IMPACT OF DIFFERENCES IN ECONOMIC DEVELOPMENT AND SOCIOECONOMIC STABILITY ON BENZODIAZEPINE EXPOSURE BETWEEN THE THREE BALKANS COUNTRIES

Introduction: Anxiety disorders are among the most common mental disorders. Benzodiazepines belong to the group of anxiolytic sedatives and the most prescribed drugs in the world. The aim in ours study was to evaluate the differences in the exposure of the population to benzodiazepines (in period from 2014-2018) be tween Serbia, Slovenia and Croa tia, the three countries of th e Southwestern Balkans with varying degrees of socioeconomic development. Study design: A academic investigator initiated, pharmacoepidemiological difference-in -difference time se ries analysis of population exposure to benzodiazepines between the three, geogr aphically close Balkans countries (Slovenia, Serbia, Croatia) wi th varying degrees of socioeconomic developmen t has been carried out. Study was conducted as academic investigator initiated, in a retrospective manner on monthly basis international data set from January 2014 to December 2018. Results: At the annual level, during the study period from Januar y 2014 to December 2018, compared to Slovenia, Serbia and Croatia had higher DIDs, from 5 fold (Croatia) to 6 fold (Serbi a), for all benzodiazepines in total. By analyzing the differenc es-in- difference, we have shown that influence of both time (month) and c ountry on DIDs is significant as well as their mutual intera ction (the country × month) for all benzodiazepines in total. Conclusion: Serbia and Croatia must implement exp licit measures of reduci ng benzodiazepine prescription in health primary care based on evidence-based recommendations in the indicati ons where general medicine practitioners/family doctors most commonly prescribe these medicines. Without providing a realistic supplement/alternative to benzodiazepines such as increasing the availability of psychotherapy and impr oving the structure of psyc hiatric professionals in healthcare settings, implicit measures are not recommended for reducing pr escription, implementing accountability measures for prolonged prescription of benzodiazepines, and in particular for“ masked ”somatic diseases . All this comes to the fore by raising economic development and socioeconomic stabilit

IMPACT OF DIFFERENCES IN ECONOMIC DEVELOPMENT AND SOCIOECONOMIC STABILITY ON BENZODIAZEPINE EXPOSURE BETWEEN THE THREE BALKANS COUNTRIES

Introduction: Anxiety disorders are among the most common mental disorders. Benzodiazepines belong to the group of anxiolytic sedatives and the most prescribed drugs in the world. The aim in ours study was to evaluate the differences in the exposure of the population to benzodiazepines (in period from 2014-2018) be tween Serbia, Slovenia and Croa tia, the three countries of th e Southwestern Balkans with varying degrees of socioeconomic development. Study design: A academic investigator initiated, pharmacoepidemiological difference-in -difference time se ries analysis of population exposure to benzodiazepines between the three, geogr aphically close Balkans countries (Slovenia, Serbia, Croatia) wi th varying degrees of socioeconomic developmen t has been carried out. Study was conducted as academic investigator initiated, in a retrospective manner on monthly basis international data set from January 2014 to December 2018. Results: At the annual level, during the study period from Januar y 2014 to December 2018, compared to Slovenia, Serbia and Croatia had higher DIDs, from 5 fold (Croatia) to 6 fold (Serbi a), for all benzodiazepines in total. By analyzing the differenc es-in- difference, we have shown that influence of both time (month) and c ountry on DIDs is significant as well as their mutual intera ction (the country × month) for all benzodiazepines in total. Conclusion: Serbia and Croatia must implement exp licit measures of reduci ng benzodiazepine prescription in health primary care based on evidence-based recommendations in the indicati ons where general medicine practitioners/family doctors most commonly prescribe these medicines. Without providing a realistic supplement/alternative to benzodiazepines such as increasing the availability of psychotherapy and impr oving the structure of psyc hiatric professionals in healthcare settings, implicit measures are not recommended for reducing pr escription, implementing accountability measures for prolonged prescription of benzodiazepines, and in particular for“ masked ”somatic diseases . All this comes to the fore by raising economic development and socioeconomic stabilit

Redox component in the adaptation of the microalga Chlorella sorokiniana to Ni(II) excess

Nickel is utilized by microalgae as a co-factor of urease. On the other hand, this transition metal represents an important pollutant of aquatic ecosystems. The effects of Ni(II) excess on microalgae and the mechanisms of adaptation are poorly understood. Redox processes represent an important component of the mechanisms of interaction of microalgae with transition metals. Pertinent to this, we analyzed redox changes in Chlorella sorokiniana culture that are induced by high levels of Ni(II). The intracellular level of reactive oxygen species (ROS) showed a rapid two-phase increase that took place prior to Ni accumulation in the cell. This was accompanied by oxidation of thiols and drastic deglutathyonilation of proteins. PAM fluorimetry showed that Ni excess induced an increase in the efficiency of photosystem II and promoted electron flow in chloroplasts, which is most likely responsible for ROS rise. In addition, a rising trend in the chlorophyll level was observed. On the other hand, the level of lipid peroxidation and activities of key antioxidative enzymes were not increased, which implies that oxidative stress is not an important player in Ni adaptation/toxicity. After prolonged exposure the efficiency of photosystem II drops, nickel is accumulated in the cells, and new redox balance is established. Our results imply that redox signalling is involved in Ni-induced metabolic activation and that key changes take place in photosynthetic machinery

The effects of ionizing irradiation on growth and lipid production in Chlorella sorokiniana

The impact of ionizing radiation on microalgae represents an important biotechnological and environmental issue. However, it has not been sufficiently investigated. Herein, we analyzed the effects of lowdose X-radiation on the growth, lipid production, and chlorophyll (Chl) and carotenoids content in Chlorella sorokiniana (CCAP 211/8K), which is both, a model and biotechnologically relevant species. C. sorokiniana culture was grown in 3N-BBM+V medium, at 22°C with a continuous photon flux of 120 μmol m−2 s−1. X-ray irradiation was applied in the early exponential phase of growth, at different doses (1, 2, 5, 10, 20 Gy) and rates (0.06, 0.24, 0.55 Gy/min). Parameters were monitored for 30 days. The exposure to 2 Gy and 5 Gy had a positive impact on biomass production. Dry weight was significantly higher in treated cultures than controls at days 25 and 30. Total lipid content (according to Nile Red fluorescence assay) was increased at day 30 in cultures exposed to 1 Gy (0.06 Gy/min) and 5 Gy (0.24 Gy/min). Chl content was increased for these doses in the exponential phase of growth. Chl b and carotenoids content was not significantly affected by irradiation. It is noteworthy that higher doses (10 and 20 Gy) had suppressing effects on growth and lipid production. The positive effects of ionizing radiation on biomass and lipid production can be attributed to the phenomenon of radiation hormesis (beneficial effects of low dose radiation on different biological parameters). Radiation hormesis has been shown previously documented on a number of plant species, and can be potentially employed in microalgae industry. On the other hand, microalgae are exposed to increased levels of ionizing irradiation in aquatic systems that are infested with radionuclides either naturally or by anthropogenic activity. Our results may add to the understanding of eco-physiology of microalgae in such systems.7th European Phycological Congress, 25-30 August 2019, Zagre

Comparative impact of Mn2+ and Ni2+ on the microalga Chlorella sorokiniana

METHODS The impact of a set of concentrations of Mn2+ and Ni2+ on growth of C. sorokiniana culture in 3N-BBM+V medium in the early stationary phase was evaluated by changes in optical density at 750 nm and biomass during 7 days treatment. Mucilage release was analyzed using SEM microscopy. Redox settings were analyzed by oxidation-sensitive fluorescent probe and assays for thiols. RESULTS Ni was more toxic than Mn and affected culture growth at lower concentrations. Microalgal cells started releasing mucilage polymers within 1 h of exposure to 1 mM Mn2+, whereas no mucilage was observed even at 24 h of treatment with equimolar Ni2+. The peak of reactive oxygen species production was reached faster for Ni2+ than Mn2+. Mn-induced drops in the concentration of reduced thiols showed a recovery after 1 h and 24 h. Ni2+-induced drop was irreversible. The observed differences between the impact of Mn and Ni may be related to different redox and coordinative properties and to higher capacities of microalgae to sequester Mn in relation to higher quotas than Ni that are required for normal functi

Mechanisms of detoxification of high manganese concentrations by the microalga Chlorella sorokiniana

Many neutrophilic and acidophilic microalgal species tolerate high metal concentrations and can survive or colonize metal-polluted waters. They show significant biotechnological potential for the remediation and wastewaters processing. On the other hand, negative effects of metal pollution on microalgae may affect the function of aquatic ecosystems because these photosynthetic microorganisms represent the primary producers of O2 and biomass. However, adaptive mechanisms that microalgae employ to detoxify metal excess are largely unknown. Herein we analyzed the response of the freshwater microalga Chlorella sorokiniana to high but non-toxic levels of Mn2+. Manganese is a key metal pollutant, with five possible oxidation forms that can bind to a variety of different ligands. At pH below 7, it is predominantly present in Mn2+ form. Scanning electron microscopy showed that in response to 1 mM Mn2+, C. sorokiniana released mucilage polymers within 1 h. Electron paramagnetic resonance spectroscopy (EPR) showed that the early response involved loose Mn2+ binding to mucilage and/or the cell wall. The amount of loosely bound Mn2+ was significantly decreased after 24 h, whereas biomass showed significant accumulation of Mn, O and P, as determined by energy dispersive X-ray spectrometry, indicating the production of polyphosphates, which may sequester Mn. Further, it was found that the exposure to Mn2+ resulted in rapid and transient decrease of total free glutathione concentration; the drop was observed after 1 h, and the concentration returned to initial values after 24 h. EPR measurements showed a similar trend in the level of reduced thiols. The observed changes can be explained either by the synthesis of phytochelatins – sulfurrich short-chain peptides that sequester metals, or by glutathionylation of proteins. Reduced thiols could not be detected in the extracellular space, indicating that C. sorokiniana did not release thiols in response to high Mn. These results demonstrate that the adaptive response of C. sorokiniana to high Mn levels involves multiple components and time phases. The early phase involves mucilage release, phytochelatins and/or protection of protein thiols, whereas the successive phase involves Mn coordination by polyphosphates and other mechanisms that remain to be resolved

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

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