New records and noteworthy data of plants, algae and fungi in SE Europe and adjacent regions, 12

This paper presents new records and noteworthy data on the following taxa in SE Europe and adjacent regions: red algae Hildenbrandia rivularis, saprotrophic fungus Cryptomarasmius corbariensis, lichenised fungi Lecanora stenotropa, Micarea misella and Sticta sylvatica, liverworts Fossombronia caespitiformis and Peltolepis quadrata, mosses Dicranoweisia cirrata and Fissidens exilis, horsetail Equisetum × moorei, gymnosperm Juniperus virginiana, monocots Galanthus reginae-olgae subsp. vernalis and Spiranthes spiralis and dicots Linaria pelisseriana, Parthenocissus quinquefolia, Pilosella rhodopea and Taraxacum erythrospermum are given within SE Europe and adjacent regions

GM-CSF-Producing Th Cells in Rats Sensitive and Resistant to Experimental Autoimmune Encephalomyelitis

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 efficacy of several mechanisms controlling (auto) reactive CD4+ lymphocyte expansion/differentiation into the cells with pathogenic phenotype and migration of the latter to the SC contribute to AO rat resistance to EAE

Sex Bias in Pathogenesis of Autoimmune Neuroinflammation: Relevance for Dimethyl Fumarate Immunomodulatory/Anti-oxidant Action

In the present study, upon showing sexual dimorphism in dimethyl fumarate (DMF) efficacy to moderate the clinical severity of experimental autoimmune encephalomyelitis (EAE) in Dark Agouti rats, cellular and molecular substrate of this dimorphism was explored. In rats of both sexes, DMF administration from the day of immunization attenuated EAE severity, but this effect was more prominent in males leading to loss of the sexual dimorphism observed in vehicle-administered controls. Consistently, in male rats, DMF was more efficient in diminishing the number of CD4+ T lymphocytes infiltrating spinal cord (SC) and their reactivation, the number of IL-17+ T lymphocytes and particularly cellularity of their highly pathogenic IFN-gamma+GM-CSF+IL-17+ subset. This was linked with changes in SC CD11b+CD45+TCR alpha beta- microglia/proinflammatory monocyte progeny, substantiated in a more prominent increase in the frequency of anti-inflammatory phygocyting CD163+ cells and the cells expressing high surface levels of immunoregulatory CD83 molecule (associated with apoptotic cells phagocytosis and implicated in downregulation of CD4+ T lymphocyte reactivation) among CD11b+CD45+TCR alpha beta- cells in male rat SC. These changes were associated with greater increase in the nuclear factor (erythroid-derived 2)-like 2 expression in male rats administered with DMF. In accordance with the previous findings, DMF diminished reactive nitrogen and oxygen species generation and consistently, SC level of advanced oxidation protein products, to the greater extent in male rats. Overall, our study indicates sex-specificity in the sensitivity of DMF cellular and molecular targets and encourages sex-based clinical research to define significance of sex for action of therapeutic agents moderating autoimmune neuroinflammation-/oxidative stress-related nervous tissue damage

Aging diminishes the resistance of AO rats to EAE: putative role of enhanced generation of GM-CSF Expressing CD4+T cells in aged rats

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) and CD45(hi) (mainly proinflammatory dendritic cells and macrophages) cells was retrieved from aged than young rat spinal cord. Besides, expression of mRNA for SOCS1, a negative regulator of proinflammatory cytokine expression in innate immunity cells, was downregulated in aged rat spinal cord mononuclear cells. Conclusions: The study revealed that aging may overcome genetic resistance to EAE, and indicated the cellular and molecular mechanisms contributing to this phenomenon in AO rats

PCR detection of genetically modified soy bean and maize in food/feed stuffs

Potencijalni štetni efekti koje na zdravlje ljudi i životinja može imati upotreba hrane poreklom od genetički modifikovanih organizama (GMO), kao i eventualni neželjeni efekti koje gajenje genetički modifikovanih biljaka može imati na životnu sredinu, doveli su do potrebe da se u EU strogo ograniči distribucija GMO. Aktima Komisije EU, 49/2000 i 50/2000, propisano je da se prehrambeni artikli proizvedeni od GMO ili koji sadrže GMO, moraju jasno obeležiti. Ovoj obavezi ne podležu oni proizvodi koji sadrže manje od 1% genetički modifikovanog materijala. Prisustvo GMO u hrani za humanu i animalnu upotrebu može biti ustanovljeno bilo detekcijom DNK kojom je urađena modifikacija (PCR metode), bilo detekcijom proteina kodiranog ovom DNK (imunološke metode). U Institutu za molekularnu genetiku i genetičko inženjerstvo standardizovane su kvalitativna i kvantitativna metoda zasnovane na tehnici PCR. Da li uzorak sadrži GMO ili ne određuje se detektovanjem CaMV35S promotora, dok se za detektovanje specifičnih genetičkih modifikacija u RoundUp Ready soji tolerantnoj na herbicid RoundUp i Bt kukuruzu otpornom na insekte, koriste prajmeri dizajnirani iz odgovarajućih specifičnih transgena - gena EPSPS iz A. tumefaciens (za soju) i gena CrylA iz B. thuringiensis (za kukuruz). Eksperimentalno utvrdjena granica osetljivosti metoda je 1pg DNK, što omogućava detektovanje 0,01% GM DNK u testiranom uzorku i odgovara kriterijumima evropskih laboratorija koje su ovlašćene da izdaju sertifikat.Public concern about possible negative health effects due to consumption of GM plants, together with concerns about possible environmental dangers, have led the European Commission to limit strictly the distribution of GM plants in the EU. The EU regulations No 49 and 50/2000 require the food industry to label, as GMO containing, those food products not certified free of transgenic plant material. Foods are considered "clean" and may be left unlabeled, if the GM content does not exceed 1%. The presence of GM in food and feed products can be evaluated by detecting the transgenic DNA (PCR technique), or by detecting proteins derived from this DNA (immunological methods). At the Institute of Molecular Genetics and Genetic Engineering both qualitative and quantitative PCR-based tests are standardized. Commonly used primers are designed from the CaMV35S promoter for the answer yes/no in general GMO screening procedure. Specific modifications, as in herbicide tolerant RoundUp Ready Soybean or insect resistant maize (Bt176 corn), are identified by primers designed from specific genes e.g. the EPSPS - gene from A.tumefaciens (for soybean) and the CrylA gene from B.thuringiensis (for maize). The experimentally determined detection limit is 1pg, which allows detection of O,01% GM DNA in test-samples and meets criteria prescribed for the EU laboratories accredited to issue a certificate

Aging oppositely affects TNF-alpha and IL-10 production by macrophages from different rat strains

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

Sex Difference in Oxidative Stress Parameters in Spinal Cord of Rats with Experimental Autoimmune Encephalomyelitis: Relation to Neurological Deficit

The study examined (a) whether there is sex difference in spinal cord and plasma oxidative stress profiles in Dark Agouti rats immunised for experimental autoimmune encephalomyelitis (EAE), the principal experimental model of multiple sclerosis, and (b) whether there is correlation between the oxidative stress in spinal cord and neurological deficit. Regardless of rat sex, with the disease development xanthine oxidase (XO) activity and inducible nitric oxide synthase (iNOS) mRNA expression increased in spinal cord, whereas glutathione levels decreased. This was accompanied by the rise in spinal cord malondialdehyde level. On the other hand, with EAE development superoxide dismutase (SOD) activity decreased, while O-2 (-) concentration increased only in spinal cord of male rats. Consequently, SOD activity was lower, whereas O-2 (-) concentration was higher in spinal cord of male rats with clinically manifested EAE. XO activity and iNOS mRNA expression were also elevated in their spinal cord. Consistently, in the effector phase of EAE the concentration of advanced oxidation protein product (AOPP) was higher in spinal cord of male rats, which exhibit more severe neurological deficit than their female counterparts. In as much as data obtained in the experimental models could be translated to humans, the findings may be relevant for designing sex-specific antioxidant therapeutic strategies. Furthermore, the study indicated that the increased pro-oxidant-antioxidant balance in plasma may be an early indicator of EAE development. Moreover, it showed that plasma AOPP level may indicate not only actual activity of the disease, but also serve to predict severity of its course

Androgens Contribute to Age-Associated Changes in Peripheral T-Cell Homeostasis Acting in a Thymus-Independent Way

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, Base