Search

Elderly patients with rheumatoid arthritis, psoriasis and psoriatic arthritis encompass those with elderly-onset disease, over 60 years of age, but also those with earlier disease onset who entered old age. Considering the age-related changes of the immune system, possible frailty, susceptibility to infection and concomitant comorbidity that implies multiple medicines, the treatment of these diseases in elderly patients can be challenging. Interleukin inhibitors have been shown to be an efficient and safe treatment for these diseases. However, elderly patients with these diseases were often included in the pivotal clinical trials for interleukin inhibitors in numbers insufficient to determine whether they responded differently from younger subjects. The aim of this paper was to review the findings on the efficacy and safety of interleukin inhibitor treatment in elderly patients with rheumatoid arthritis, psoriasis, and psoriatic arthritis.The findings suggest that, for all the interleukin inhibitors reviewed herein, used in elderly patients with rheumatoid arthritis, or with psoriasis and psoriatic arthritis, the efficacy was comparable to younger patients. Furthermore, the incidence of reported adverse events was similar in these two age groups. Severe adverse events, which were related to sarilumab treatment for rheumatoid arthritis and secukinumab treatment for psoriasis, were higher in elderly patients.The reviewed findings suggest that the interleukin inhibitors approved and currently in use in clinical practice for the treatment of rheumatoid arthritis, psoriasis, and psoriatic arthritis can be considered a safe and efficient option for these diseases in elderly patients. Stariji pacijenti koji boluju od reumatoidnog artritisa, psorijaze ili psorijaznog artritisa uključuju one kod kojih je bolest imala kasni početak, nakon 60. godine starosti, ali takođe i one pacijente kod kojih je bolest počela ranije, a koji su ušli u staro doba. Imajući u vidu starenjem uslovljene promene imunskog sistema, moguću slabost starijih, podložnost infekcijama, prateći komorbiditet, koji uključuje i uzimanje više lekova, terapija ovih bolesti kod starijih pacijenata može predstavljati izazov. Inhibitori interleukina su se pokazali kao efikasna i bezbedna terapija ovih poremećaja. Međutim, stariji pacijenti sa ovim bolestima su često bili nedovoljno zastupljeni u ključnim kliničkim ispitivanjima inhibitora interleukina da bi se moglo sa sigurnošću utvrditi da postoji razlika u terapijskom odgovoru kod ovih pacijenata u odnosu na isti kod mlađih pacijenata. Cilj ovog rada bio je da prikaže nalaze od značaja za bezbednost i efikasnost terapije inhibitorima interleukina kod starijih pacijenata sa reumatoidnim artritisom, psorijazom ili psorijaznim artritisom. Nalazi ukazuju da je efikasnost inhibitora interleukina, prikazanih u ovom radu, uporediva kod starijih i mlađih pacijenata. Osim toga, incidencija neželjenih događaja se nije razlikovala između ove dve starosne grupe. Veća incidencija teških neželjenih događaja kod starijih pacijenata u odnosu na mlađe bila je zabeležena u terapiji reumatoidnog artritisa sarilumabom i psorijaze secukinumabom. Terapija reumatoidnog artritisa, psorijaze i psorijaznog artritisa inhibitorima interleukina može se smatrati efikasnom i bezbednom u populaciji starih pacijenata.

Monocytes' plasticity has an important role in the development of rheumatoid arthritis (RA), an autoimmune disease exhibiting greater prevalence in women. Contribution of this phenomenon to sex bias in RA severity was investigated in rat collagen-induced arthritis (CIA) model of RA. The greater severity of CIA in females (exhibiting signs of bone resorption) was accompanied by the higher blood level of advanced oxidation protein products and a more pro-oxidant profile. Consistently, in females, the greater density of giant multinuclear cells (monocytes/macrophages and osteoclasts) in inflamed joint tissue was found. This correlated with the higher frequencies of CCR2- and CX3CR1- expressing cells (precursors of inflammatory monocytes/macrophages and osteoclasts) among CD11b+ splenocytes. This in conjunction with the enhanced migratory capacity of CD11b+ monocytic cells in females compared with males could be linked with the higher frequencies of CCR2+CX3CR1-CD43(low)CD11b+ and CCR2-CX3CR1+CD43(hi)CD11b+ cells (corresponding to "classical" and "non-classical" monocytes, respectively) and the greater density of CD68+ cells (monocytes/macrophages and osteoclast precursors/osteoclasts) in blood and inflamed paws from female rats, respectively. Consistently, the higher levels of GM-CSF, TNF-alpha and IL-6, IL-1 beta (driving Th17 cell differentiation), and IL-17 followed by the lower level of IL-10 were measured in inflamed paw cultures from female compared with male rats. To the greater IL-17 production (associated with enhanced monocyte immigration and differentiation into osteoclasts) most likely contributed augmented Th17 cell generation in the lymph nodes draining arthritic joints from female compared with male rats. Overall, the study suggests the sex-specific contribution of monocytic lineage cells to CIA, and possibly RA development.

The study examined germinal centre (GC) reaction in lymph nodes draining inflamed joints and adjacent tissues (dLNs) in male and female Dark Agouti rat collagen type II (CII)-induced arthritis (CIA) model of rheumatoid arthritis. Female rats exhibiting the greater susceptibility to CIA mounted stronger serum CII-specific IgG response than their male counterparts. This correlated with the higher frequency of GC B cells in female compared with male dLNs. Consistently, the frequency of activated/proliferating Ki-67+ cells among dLN B cells was higher in females than in males. This correlated with the shift in dLN T follicular regulatory (Tfr)/T follicular helper (Tfh) cell ratio towards Tfh cells in females, and greater densities of CD40L and CD40 on their dLN T and B cells, respectively. The higher Tfh cell frequency in females was consistent with the greater dLN expression of mRNA for IL-21/27, the key cytokines involved in Tfh cell generation and their help to B cells. Additionally, in CII-stimulated female rat dLN cell cultures IFN-γ/IL-4 production ratio was shifted towards IFN-γ. Consistently, the serum IgG2a(b)/IgG1 CII-specific antibody ratio was shifted towards an IgG2a(b) response in females. Thus, targeting T-/B-cell interactions should be considered in putative further sex-based translational pharmacology research.

The study explored influence of biological sex on development of humoral immune response to seasonal trivalent whole inactivated virus (WIV) and split virus (SV) influenza vaccines in outbred Swiss mouse model. To this end, mice of both sexes were immunized with WIV (WIV mice) and SV vaccines (SV mice) and examined for specific antibody response. Irrespective of sex, total IgG and neutralizing antibody responses to distinct virus strains were weaker in SV than in WIV mice. In WIV mice of both sexes, irrespective of strain specificity, IgG isotype response was dominated by IgG2a antibodies, while in SV mice nearly equal representation of IgG2a and IgG1 antibodies was found. The analyses of sex differences showed higher titers of H1N1-specific and both H1N1- and H3N2-specific total IgG and neutralizing antibodies in female WIV and SV mice, respectively. Additionally, sexual dimorphism in IgG subclass profile depended on vaccine type. Specifically, compared with males, in females WIV shifted IgG2a/IgG1 antibody ratio towards IgG2a isotype on the account of weaker IgG1 response, whereas in SV mice, irrespective of virus strain, IgG2a and IgG1 isotypes were equally represented in both sexes. These findings indicate the vaccine type-dependent sex bias in antibody response to inactivated influenza vaccines.

To close the gap in our knowledge of sex influence on age-related changes in inflammation-oxidation state in spinal cord (SC) relevant to inflammation/oxidative-stress associated neuropathologies, 2-3 month-old (young) and 18-20 month-old (old) rats, exhibiting increased level of IL-6, a commonly used marker of inflamm-aging, were examined for inflammatory/redox status, and the underlying regulatory networks' molecules expression. With age, rat SC microglia became sensitized ("primed"), while SC tissue shifted towards mild inflammatory state, with increased levels of proinflammatory IL-1 beta (key marker of microglial systemic inflammation-induced neurotoxicity), which was more prominent in males. This, most likely, reflected age- and sex-related impairment in the expression of CX3CR1, the receptor for fractalkine (CX3CL1), the soluble factor which regulates microglial activation and diminishes production of IL-1 beta (central for fractalkine neuroprotection). Considering that (i) age-related changes in SC IL-1 beta expression were not followed by complementary changes in SC IL-6 expression, and (ii) the reversal in the direction of the sex bias in circulating IL-6 level and SC IL-1 beta expression, it seems obvious that there are tissue-specific differences in the proinflammatory cytokine profile. Additionally, old male rat SC exhibited greater oxidative damage than female, reflecting, most likely, their lower capacity to maintain the pro-oxidant-antioxidant balance. In conclusion, these findings, apart from highlighting the significance of sex for age-associated changes in SC inflammation-oxidation, may be relevant for understating sex differences in human inflammation/oxidative-stress related SC diseases, and consequently, for optimizing their prevention/therapy.

Considering variability in vaccine responsiveness across human populations, in respect to magnitude and quality, and importance of vaccines in the elderly, the influence of recipient genetic background on the kinetics of age-related changes in the serum IgG antibody responses to seasonal trivalent inactivated split-virus influenza bulk (TIV) was studied in BALB/c and C57BL/6 mice showing quantitative and qualitative differences in this responses in young adult ages. With ageing the total serum IgG response to influenza viruses declined, in a strain-specific manner, so the strain disparity observed in young adult mice (the greater magnitude of IgG response in BALB/c mice) disappeared in aged mice. However, the sexual dimorphisms in this response (more prominent in females of both strains) remained in aged ones. The strain-specific differences in age-related decline in the magnitude of IgG response to TIV correlated with the number of germinal centre (GC) B splenocytes. The agerelated decline in GC B cell number was consistent with the decrease in the proliferation of B cells and CD4 + cells in splenocyte cultures upon restimulation with TIV. Additionally, the age-related decrease in the magnitude of IgG response correlated with the increase in follicular T regulatory (fTreg)/follicular T helper (fTh) and fTreg/GC B splenocyte ratios (reflecting decrease in fTh and GC B numbers without changes in fTreg number), and the frequency of CD4 + splenocytes producing IL-21, a key factor in balancing the B cell and fTreg cell activity. With ageing the avidity of virus influenza-specific antibody increased in females of both strains. Moreover, ageing affected IgG2a/IgG1 and IgG2c/IgG1 ratios (reflecting Thl/Th2 balance) in male BALB/c mice and female C57BL/6 mice, respectively. Consequently, differently from young mice exhibiting the similar ratios in male and female mice, in aged female mice of both strains IgG2a(c)/IgG1 ratios were shifted towards a less effective IgG1 response (stimulated by IL-4 cytokines) compared with males. The age-related alterations in IgG subclass profiles in both strains correlated with those in IFN-gamma/IL-4 production level ratio in splenocyte cultures restimulated with TIV. These findings stimulate further research to formulate sex-specific strategies to improve efficacy of influenza vaccine in the elderly.

The study investigated influence of sex and age on splenic myeloid dendritic cells (DCs) from Dark Agouti rats. Freshly isolated DCs from young males exhibited less mature phenotype and greater endocytic capacity compared with those from age-matched females. Upon LPS stimulation in vitro they were less potent in stimulating allogeneic CD4+ cells in mixed leukocyte reaction (MLR), due to lower expression of MHC II, and greater NO and IL-10 production. In accordance with higher TGF-beta production, young male rat DCs were less potent in stimulating IL-17 production in MLR than those from young females. Irrespective of sex, endocytic capacity and responsiveness of DCs to LPS stimulation in culture, judging by their allostimulatory capacity in MLR decreased with age, reflecting decline in MHC II surface density followed by their greater NO production; the effects more prominent in females. Additionally, compared with LPS-stimulated DCs from young rats, those from sex-matched aged rats were more potent in stimulating IL-10 production in MLR, whereas capacity of DCs from aged female and male rats to stimulate IL-17 production remained unaltered and decreased, respectively. This reflected age-related shift in IL-6/TGF-beta production level ratio in LPS-stimulated DC cultures towards TGF-beta, and sex-specific age-related remodeling CD4+ cell cytokine pathways. Additionally, compared with LPS-stimulated DCs from young rats, those cells from sex-matched aged rats were less potent in stimulating IFN-gamma production in MLR, the effect particularly prominent in MLRs encompassing male rat DCs. The study showed that stimulatory and polarizing capacity of DCs depends on rat sex and age.

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.

The study examined sex-specificities in age-related changes in BALB/c mice IgG antibody responses to immunisation with trivalent inactivated split-virus influenza bulk. Aging diminished the total serum IgG antibody responses to H1N1 and H3N2 and B influenza virus antigens in mice of both sexes, but they remained greater in aged females. This sex difference in aged mice correlated with the greater post-immunisation increase in the frequency of spleen germinal centre (GC) B cells and more favourable T follicular regulatory (Tfr)/GC B cell ratio, as Tfr cells are suggested to control antibody production through suppression of glycolysis. The greater post-immunisation GC B cell response in aged females compared with males correlated with the greater proliferation of B cells and CD4+ cells in splenocyte cultures from aged females restimulated with inactivated split-virus influenza from the bulk. To support the greater post-immunisation increase in the frequency GC B cell in aged females was more favourable Tfr/T follicular helper (Tfh) cell ratio. Additionally, compared with aged males, in age-matched females the greater avidity of serum IgG antibodies was found. However, in aged females IgG2a/IgG1 antibody ratio, reflecting spleen Th1/Th2 cytokine balance, was shifted towards IgG1 when compared with age-matched male mice. This shift was ascribed to a more prominent decline in the titres of functionally important IgG2a antibodies in females with aging. The study suggest that biological sex should be considered as a variable in designing strategies to manipulate with immune outcome of immunisation in aged animals, and possibly, at very long distance, humans.

Collagen type II-induced arthritis (CIA) in Dark Agouti rats, a model of rheumatoid arthritis (RA), reproduces sexual dimorphism in the incidence and severity of the human disease. Th17 cells are central in the induction/propagation of autoimmune inflammation in CIA and RA. To assess mechanisms underlying this dimorphism in CIA rats, in lymph nodes draining inflamed joints and adjacent tissues (dLNs) from CIA rats of both sexes Th17/CD25 + Foxp3 + CD4 + T-regulatory cell (Treg) ratio, Th17 cell redifferentiation in functionally distinct subsets and Treg transdifferentiation into IL-17-producing cells (exTregs) were examined. In female rats (developing more severe CIA than their male counterparts) the higher frequency of all Th17 cells (reflecting partly their greater proliferation), followed by the higher frequency of highly pathogenic IFN-gamma/GM-CSF-co-producing cells, but lower frequency of less pathogenic/immunoregulatory IL-10-producing cells among them was found. Additionally, compared with male rats, in female rats the lower frequency of Tregs was observed. Moreover, Tregs from female rats exhibited diminished proliferative and suppressive capacity (judging by PD-1 expression) and enhanced conversion into IL-17-producing cells. Given that TGF-beta concentration was comparable in collagen-type II-stimulated dLN cell cultures from female and male rats, the shift in Th17/Treg ratio followed by augmented Th17 cell redifferentiation into IFN-gamma/GM-CSF-co-producing cells and Treg transdifferentiation into IL-17-producing cells in female rats was associated with increased concentration of IL-6 in female rat dLN cell cultures, and the higher frequency of IL-1 beta- and IL-23-producing cells among their dLN cells. The lower frequency of IL-10-producing B cells, presumably B regulatory cells (Bregs) could also contribute to the shift in Th17/Treg ratio in female rat compared with male rat dLNs. Consistently, the lower expression of IL-35 (the cytokine promoting Treg expansion directly and indirectly, by favoring Breg expansion and conversion into IL-10/IL-35-producing cells) in female rat dLN cells was detected. Thus, the study identified putative cellular and molecular substrates of the sexual dimorphism in the immunopathogenesis and clinical outcome of CIA and suggested mechanisms to be targeted in females to improve control of Th17 response, and consequently clinical outcome of CIA, and possibly RA.

The study investigated strain specificities in age-related differences in CD8+ T cell-and microglial cell-mediated mechanisms implicated in induction/perpetuation and/or control of neuroinflammation in experimental autoimmune encephalomyelitis (EAE) in Albino Oxford (AO) and Dark Agouti (DA) rats exhibiting age-related changes in the susceptibility to EAE in the opposite direction (increase in relatively resistant AO rats vs decrease in DA rats). In the inductive phase of EAE, the greater number of fully differentiated effector CD8+ T lymphocytes was found in draining lymph nodes (dLNs) from aged rats of both strains than in strain-matched young rats, but this was particularly prominent in AO rats, which exhibited milder EAE of prolonged duration compared with their DA counterparts. Consistently, dLN IFN-gamma+ and IL-17+ CD8+ T cell counts were greater in aged AO than in DA rats. Additionally, the magnitudes of myelin basic protein (MBP)-induced rise in the frequency of IFN-gamma+ and IL-17+ CD8+ T cells (providing important help to neuroantigen-specific CD4+ T cells in EAE models characterized by clinically mild disease) were greater in dLN cell cultures from aged AO rats. Consistently, the magnitudes of MBP-induced rise in the frequency of both IFN-gamma+ and IL-17+ CD8+ T cells were greater in spinal cord mononuclear cell cultures from aged AO rats compared with their DA counterparts. Besides, with aging CD4+ CD25+ Foxp3+/CD8+ CD25+ Foxp3+ regulatory T cell ratio changed in spinal cord in the opposite direction. Consequently, in aged AO rats it was shifted towards CD8+ CD25+ Foxp3+ regulatory T cells (exhibiting lower suppressive capacity) when compared with DA rats. Moreover, the frequency of CX3CR1+ cells among microglia changed with aging and the disease development. In aged rats, in the effector phase of EAE it was lower in AO than in DA rats. This was accompanied by higher frequency of cells expressing IL-1 beta (whose down-regulation is central for CX3CR1-mediated neuroprotection), but lower that of phagocyting cells among microglia from aged AO compared their DA counterparts. The study indicates the control points linked with strain differences in age-related changes in EAE pathogenesis.

Aims The study examined the influence of sex and mouse strain on germinal center (GC) reaction and antibody responses to seasonal split trivalent influenza vaccine (TIV). Main methods C57BL/6 and BALB/c mice of both sexes were immunized with TIV and examined for specific antibody response by ELISA. Splenic T follicular regulatory (Tfr), T follicular helper (Tfh) and GC B cells are detected by flow cytometry. The proliferative response of splenocytes, and concentrations of IFN-γ and IL-4 upon restimulation with vaccine antigens were examined by 7-AAD staining and ELISA, respectively. Key findings BALB/c mice developed more robust IgG responses to vaccine type A antigens than their sex-matched C57BL/6 counterparts, while that to B antigen did not differ between strains. In both strains IgG responses against type A vaccine antigens were greater in females than in males. The greater IgG responses correlated with lower splenic Tfr/Tfh and Tfr/GC B cell ratios and greater vaccine antigen-specific proliferative responses of CD4+ and B cells in splenocyte cultures. In both mouse strains IgG2a(c)/IgG1 ratios were comparable between sexes, but lower in BALB/c than in C57BL/6 mice indicating a shift in Th1/Th2 balance towards Th2 response in BALB/c ones. Consistently, splenocytes from BALB/c mice produced more IL-4 and less IFN-γ than those from C57BL/6 mice. Significance The study indicated that magnitude of humoral response to influenza type A haemagglutinins depends on mouse strain and sex, and thereby set background for the vaccination strategies taking into account biological sex, and in a longterm perspective individual differences in immune reactivity.

Collagen-induced arthritis (CIA) is a frequently used animal model of rheumatoid arthritis, human autoimmune disease that exhibits clear sex bias in incidence and clinical course. Female Dark Agouti rats immunized for CIA showed also greater incidence and higher arthritic score than their male counterparts. The study investigated sex differences in mechanisms controlling the primary immune responses in draining lymph nodes (dLNs), as a factor contributing to this dimorphism. The higher frequencies of CD4 + CD25 + Foxp3- cells, presumably activated effector T (Teff) cells, and IL-17+, IFN-gamma + and IL-17 + IFN-gamma + T cells were found in female compared with male rat dLNs. However, the frequency of CD4 + CD25 + Foxp3 + T regulatory cells (Treg) did not differ between sexes. Thus, CD4 + Teff cells/Treg ratio, and IL-17 + T cells/Treg and IFN-gamma + T cells/Treg ratios were higher in female than in male rats, and among them was found lower frequency of PD-1+ cells. This suggested less efficient control of (auto)immune Th1/Th17 cell responses in female rat dLNs. On the contrary, the frequency of IL-4 + T cells was lower in female than in male rat dLNs. Consistently, the ratio of serum levels of collagen-specific IgG2a (IFN-gamma-dependent, with an important pathogenic role in CIA) and IgG1 (IL-4-dependent) was shifted towards IgG2a in female compared with male rats. As a whole, the study suggests that sexual dimorphism in the control of T cell activation/polarization could contribute to sex bias in the susceptibility to CIA. Moreover, the study advises the use of animals of both sexes in the preclinical testing of new drugs for rheumatoid arthritis.

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 immunoregulatory properties. Compared with young rats, following short-term PMA and ionomycin stimulation in vitro, the frequency of IL-17+ and IFN-gamma+CD4+ T lymphocytes among the spinal cord mononuclear cells from aged rats and the cytokine expression density on a per lymphocyte basis were reduced. Additionally, the increase in the proportion of autoregulatory IL-17+IL-10+ cells on the account of proinflammatory IL-17+IFN-gamma+ cells within IL-17+ lymphocytes suggested their lower pathogenic capacity in aged rats. This most likely reflected alterations in the aged rat spinal cord cytokine milieu, which were mirrored in a diminished expression of IL-1 beta mRNA followed by an enhanced expression of IL-6 and TGF-beta mRNA. Overall, the study points to age-related changes in T lymphocytes and other cells from the spinal cord infiltrate which could contribute to the decreased susceptibility of aged rats to the induction of EAE. (C) 2014 Elsevier Inc. 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 naïve and memory/activated cells substantially differed. Compared with age-matched controls, in aged ORX rats the numbers of CD4+ mature naïve 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.

In the light of the increasing resistance of bacterial pathogens to antibiotics, one of the main global strategies in applied science is development of alternative treatments, which would be safe both for the host and from the environmental perspective. Accordingly, the aim of this study was to test whether two lactic acid bacteria (LAB) strains, Lactococcus lactis BGBU1-4 and Lactobacillus salivarius BGHO1, could be applied as safe supplements for Listeria infection. Two major research objectives were set: to compare the effects of BGBU1-4 and BGHO1 on early immune response in gut tissue of Wistar rats co-administered with Listeria monocytogenes ATCC19111 and next, to test how this applies to their usage as therapeutics in acute ATCC19111 infection. Intestinal villi (IV), Peyer’s patches (PP) and mesenteric lymph nodes (MLN) were used for the analysis. The results showed that BGHO1 increased the mRNA expression of innate immune markers CD14, interleukin (IL)-1β and tumour necrosis factor (TNF)-α in PP and IV, and, in parallel, caused a decrease of listeriolysin O (LLO) mRNA expression in same tissues. In MLN of BGHO1 treated rats, LLO expression was increased, along with an increase of the expression of OX-62 mRNA and CD69, pointing to the activation of adaptive immunity. On the other hand, in BGBU1-4 treated rats, there was no reduction of LLO mRNA expression and no induction of innate immunity markers in intestinal tissue. Additionally, CD14 and IL-1β, as well as LLO, but not OX-62 mRNA and CD69 expression, were elevated in MLN of BGBU1-4 treated rats. However, when applied therapeutically, both, BGBU1-4 and BGHO1, lowered Listeria count in spleens of infected rats. Our results not only reveal the potential of LAB to ameliorate Listeria infections, but suggest different immunological effects of two different LAB strains, both of which could be effective in Listeria elimination.

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 examined the influence of age, sex and peripubertal gonadectomy on a set of T-cell phenotypic parameters. Rats of both sexes were gonadectomised at the age of 1 month and peripheral blood and spleen T lymphocytes from non-gonadectomised and gonadectomised 3- and 11-month-old rats were examined for the expression of differentiation/activation (CD90/CD45RC) and immunoregulatory markers. Peripheral blood T lymphocytes from non-gonadectomised rats showed age-dependent sexual dimorphisms in (1) total count (lower in female than male 11-month-old rats); (2) CD4+:CD8 + cell ratio (higher in female than male rats of both ages); (3) the proportion of recent thymic emigrants in CD8 + T cells (lower in female than male 3-month-old rats) and (4) the proportions of mature na lt ve and memory/activated cells (irrespective of age, the proportion of na lt ve cells was higher, whereas that of memory/activated cells was lower in females). Gonadectomy influenced magnitudes or direction of these sex differences. Additionally, sex differences in peripheral blood T-lymphocyte parameters did not fully correspond to those observed in T-splenocyte parameters, suggesting the compartment-specific regulation of the major T-cell subpopulations' and their subsets' composition. Furthermore, there was no sexual dimorphism in the proportion of either CD25 + Foxp3 + cells among CD4 + or CD161+ (NKT) cells within CD8 + T lymphocytes. However, there was gonadal hormone-independent age-associated sexual dimorphism in the proportion of CD161 + cells (NKT cells) in CD8 + T splenocytes. Overall, the study revealed age-dependent variations in sexual dimorphisms in T-cell parameters relevant for immune response efficacy and showed that they are T-cell compartment-specific and partly gonadal hormone-related.

Leflunomide is an immunosuppressive drug effective in experimental models of transplantation and autoimmune diseases and in the treatment of active rheumatoid arthritis (RA). Having in mind that it has been shown that some other immunosuppressive drugs (glucocorticoids, mycophenolate mofetil, sirolimus etc.) impair dendritic cell (DC) phenotype and function, we investigated the effect of A77 1726, an active metabolite of leflunomide, on the differentiation and function of human monocyte-derived dendritic cells (MDDC) in vitro. Immature MDDC were generated by cultivating monocytes in medium supplemented with GM-CSF and IL-4. To induce maturation, immature MDDC were cultured for 2 additional days with LPS. A77 1726 (100 μM) was added at the beginning of cultivation. Flow cytometric analysis showed that MDDC differentiated in the presence of A77 1726 exhibited an altered phenotype, with a down-regulated surface expression of CD80, CD86, CD54 and CD40 molecules. Furthermore, the continuous presence of A77 1726 during differentiation and maturation prevented successful maturation, judging by the decreased expression of maturation marker CD83, costimulatory and adhesive molecules on A77 1726-treated mature MDDC. In addition, A77 1726-pretreated MDDC exhibited a poor stimulatory capacity of the allogeneic T cells and a low production of IL-10 and IL-18. These data suggest that leflunomide impairs the differentiation, maturation and function of human MDDC in vitro, which is an additional mechanism of its immunosuppressive effect.

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. Collectively, the findings indicate that estrogen directly acting on OX62+ DCs, may affect CD4+ lymphocyte-dependent immune response in aged female rats. (C) 2014 Elsevier B.V. All rights reserved.

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.

Compared with females, male Dark Agouti (DA) rats immunized for experimental autoimmune encephalomyelitis (EAE) with rat spinal cord homogenate in complete Freund's adjuvant (CFA) exhibited lower incidence of the disease, but the maximal neurological deficit was greater in the animals that developed the disease. Consistently, at the peak of the disease greater number of reactivated CD4+CD134+CD45RC- T lymphocytes was retrieved from male rat spinal cord. Their microglia/-macrophages were more activated and produced greater amount of prototypic proinflammatory cytokines in vitro. Additionally, oppositely to the expression of mRNAs for IL-12/p35, IL-10 and IL-27/p28, the expression of mRNA for IL-23/p19 was upregulated in male rat spinal cord mononuclear cells. Consequently, the IL-17+:IFN-gamma+ cell ratio within T lymphocytes from their spinal cord was skewed towards IL-17+ cells. Within this subpopulation, the IL-17+IFN-gamma+:IL-1 7+IL-10+ cell ratio was shifted towards IL-17+IFN-gamma+ cells, which have prominent tissue damaging capacity. This was associated with an upregulated expression of mRNAs for IL-1 beta and IL-6, but downregulated TGF-beta mRNA expression in male rat spinal cord mononuclear cells. The enhanced GM-CSF mRNA expression in these cells supported the greater pathogenicity of IL-17+ T lymphocytes infiltrating male spinal cord. In the inductive phase of the disease, contrary to the draining lymph node, in the spinal cord the frequency of CD 134+ cells among CD4+ T lymphocytes and the frequency of IL-17+ cells among T lymphocytes were greater in male than in female rats. This most likely reflected an enhanced transmigration of mononuclear cells into the spinal cord (judging by the lesser spinal cord CXCL12 mRNA expression), the greater frequency of activated microglia/macrophages and the increased expression of mRNAs for Th17 polarizing cytokines in male rat spinal cord mononuclear cells. Collectively, the results showed cellular and molecular mechanisms underlying the target organ specific sexual dimorphism in the T lymphocyte-dependent immune/inflammatory response, and suggested a substantial role for the target organ in shaping the sexually dimorphic clinical outcome of EAE. (C) 2015 Elsevier Inc. All rights reserved.

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 TCRαβ + 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 CD4-CD8+TCRαβhigh 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.

An outbred mouse model was used to determine if antibody response to immunization with whole-virus trivalent inactivated influenza vaccine (TIV) differs between the sexes. The antibody response was examined one (serum titer of IgM antibodies), and three and six weeks post-immunization (serum titer of neutralizing and total IgG antibodies and IgG subclass profile). Compared with male in female mice was found (i) the more robust IgM response against all influenza strains included in TIV and (ii) more vigorous neutralizing antibody and total IgG responses against H1N1 influenza virus at both the examined time points post-immunization. The total IgG antibody response against H3N2 and B influenza viruses was comparable between female and male mice three weeks post-immunization, but significantly greater in female mice six weeks post-immunization. The neutralizing antibody response against H3N2 and B influenza viruses did not significantly differ between sexes at both the examined points post-immunization. Finally, three weeks post-immunization subclass profile of IgG specific to the influenza strains included in TIV differed between female and male mice, reflecting the lower titer of IgG1 antibodies in female ones, so that IgG2a (contributing mainly to the total IgG) to IgG1 ratio in mice of this sex was shifted toward the former. In agreement with this shift, compared with male mice, Th1/Th2 balance in female mice was shifted toward Th1, as shown by ELISPOT. Collectively, the results showed influenza virus strain-dependent sexual dimorphism in the magnitude, dynamics and characteristics of antibody response in outbred mice immunized with TIV. (C) 2015 Elsevier Ltd. All rights reserved.

The study was undertaken to assess the influence of functional -308G/A TNF-α (rs 1800629) and -174G/C IL-6 (rs1800795) promoter polymorphisms on the therapeutic response to etanercept, a TNF-α blocker, in patients with rheumatoid arthritis (RA).Seventy-three patients suffering from active RA were studied, at baseline and 6 and 12 months after therapy. The therapeutic response was estimated according to the European League Against Rheumatism response criteria. Patients were genotyped for -308G/A TNF-α and -174G/C IL-6 polymorphisms by the PCR-RFLP method, and the influence of genotype on etanercept response was assessed.No difference in the percentage of responders (patients who had DAS28 improvement1.2) between patients with the TNF-α-308GG and GA and AA genotype was detected after 6 and 12 months of treatment. After 12 months of treatment the percentage of responders was significantly increased in patients with the IL-6 -174GG genotype compared with those with the GC or CC genotype (p=0.006 by Chi-square test). Evaluation of the patients according to their combined IL-6/TNF-α genotypes showed that patients with the IL-6 -174GG / TNF-α-308GG genotype were more frequent among the responders compared to those with other combined genotypes (p=0.022 by Chi-square test). More precisely, all patients with the combined IL-6 -174GG / TNF-α-308GG genotype were responders after 12 months of etanercept treatment.The study suggests that patients who are genetically low TNF-α and IL-6 producers are the best responders to etanercept therapy.Genetički faktori su značajni za predviđanje ishoda lečenja bolesnika sa reumatoidnim artritisom (RA). Cilj studije bio je da se ispita uticaj -308G/A TNF-α (rs 1800629) i -174G/C IL-6 (rs1800795) promotorskih polimorfizama na terapijski odgovor na etanercept.U studiju su bila uključena 73 pacijenta sa aktivnim RA. Terapijski odgovor je procenjivan posle 6 i 12 meseci terapije po kriterijumima Evropske lige protiv reumatizma. Genotipizacija pacijenata za polimorfizme -308G/A TNF-α i -174G/C IL-6 urađena je PCR-RFLP metodom i procenjivan je uticaj genotipova na odgovor na etanercept.Nije bilo razlike u procentu respondera (pacijenti kod kojih se DAS28 popravio1,2) između pacijenata sa TNF-α-308GG, GA i AA genotipom ni posle 6 ni posle 12 meseci tretmana. Posle 12 meseci lečenja procenat respondera je bio značajno veći kod pacijenata sa IL-6 -174GG u odnosu na pacijente sa GC ili CC genotipom (p=0,006, χStudija pokazuje da bolesnici sa genotipovima koji se povezuju sa manjom produkcijom TNF-α i IL-6 najbolje odgovaraju na terapiju etanerceptom.

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 3month-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-α 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+:CD4DC 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-α 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 IL-1β (positive IL-17 regulator), whereas down-regulated the expression of IL-10 (negative IL-17 regulator) when compared with young strain-matched rats. In DA rats aging incresed IL-23p19 mRNA expression in LPS-stimulated DCs, whereas exerted the opposing effects on the expression of mRNAs for IL-10 and IL-1β compared to AO rats. Irrespective of the rat strain, aging did not influence mRNA expression for IL-12p35 (driving Th1 polarization) in DCs. Overall, results suggest role of changes in the expression of genes encoding proinflammatory and immunosuppressive cytokines in development of age-related alterations in rat susceptibility to EAE induction. [Projekat Ministarstva nauke Republike Srbije, br. 175050]

The present study, through quantification of tyrosine hydroxylase (TH) expression and catecholamine (CA) content in the presence and in the absence of alpha-methyl-p-tyrosine (AMPT), a TH inhibitor, in adult thymic organ (ATOC) and thymocyte culture, demonstrated that thymic cells produce CAs. In addition, in ATOC an increase in beta(2)-adrenoceptor (AR) mRNA expression and beta(2)-AR thymocyte surface density was registered. Furthermore, AMPT (10(-4) M), as propranolol (10(-4) M), augmented thymocyte apoptosis and diminished thymocyte proliferation in ATOC. Propranolol exerted these effects acting on CD3(high) thymocytes. However, in thymocyte cultures, propranolol (10(-6) M) acting on the same-thymocyte subset exerted the opposing effect on thymocyte apoptosis and ConA-stimulated proliferation. This suggested that, depending on thymocyte microenvironment, differential effects can be induced through the same type of AR. Additionally, arterenol (10(-8) to 10(-6) M), similar to propranolol, diminished apoptosis, but increased ConA-stimulated thymocyte proliferation in thymocyte culture. However, differently from propranolol, arterenol affected manly CD3- thymocyte subset, which harbors majority of alpha(1)-AR+ thymocytes. Additionally, arterenol showed a dose-dependent decrease in efficiency of thymocyte apoptosis and proliferation modulation with the rise in its concentration. Considering greater affinity of arterenol for alpha(1)-ARs than for beta(2)-ARs, the previous findings could be attributable to increased engagement of beta(2)-ARs with the rise of arterenol concentration. Consistently, in the presence of propranolol (10(-6) M), a beta-AR blacker, the arterenol (10(-8) M) effects on thymocytes were augmented. In conclusion, thymic endogenous CAs, acting through distinct AR types and, possible, the same AR type (but in different cell microenvironment) may exert the opposing effects on thymocyte apoptosis/proliferation. (C) 2014 Elsevier B.V. All rights reserved.

It is well-established that almost all cellular components of innate and adaptive immunity undergo age-related remodelling. The findings on age-related changes in both human and mouse dendritic cells (DCs) are conflicting, whereas there are no data on the influence of aging on rat DCs. In an attempt to fill this gap, freshly isolated splenic DCs expressing CD103 (alpha(OX-62) integrin), a DC specific marker recognized by MRC OX62 monoclonal antibody, from 3- (young) and 26-month-old (aged) Albino Oxford rats were examined for subset composition, expression of activation/differentiation markers (CD80, CD86 and CD40 and MHC II molecules) and endocytic capacity using flow cytometric analysis (FCA). In addition, splenic OX62+ DCs cultured in the presence or absence of LPS were analysed for the activation marker and TNF-alpha, IL-6, IL-12, IL-23, TGF-beta 1, IL-10 expression using FCA, RT-PCR and ELISA, respectively. Moreover, the allostimulatory capacity of OX62+ DCs and IFN-gamma, IL-4 and IL-17 production by CD4+ T cells in mixed leukocyte reaction was quantified using FCA and ELISA, respectively. It was found that aging: i) shifts the CD4+:CD4- subset ratio in the OX62+ DCs population towards the CD4- subset and ii) influences DCs maturation (judging by activation marker expression and efficiency of endocytosis) by affecting the expression of intrinsic (TNF-alpha and IL-10) and extrinsic maturation regulators. Furthermore, in LPS-matured OX62+ DCs from aged rats expression of TNF-alpha, IL-12, IL-23 and IL-6 was increased, whereas that of IL-10 was diminished compared with the corresponding cells from young rats. Moreover, in MLR, OX62+ DCs from aged rats exhibited enhanced Th1/Th17 driving force and diminished allostimulatory capacity compared with those from young rats.

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.

Purpose: Experimental autoimmune myocarditis (EAM) in rats is an animal model of human giant cell myocarditis and post-myocarditis dilated cardiomyopathy. The pathogenesis of EAM has not been elucidated, but there is accumulating evidence that cytokines secreted from monocytes/macrophages and T cells play a crucial role in the induction and progression of disease. Flavonoids are a large group of polyphenolic compounds abundantly present in the human diet, which scavenge oxygen radicals and have anti-inflammatory activities. Having in mind in vivo beneficial effects of flavonoid quercetin in different animal models of immunoinflammatory diseases such as experimental autoimmune encephalomyelitis and adjuvant arthritis, on the one side, and its in vitro suppressive effect on production of tumor necrosis factor–alpha (TNF-alpha on the other side, we investigated the effects of quercetin on EAM in rats. Methods: Myocarditis was induced in Dark Agouti (DA) rats by injection of porcine cardiac myosin and quercetin at doses of 10 or 20 mg/kg was orally administered from days 0 to 21 after induction of disease. The severity of myocarditis was evaluated by determination of heart weight / body weight ratio (Hw/Bw) and histopathological examination of hearts. The levels of cytokines (TNF-alpha, IL-12, IL-17 and IL-10) in serum and lymph node cells (LNC) culture supernatants were measured by ELISA. Results: The rats treated with 20 mg/kg of quercetin had significantly decreased incidence of EAM, Hw/Bw, macroscopic and microscopic scores of hearts. Further, in EAM rats treated with quercetin levels of TNF-alpha and IL-17 were significantly lower, while the level of IL-10 was significantly higher both in serum and culture supernatants of LNC stimulated with concanavalin A compared with vehicle-treated animals. Conclusions: The present study suggests that quercetin ameliorates EAM, at least in part, by interfering production of proinflammatory (TNF-alpha and IL-17) and/or anti-inflammatory (IL-10) cytokines.

Acetylsalicilyc acid (aspirin, ASA) is a well known anti-inflammatory drug with immunomodulatory properties. NO-releasing aspirins (NO-ASA) are new compounds with anti-inflammatory properties. We studied the effects of ASA and two NO-ASA (NCX 4016 and NCX 4040) on human monocyte-derived dendritic cells (MoDC). Immature MoDC were generated in vitro from monocytes in the presence of recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4. Mature MoDC were obtained by adding lipopolysaccharide (LPS) in cultures of immature MoDC. As we found that ASA at 4-8 mM, NCX 4016 at 400-800 mu M and NCX 4040 at 4-8 mu M stimulated apoptosis of monocytes and immature MoDC, sub-apoptotic concentrations of ASA (2 mM), NCX 4016 (200 mu M) and NCX 4040 (2 mu M) were used in experiments. Examined substances were added at the beginning of MoDC cultivation. MoDC differentiated in the presence of examined compounds had lower expression of HLA-DR, CD80, CD40 and CD54, decreased allostimulatory activity and lower production of IL-12 p40. ASA and NCX 4016 decreased production of IL-10, whereas NCX 4040 had the opposite effect. ASA inhibited the expression of CD1a and prevented downregulation of 014, NCX 4016 stimulated the differentiation of CD1a(+)CD14(+) and CD1a(-)CD14(+) cells, whereas NCX 4040 decreased the proportion of CD1a(+)CD14(-) and increased the frequency of CD1a(+)CD14(+) cells, compared to control. Maturation, both in ASA and NO-ASA treated MoDC was characterized by decreased allostimulatory activity, lower expression of CD83, HLA-DR, costimulatory molecules and CD54 and decreased production of IL-10 and IL-12 p40. In conclusion, we confirmed that ASA impairs differentiation, maturation and function of MoDC and found that NCX 4016 and NCX 4040 exerted similar, but not identical effects at about 10- and 1000-fold lower concentrations, respectively, compared to ASA.

Background. Fumaric acid esters (FAE) have been proven to be effective for the systemic treatment of psoriasis and multiple sclerosis, Thl cell-mediated chronic inflammatory diseases, but their effect on autoimmune rnyocarditis has not yet been addressed. We investigated the effect of dimethyl fumarate (DMF) on myosin-induced experimental autoimmune myocarditis (EAM). Methods. Dark Agouti (DA) rats immunized with porcine cardiac myosin were orally treated with 5 and 15 mg/kg body weight (bw) DMF either from days 0-10 (early treatment groups) or from days 10-21 (late treatment groups) after induction of EAM. All rats were sacrificed on day 21 after immunization and hearts were evaluated macroscopically and microscopically. Levels of TNF-alpha and IL-10 in serum and lymph node cells culture supernatants were detected by ELISA. Results. Both early and late treatment with 15 mg/kg body weight (bw) DMF markedly C, reduced the severity of myocarditis by comparing the incidence, heart weight bw ratio, macroscopic and microscopic scores, and number of OX-6+ cells in the myocardium. Further, levels of tumor necrosis factor-alpha (TNF-alpha) in serum and culture supernatants of lymph node cells stimulated with ConA or myosin were significantly lower in DNIF-treated EAM animals compared with vehicle-treated EAM rats. There was no significant difference in serum levels of interleukin-10 between DMF- and vehicle-treated EAM rats. Conclusions. These results show for the first time that DMF ameliorates experimental autoimmune rnyocarditis and may be acted, at least in part, by interfering with the production of TNF-alpha.

Background. Dendritic cells (DC) have been used for immunotherapy of malignant tumors, different kinds of infections, and other clinical conditions. For that purpose, optimal conditions for the generation of functionally mature DC in vitro are required. Two different protocols for the induction of maturation of monocyte-derived DC (MDDC) were compared in this study. Methods. MDDC were generated in vitro by cultivating adherent monocytes of healthy volunteers with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) during 6-days period. The immature DC thus prepared were induced to mature using two protocols. DC were stimulated for 2 days with lipopolysaccharide (LPS), or with a cocktail of proinflammatory mediators (PM) containing IL-1b, IL-6, tumor necrosis factor α (TNFα), and prostaglandin E2 (PGE2), respectively. Phenotypic characteristics of MDDC and their endocytic activity were studied by flow cytometry. Allostimulatory activity of these cells was tested in the mixed leukocyte reaction (MLR), whereas the production of cytokines was determined by ELISA kits. Results. MDDC matured with PM (PM-DC) were predominantly non-adherent cells, while about 30% of LPS-matured DC were adherent cells. In comparison with LPS-DC, PM-DC expressed higher levels of CD86 and CD83, had lower endocytic activity, produced higher levels of IL-10 and lower levels of IL-12, and more strongly stimulated proliferation of allogeneic lymphocytes. Conclusion The protocol based on the combination of proinflammatory cytokines and PGE2 is better for the induction of maturation of human MDDC in vitro than the protocol using LPS alone. Uvod. Dendritične ćelije (DC) se koriste u imunoterapiji malignih tumora, različitih vrsta infekcija i drugih oboljenja. U tom cilju neophodni su optimalni uslovi za dobijanje funkcionalno zrelih DC in vitro. U ovom radu smo poredili dva različita protokola za indukciju maturacije DC monocitnog porekla (MDDC). Metode. MDDC su dobijene in vitro kultivisanjem adherentnih monocita zdravih dobrovoljnih davalaca krvi pomoću faktora stimulacije granulocitno-makrofagnih kolonija (GM-CSF) i interleukina-4 (IL-4) u toku 6 dana. Tako pripremljene nezrele DC su indukovane na sazrevanje pomoću dva protokola. DC su stimulisane u toku 2 dana lipopolisaharidom (LPS) ili koktelom proinflamatornih medijatora (PM) koji je sadržavao IL-1b, IL-6, faktor nekroze tumora α (TNFα) i prostaglandin E2 (PGE2). Fenotipske karakteristike MDDC i njihova endocitozna aktivnost su ispitivani pomoću protočne citometrije. Alostimulatorna aktivnost ovih ćelija je ispitivana u testu mešane leukocitne reakcije (MLR), dok je stvaranje citokina određivano ELISA kompletima. Rezultati. MDDC koje su sazrevale u prisustvu PM (PM-DC) su bile predominantno neadherentne ćelije, dok su oko 30% DC koje su sazrevale pod uticajem LPS (LPS-DC) bile adherentne. U poređenju sa LPS-DC, PM-DC su ispoljavale više nivoe CD86 i CD83 molekula, imale slabiju endocitoznu aktivnost, produkovale više IL-10, a manje IL-12 i snažnije stimulisale proliferaciju alogenih limfocita. Zaključak. Protokol koji se bazira na primeni kombinacije proinflamatornih citokina i PGE2 je bolji za indukciju maturacije humanih MDDC in vitro nego LPS protokol.

In the present study, possibilities for using novel nanocomposites based on alginate and silver nanoparticles for wound treatment were investigated in a second-degree thermal burn model in Wistar rats. Silver nanoparticles (AgNPs) were electrochemically synthesized in alginate solutions that were further utilized to obtain the Ag/alginate solution and microfibers for subsequent in vivo studies. Daily applications of the Ag/alginate colloid solution, containing AgNPs, alginate and ascorbic acid (G3), wet Ag/alginate microfibers containing AgNPs (G5) and dry Ag/alginate microfibers containing AgNPs (G6) were compared to treatments with a commercial cream containing silver sulfadiazine (G2) and a commercial Ca-alginate wound dressing containing silver ions (G4), as well as to the untreated controls (G1). Results of the in vivo study have shown faster healing in treated wounds, which completely healed on day 19 (G4, G5 and G6) and 21 (G2 and G3) after the thermal injury, while the period for complete reepitelization of untreated wounds (G1) was 25 days. The macroscopic analysis has shown that scabs fell off between day 10 and 12 after the thermal injury induction in treated groups, whereas between day 15 and 16 in the control group. These macroscopic findings were supported by the results of histopathological analyses, which have shown enhanced granulation and reepithelization, reduced inflammation and improved organization of the extracellular matrix in treated groups without adverse effects. Among the treated groups, dressings based on Ca-alginate (G4-G6) induced enhanced healing as compared to the other two groups (G2, G3), which could be attributed to additional stimuli of released Ca2+. The obtained results indicated potentials of novel nanocomposites based on alginate and AgNPs for therapeutic applications in wound treatments.