-AR-mediated signaling, we examined the effects of orchidectomy and 14-day-long propranolol (PROP) treatment in orchidectomized (ORX) and sham-ORX rats on thymic norepinephrine (NE) concentration and metabolism and beta(2)-AR expression. Orchidectomy, despite an increase in the average NE amount per thymocyte and total thymocyte NE content diminished thymic NE concentration. This decrease reflected the diminished density of CA-synthesizing nerve fibers, CD68 + macrophages, cortical (aminopeptidase A+), and medullary (UEA-1+) thymic epithelial cells (TECs) and their CA content (probably due to lessened TH expression accompanied by increased MAO-A expression). Moreover, orchidectomy decreased the surface beta(2)-AR expression on thymocytes, CD68+ macrophages and OX-62+ dendritic cells, but increased its expression on the TECs. In sham-ORX rats, PROP reduced thymic NE concentration by diminishing TH expression in the thymic cells. Additionally, PROP in thymocytes and thymic stromal cells diminished and enhanced the beta(2)-AR mRNA expression, respectively. However, in ORX rats PROP did not significantly affect CA(NE):beta(2)-AR complex components. This indicated that prepubertal or

Glucocorticoids have been shown to modulate the expression of noradrenaline metabolizing enzymes and beta(2)- and alpha(1B)-adrenoceptors in a tissue- and cell- specific manner. In the thymus, apart from extensive sympathetic innervation, a regulatory network has been identified that encompasses catecholamine-containing non-lymphoid and lymphoid cells. We examined a putative role of adrenal- and thymus-derived glucocorticoids in modulation of rat thymic noradrenaline levels and adrenoceptor expression. Seven days postadrenalectomy, the thymic levels of mRNAs encoding tyrosine hydroxylase, dopamine beta-hydroxylase, monoamine oxidase-A and, consequently, noradrenaline were decreased. Catecholamine content was diminished in autofluorescent nerve fibres (judging by the intensity of fluorescence) and thymocytes (considering HPLC measurements of noradrenaline and the frequency of tyrosine hydroxylase-positive cells), while it remained unaltered in non-lymphoid autofluorescent cells. In addition, adrenalectomy diminished the thymocyte expression of beta(2)- and alpha(1B)-adrenoceptors at both mRNA and protein levels. Administration of ketoconazole (an inhibitor of glucocorticoid synthesis/action; 25 mg kg(-1) day(-1), s.c.) to glucocorticoid-deprived rats increased the thymic levels of tyrosine hydroxylase, dopamine beta-hydroxylase and, consequently, noradrenaline. The increased intensity of the autofluorescent cell fluorescence in ketoconazole-treated rats indicated an increase in their catecholamine content, and suggested differential glucocorticoid-mediated regulation of catecholamines in thymic lymphoid and non-lymphoid cells. In addition, ketoconazole increased the thymocyte expression of alpha(1B)-adrenoceptors. Thus, this study indicates that in the thymus, as in some other tissues, glucocorticoids not only act in concert with cateholamines, but they may modulate catecholamine action by tuning thymic catecholamine metabolism and adrenoceptor expression in a cell-s

There is evidence that the major mediators of stress, i.e., catecholamines and glucocorticoids, play an important role in modulating thymopoiesis and consequently immune responses. Furthermore, there are data suggesting that glucocorticoids influence catecholamine action. Therefore, to assess the putative relevance of glucocorticoid-catecholamine interplay in the modulation of thymopoiesis we analyzed thymocyte differentiation/maturation in non-adrenalectomized and andrenalectomized rats subjected to treatment with propranolol (0.4 mg.100 g body weight(-1).day(-1)) for 4 days. The effects of beta-adrenoceptor blockade on thymopoiesis in non-adrenalectomized rats differed not only quantitatively but also qualitatively from those in adrenalectomized rats. In adrenalectomized rats, besides a more efficient thymopoiesis [judged by a more pronounced increase in the relative proportion of the most mature single-positive TCR alpha beta(high) thymocytes as revealed by two-way ANOVA; for CD4(+)CD8(-)F (1,20) = 10.92, P lt 0.01; for CD4(-)CD8(+)F (1,20) = 7.47, P lt 0.05], a skewed thymocyte maturation towards the CD4(-)CD8(+) phenotype, and consequently a diminished CD4(+)CD8(-)/CD4(-)CD8(+) mature TCR alpha beta(high) thymocyte ratio (3.41 +/- 0.21 in non-adrenalectomized rats vs 2.90 +/- 0.31 in adrenalectomized rats, P lt 0.05) were found. Therefore, we assumed that catecholaminergic modulation of thymopoiesis exhibits a substantial degree of glucocorticoid-dependent plasticity. Given that glucocorticoids, apart from catecholamine synthesis, influence adrenoceptor expression, we also hypothesized that the lack of adrenal glucocorticoids affected not only beta-adrenoceptor- but also alpha-adrenoceptor-mediated modulation of thymopoiesis.

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.

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.

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.

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.

The study was aimed to define the putative role of ovarian hormones in shaping thymocyte apoptosis and proliferation during thymic involution. Thymocytes from young adult and middle-aged rats ovariectomized (Ox) before puberty were examined for apoptosis and proliferation. Apoptosis and proliferation were measured in fresh thymocyte suspensions and in their 18-hour cultures, and fresh thymocyte suspensions, respectively. The thymocyte population and the major thymocyte subsets were analyzed following triple staining using anti-CD4 and anti-CD8 monoclonal antibodies and 7-AAD to label apoptotic or proliferating cells. The frequency of apoptotic cells was lower in thymocyte suspensions and cultures from Ox rats of both ages. This reflected in a diminished frequency of apoptotic cells amongst CD4+CD8+ double positive (DP) and CD4+CD8- single positive (SP), and DP cells in young and middle-aged Ox rats, respectively. Additionally, in thymocyte cultures from Ox rats the frequency of apoptotic cells amongst CD4+CD8- and CD4-CD8+ SP cells decreased with age, but increased within DP and CD4-CD8- double negative (DN) subsets, reaching in the former subset from middle-aged Ox rats higher values than in age-matched controls. The frequency of proliferating cells was also lower in Ox rats than in controls. This reflected the lower frequency of cycling cells amongst CD4+CD8- SP and CD4-CD8+ SP thymocytes in young rats, and DP and CD4-CD8+ SP thymocytes in middle-aged rats. Besides, in both SP and DP thymocyte subsets from Ox rats the frequency of proliferating cells declined with age. In conclusion, thymocyte apoptosis and proliferation exhibit ovarian hormone-dependent thymocyte subset specific alterations during thymic involution., Cilj istraživanja je bio da se definiše značaj hormona ovarijuma za razvoj promena u apoptozi i proliferaciji timocita tokom involucije timusa. U tom cilju apoptoza i proliferacija timocita ispitivana je kod prepubertetno ovariektomisanih (Ox) mladih (uzrasta 2 meseca) i sredovečnih pacova (uzrasta 11 meseci). Apoptoza je određivana u suspenziji sveže izolovanih timocita i nakon njihove 18- časovne kultivacije, a proliferacija u suspenziji sveže izolovanih timocita. Procenat apoptotičnih i proliferišućih ćelija je određivan u celokupnoj populaciji timocita, i unutar glavnih subpopulacija ovih ćelija, koje su razdvojene na osnovu ekspresije CD4/CD8 molekula, metodom protočne fluorocitometrije, korišćenjem 7-aminoaktinomicina D (7-AAD). Procenat ćelija u apoptozi je bio značajno manji u suspenzijama svežih timocita koji su izolovani iz Ox životinja i u njihovim kulturama nego u onim izolovanim iz kontrolnih životinja. Ovaj nalaz je odražavao smanjenu učestalost ćelija u apoptozi u CD4+CD8+ dvostruko pozitivnoj (DP) i CD4+CD8- jednostruko pozitivnoj (JP) subpopulaciji timocita kod mladih i u DP subpopulaciji kod sredovečnih Ox pacova. U kulturama timocita koji su izolovani iz sredovečnih Ox pacova uočeno je smanjenje učestalosti ćelija u apoptozi unutar subpopulacija CD4+CD8- i CD4-CD8+ JP timocita, a povećanje unutar DP i CD4-CD8- dvostruko negativne (DN) subpopulacije ovih ćelija. Učestalost proliferišućih ćelija je takođe bila niža u suspenzijama timocita izolovanih iz Ox pacova nego u onim izolovanim iz kontrolnih životinja. Ovo je odražavalo smanjenu proliferaciju CD4+CD8- i CD4-CD8+ JP timocita kod mladih, a DP i CD4-CD8+ JP timocita kod sredovečnih pacova. Procentualna zastupljenost proliferišućih ćelija u subpopulacijama JP i DP timocita je bila veća kod mladih nego kod sredovečnih Ox pacova. U zaključku, tokom involucije timusa dolazi do promena u apoptozi i proliferaciji timocita koje su specifične za pojedine subpopulacije timocita i zavisne od prisustva hor

The study was undertaken to assess the effects of ovarian hormone withdrawal in prepubertal age on thymopoiesis in 2- (young) and 11-month-old (middle-aged) rats. In ovariectomized (Ox) rats, irrespective of age, thymic weight and cellularity were greater than in age-matched controls, but the values of both parameters exhibited the age-related decline. In addition, although thymopoietic efficiency was increased in both groups of Ox rats when compared with age-matched controls, thymopoiesis exhibited the age-related decline mirrored in the lower numbers of both CD4+ and CD8+ recent thymic emigrants in peripheral blood. This reflected the prethymic changes affecting bone marrow progenitor generation/entry and the thymic alterations encompassing the impaired progenitor progression through early pre-T-cell receptor developmental stages (defined by CD45RC/CD2 expression) and, possibly, a more pronounced decrease in the proliferation of the most mature thymocytes. Apart from the changes at thymocyte level, in Ox rats the age-related alterations in thymic stroma (substantiated in a prominent loss of thymic epithelial cells) were registered. Ovariectomy-induced changes in thymic lymphoid and epithelial component, most probably, influenced each other leading to the increase in thymic expression of interleukin-6 and interleukin-7 mRNAs along with time after ovariectomy. Collectively, the study showed that the withdrawal of ovarian hormones in prepubertal age increases the efficiency of thymopoiesis in young adult rats, but does not prevent decline in thymopoiesis occurring with age.