Sezonska horizontalna distribucija i abundancija vrsta familija Temoridae u Bokokotorskom zalivu

U Bokokotorskom zalivu registrovane su dvije vrste Copepoda iz familije Temoridae: Temora stylifera (Dana, 1849) i Temora longicornis (Müller 1792). U radu dajemo podatke o horizontalnoj distribuciji i abundanciji ovih vrsta tokom 2007. godine. Temora stylifera ima široko rasprostranjenjeu obalnom I pučinskim vodama Jadrana, dok je Temora longicornis tipičan predstavnik estuarsko-neritičkih voda sjevernog Jadrana, Kvarnerskog područja i zapadno uz italijansku obalu. Njihova abundancija je bila posebno izražena u proleće i leto

Role of the End-Point Mediators of Sympathoadrenal and Sympathoneural Stress Axes in the Pathogenesis of Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis

The role of stress effector systems in the initiation and progression of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), the most commonly used experimental model of MS, has strongly been suggested. To corroborate this notion, alterations in activity of the sympathoadrenal and sympathoneural axes of sympathoadrenal system (a major communication pathway between the central nervous system and the immune system), mirrored in altered release of their end-point mediators (adrenaline and noradrenaline, respectively), are shown to precede (in MS) and/or occur during development of MS and EAE in response to immune cell activation (in early phase of disease) and disease-related damage of sympathoadrenal system neurons and their projections (in late phase of disease). To add to the complexity, innate immunity cells and T-lymphocytes synthesize noradrenaline that may be implicated in a local autocrine/paracrine self-amplifying feed-forward loop to enhance myeloid-cell synthesis of proinflammatory cytokines and inflammatory injury. Furthermore, experimental manipulations targeting noradrenaline/adrenaline action are shown to influence clinical outcome of EAE, in a disease phase-specific manner. This is partly related to the fact that virtually all types of cells involved in the instigation and progression of autoimmune inflammation and target tissue damage in EAE/MS express functional adrenoceptors. Although catecholamines exert majority of immunomodulatory effects through beta(2)-adrenoceptor, a role for alpha-adrenoceptors in EAE pathogenesis has also been indicated. In this review, we summarize all aforementioned aspects of immunopathogenetic action of catecholamines in EAE/MS as possibly important for designing new strategies targeting their action to prevent/mitigate autoimmune neuroinflammation and tissue damage

Natural killer cells as participants in pathogenesis of rat experimental autoimmune encephalomyelitis (EAE): lessons from research on rats with distinct age and strain

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

Dalji pravci razvoja terapijskih monoklonskih antitela: novi formati i nove funkcije

Monoclonal antibody therapeutics have dramatically changed the landscape of medicine and human health. In 2022, thirty-six years on from the approval of a first monoclonal antibody, about 100 monoclonal antibodies for treatment of cancer and noncancer indications had secured United States Food and Drug Administration approval. The idea that antibodies could serve as therapeutics emerged over century ago, but it became a reality when scientists developed hybridoma technology for antibody generation in the laboratory. To avoid the shortcomings of the earliest therapeutic monoclonal antibodies of murine origin, which tended to be immunogenic in humans, therapeutic antibody constructs that were more human-compatible (chimeric and humanized) have been produced using genetic engineering technology. Finally, development of phage display, the human antibody mouse, and single B cell antibody innovative technologies, have enabled production of fully human therapeutic antibodies. The successful application of full-size monoclonal antibodies over the last decades has motivated the pharmaceutical company to develop various types of antibody formats in order to improve their efficacy and lower adverse effects. By using strategies to miniaturize and multifunctionalize antibody molecules new classes of antibody therapeutics, such as antibody derivatives (e.g., antibody–drug conjugates and immunocytokines), bispecific/multispecific antibodies, antibody fragments, were developed. In the future, with the progress of modern biotechnology, it can be expected that these new-designed antibodies will finally pave the way for successful treatments of various diseases.Otkriće monoklonskih antitela i njihova primena u terapiji je značajno uticala na lečenje i zdravlje ljudi širom sveta. Danas, tri i po decenije nakon što je prvo monoklonsko antitelo odobreno za upotrebu od strane Agencije za hranu i lekove Sjedinjenih Američkih Država, više od 100 ovih terapeutika za lečenje malignih tumora i drugih bolesti se nalazi na tržištu. Iako je ideja da se antitela mogu koristiti kao lekovi stara više od jednog veka, do njene realizacije je došlo tek kada su naučnici uspostavili tehnologiju za dobijanje hibridoma ćelija čime je bila omogućena proizvodnja antitela u laboratorijskim uslovima. S obzirom da su ta prva terapijska antitela bila mišja i samim tim imunogena za čoveka, razvojem tehnologije genetskog inženjeringa omogućeno je konstruisanje antitela koja su više “humana” (tzv. himerna i humanizovana). Konačno, uspostavljanjem inovativnih platformi za prikazivanje antitela ili njihovih fragmenata na fagima (“phage display”), generisanje miševa koji sintetišu humana antitela ili izolaciju gena za antitela iz pojedinačne B ćelije omogućeno je dobijanje potpuno humanih antitela. I pored više nego uspešne primene celih molekula antitela u terapiji, da bi se prevazišla nedovoljna efikasnost nekih od njih i smanjili neželjeni efekti, farmaceutske kompanije su počele da razvijaju različite formate antitela. Korišćenjem strategije smanjenja veličine i/ili povećanja funkcionalnosti molekula antitela razvijena je potpuno nova klasa terapijskih antitela kao što su derivati antitela (npr. konjugati antitela i lekova), bispecifična/multispecifična antitela, fragmenti antitela i drugi. U budućnosti, sa daljim razvojem moderne biotehnologije, može se očekivati da će ta novodizajnirana antitela omogućiti uspešno lečenje mnogih bolesti.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Ageing Affects Thymopoiesis and Experimental Autoimmune Encephalomyelitis Development in a Strain-Dependent Manner

INTRODUCTION: Considering significance of mechanisms of central tolerance for development of autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), and suppressive influence of circulating proinflammatory cytokines and alterations in brain-thymus communication, characteristic for the central nervous system (CNS) autoimmune diseases, on thymopoiesis, the study interogated putative strain-based thymus-related specificities relevant for the opposite effects of ageing on susceptibility of Dark Agouti (DA) and Albino Oxford (AO) rats to EAE. METHODS: Quantitative and qualitative changes in thymopoiesis including underlying mechanisms were examined using flow cytometry and RT-qPCR quantification of mRNAs for molecules relevant for integrity of stroma and T-cell development, respectively. RESULTS: With ageing, differently from DA rats, in AO rats the surface density of CD90, a negative regulator of selection threshold, on thymocytes undergoing lineage commitment was upregulated (consistent with TGF-β expression downregulation), whereas the generation of natural CD4+CD25+Foxp3+ regulatory T cells (nTregs) was impaired reflecting differences in thymic expression of cytokines supporting their development. Additionally, specifically in old AO rats, in whom EAE development depends on IL-17-producing CD8+ T cells, their thymic differentiation was augmented, reflecting augmented thymic IL-4 expression. In turn, differently from old DA rats developing self-limiting EAE, in age-matched AO rats developing EAE of prolonged duration, EAE development led to impaired generation of nTregs and accumulation of proinflammatory, cytotoxic CD28-CD4+ T cells in the periphery. DISCUSSION: The study indicates that strain differences in age-related changes in the efficacy of central tolerance, in addition to enhanced thymic generation of CD8+ T cells prone to differentiate into IL-17-producing cells, could partly explain the opposite effect of ageing on DA and AO rat susceptibility to EAE induction. Additionally, it suggested that EAE development leading to a less efficient thymic output of CD4+ cells and nTregs in old AO rats than their DA counterparts could contribute to prolonged EAE duration in AO compared with DA rats. CONCLUSION: The study warns to caution when designing therapeutic interventions to enhance thymic activity in genetically diverse populations, e.g., humans, and interpreting their outcomes. Furthermore, it indicates that CNS autoimmune pathology may additionally worsen thymic involution and age-related immune changes

Propranolol diminished severity of rat EAE by enhancing immunoregulatory/protective properties of spinal cord microglia

Sympathetic dysfunction is suggested to contribute to development of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) alike. Considering important role of microglia in development/resolution of neuroinflammation, contribution of noradrenaline, the key sympathetic end-point mediator, in modulation of microglial phenotypic and functional properties in rat EAE model was examined. The study showed that noradrenaline acting in neurocrine and autocrine/paracrine way might influence microglia during EAE. Propranolol treatment over the effector phase moderated EAE course. This was associated with the increased proportion of microglia expressing CX3CR1, the key molecule in their immunomodulatory/neuroprotective action, and up regulation of CX3CR1 downstream Nrf2 gene. This correlated with the increased heme oxygenase-1 (HO-1) expression and phagocytic capacity of microglia, and their phenotypic changes mirrored in increased proportion of CD163- and CD83-expressing cells. The enhanced HO-1 expression was linked with the decreased proportion of microglial cells expressing IL-1 beta and IL-23, and possibly IL-6, followed by increased proportion of IL-10 expressing microglia, and downregulated MCP-1/CCL2 expression. Consistently, spinal cord infiltration with blood-borne myeloid and CD4 + T cells, as well as CD4 + T-cell reactivation/proliferation and differentiation into highly pathogenic IL-17 + cells co-producing IFN-gamma and GM-CSF were decreased in propranolol-treated rats compared with saline-injected controls. The in vitro investigations of the effects of noradrenaline on microglia showed that noradrenaline through beta-adrenoceptor may influence Nrf2 expression also via CX3CR1-independent route. The study suggests beta-adrenoceptor-mediated neuroinflammation-promoting role of noradrenaline in EAE via modulation of microglial Nrf2 expression, and thereby forms the basis for further translational pharmacological research to improve multiple sclerosis therapy

Strain-specific differences in age-related changes in rat susceptibility to experimental autoimmune encephalomyelitis and dendritic cell cytokine gene expression

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis, a prototype of Th1/Th17-mediated organ-specific autoimmune disease. In the rat, susceptibility to development of these diseases is shown to be strain-and age-dependent. In adult rats of distinct strains, it correlates with splenic dendritic cell (DC) subset composition, which also exhibit age-related changes. The aim of this study was to examine influence of aging on: i) Albino Oxford (relatively resistant to EAE) and Dark Agouti (susceptible to EAE) rat development of EAE and ii) their splenic conventional (OX62+) DC population in respect to its subset composition and expression of mRNAs for proinflammatory and immunosuppressive cytokines. We used 3-month-old (young) and 26-month-old (aged) rats of AO and DA strain. The rats were immunized for EAE with rat spinal cord homogenate in complete Freund's adjuvant and clinical course of the disease was followed. Fresh OX62+DCs were examined for the expression of CD4 (using flow cytometry) and genes encoding cytokines influencing DC activation/maturation (TNF-alpha and IL-6) using RT-PCR. Additionally, in vitro lipopolysaccharide (LPS) activated/matured DCs were examined for the expression of genes encoding cytokines controlling Th1/Th17 cell polarization using RT-PCR. With aging, AO rats became more susceptible, whereas DA rats largely lose their susceptibility to the induction of EAE. In AO rats aging shifted CD4+: CD4-DC ratio towards CD4- cells, producing large amount of proinflammatory cytokines, whereas in DA rats CD4+: CD4-DC ratio remained stable with aging. In fresh DCs from rats of both the strains the expression of TNF-alpha mRNA increased with aging, whereas that of IL-6 mRNA decreased and increased in DCs from AO and DA rats, respectively. Following in vitro LPS stimulation OX62+ DCs from aged AO rats up-regulated the expression of mRNA for IL-23p19 (specific subunit of IL-23; crucial for sustained IL-17 production) and IL-1 beta (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-1 beta 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

Imunonefelometrija i imunoturbidimetrija - teorijski principi, instrumenti i primena

Nephelometry and turbidimetry are methods based on the phenomenon whereby light, passing through a medium with dispersed particles is attenuated in intensity by scattering. The intensity of the scattered light is measured at right angle (usually, but not necessarily) to the incident light beam (nephelometry) or at a forward angle (turbidimetry). Based on nephelometry and turbidimetry, immunonephelometric and immunoturbidimetric assays for the measurement of proteins have developed and expanded rapidly in recent years, progressively replacing the time-honored gel precipitation techniques. In this study, the theoretical background of light scattering theory, instrumental systems for nephelometry and turbidimetry, the nature and kinetics of the antibody-antigen reaction in fluid, and the application of immunonephelometric and immunoturbidimetric assays for specific proteins in the clinical laboratory are described.Nefelometrija i turbidimetrija su metode koje sa zasnivaju na rasipanju svetlosti: pri prolasku kroz disperzionu sredinu, svetlost se rasipa na česticama disperzione faze. Intenzitet rasute svetlosti može se meriti pod nekim uglom (uobičajeno, ali ne i obavezno, pod pravim uglom), što predstavlja nefelometriju ili u pravcu upadne svetlosti, što predstavlja turbidimetriju. Primenom nefelometrije i turbidimetrije u imunologiji, razvile su se imunonefelometrija i imunoturbidimetrija, kao savremene tehnike za određivanje proteina i peptida, koje su zamenile klasične, dugotrajne, precipitacione tehnike u gelu. U ovom radu objašnjene su teorijske osnove rasipanja svetlosti, prikazane su sheme i delovi instrumenata nefelometra i turbidimetra i opisane su imunonefelometrija i imunoturbidimetrija koje su u širokoj upotrebi u savremenim kliničkim laboratorijama

Strain differences in thymic atrophy in rats immunized for EAE correlate with the clinical outcome of immunization

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

Thymic changes as a contributing factor in the increased susceptibility of old Albino Oxford rats to EAE development

The study was aimed to examine putative contribution of thymic involution to ageing-associated increase in susceptibility of Albino Oxford (AO) rats to the development of clinical EAE, and vice versa influence of the disease on the progression of thymic involution. To this end we examined (i) the parameters of thymocyte negative selection efficacy, the thymic generation of CD4+CD25+Foxp3+ T regulatory cells (Tregs) and thymic capacity to instruct/predetermine IL-17-producing T-cell differentiation, and thymopietic efficacy-associated accumulation of “inflammescent” cytotoxic CD28- T cells in the periphery, and (ii) the key underlying mechanisms in young and old non-immunised AO rats and their counterparts immunised for EAE (on the 16th day post-immunisation when the disease in old rats reached the plateau) using flow cytometry analysis and/or RT-qPCR. It was found that thymic involution impairs: (i) the efficacy of negative selection (by affecting thymocyte expression of CD90, negative regulator of selection threshold and the expression of thymic stromal cell integrity factors) and (ii) Treg generation (by diminishing expression of cytokines supporting their differentiation/maturation). Additionally, the results suggest that thymic involution facilitates CD8+ T-cell differentiation into IL-17-producing cells (previously linked to the development of clinical EAE in old AO rats). Furthermore, they confirmed that ageing-related decrease in thymic T-cell output (as indicated by diminished frequency of recent thymic emigrants in peripheral blood) resulted in the accumulation of CD28- T cells in peripheral blood and, upon immunisation, in the target organ. On the other hand, the development of EAE (most likely by increasing circulatory levels of proinflammatory cytokines) contributed to the decline in thymic output of T cells, including Tregs, and thereby to the progression/maintenance of clinical EAE. Thus, in AO rats thymic involution via multi-layered mechanisms may favour the development of clinically manifested autoimmunity, which, in turn, precipitates the thymus atrophy