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Streptokoki skupine B (GBS) so vodilni vzrok obolevnosti in umrljivosti nosečnic, ploda in novorojenčkov po vsem svetu. Preklop med asimptomatskim kolonizatorjem v invazivnega patogena še ni popolnoma znan, zagotovo pa korelira z naborom številnih virulentnih dejavnikov. Enega izmed glavnih predstavlja kapsula, s pomočjo katere GBS vežejo regulatorne komponente komplementa in se izognejo prepoznavi z imunskim sistemom gostitelja. Nadalje je dokazano, da površinski protein β z vezavo faktorja H (FH) preprečuje opsonizacijo s C3b. Slednje smo v našem poskusu preverili z označevanjem s protitelesi anti-C3b/iC3b in anti-FH ter opazovali vezavo le-teh na kapsulo bakterij, ki smo jih predhodno izpostavili humanemu serumu. Pri tem smo spremljali tudi samo preživetje bakterij v serumu. Namen raziskave je bilo ugotoviti, če in kako sevi GBS, ki pripadajo različnim serotipom in vsebujejo različne virulentne dejavnike, vežejo in aktivirajo komplement. V študiji smo uporabili virulentno različne izolate GBS ter kontrolne seve E. coli z različnim tipom LPS. Vezavo komponent C3b/iC3b in FH smo interpretirali skupaj z rezultati viabilnosti. Razlike v vezavi C3b/iC3b in FH so bile opazne tako med sevi različnih serotipov kot tudi med sevi istega kapsularnega serotipa. Vendar pa smo ugotovili, da količina vezanega FH in C3b/iC3b ne vpliva vedno na povečano možnost preživetja. Prav tako nismo dokazali močne korelacije med izražanjem β proteina ter povečano viabilnostjo. Group B Streptococcus (GBS) is a major cause of maternal, fetal and neonatal morbidity and mortality worldwide. The transition from asymptomatic colonizer to invasive pathogen is not yet known, but it certainly correlates with a number of virulence factors. One of the most important is the capsule by which GBS binds regulatory components of the complement and thereby avoids recognition by the host immune system. In addition, the surface protein β has been shown to prevent opsonization by C3b by binding factor H (FH). In our experiment, we verified the latter by labeling with anti-C3b/iC3b and anti-FH antibodies and observed their binding to the capsule of bacteria previously exposed to the human serum. We also observed the survival of the bacteria in serum. The aim of the studywas to determine whether and how GBS strains, belonging to different serotypes and containing different virulence factors, bind and activate the complement system. In the study, virulently different GBS strains and control E. coli strains with different types of LPS were used. Binding of C3b/iC3b and FH was interpreted along with viability results. Differences in C3b/iC3b and FH binding were found between different GBS serotypes as well as between strains of the same capsular serotype. However, we found that the amount of FH and C3b/iC3b bound did not always increased survival. We also did not find a strong correlation between the expression of β protein and increased viability.

Konec decembra 2019 so iz Kitajskega mesta Vuhan poročali o izbruhu atipične pljučnice neznanega izvora. Analize genoma so pokazale, da gre za novega predstavnika koronavirusov, ki so ga poimenovali virus SARS-CoV-2. V manj kot enem letu od izbruha je virus zaradi visoke stopnje kužnosti povzročil pandemijo. Za njeno obvladovanje je bil razvoj učinkovitega cepiva nujno potreben. Do danes so bila odobrena 4 cepiva proti SARS-CoV-2, ki temeljijo na mRNA in adenovirusnih vektorjih. Veliko pozornosti je bilo usmerjene tudi v razvoj DNA-cepiv proti SARS-CoV-2. Učinkovitost DNA-cepiva je pogojena z metodo vnosa v tarčno tkivo. Ena od metod je elektroporacija, s katero povečamo permeabilnost celičnih membran in olajšamo vstop DNA v citoplazmo celic. Transfekcijo DNA v tarčno celico z elektroporacijo imenujemo genski elektroprenos. Glavni namen magistrske naloge je bil primerjati imunski odziv po genskem elektroprenosu DNA-cepiva proti virusu SARS-CoV-2 v mišico in kožo eksperimentalnih miši. Za genski elektroprenos smo pripravili mešanice plazmidnih DNA z geni, ki kodirajo za N in S proteina virusa SARS-CoV-2 in citokin IL-12. Imunski odziv smo vrednotili v vzorcih odvzetih na 13. in 28. dan po cepljenju. S komercialno dostopnimi testi ELISA smo določali prisotnost in koncentracije IFNγ (nespecifična imunost), ter titer anti-N in anti-S IgG protiteles (specifična humoralna imunost). S testom tetramerov smo določali delež CD8+N+ in CD8+S+ T celic (specifična celična imunost). Dokazali smo, da genski elektroprenos DNA-cepiva izzove tako nespecifično kot specifično imunost proti N in S proteinu. Ugotovili smo, da je aktivacija imunskega odziva intenzivnejša po genskem elektroprenosu v mišico in, da je v našem primeru N protein bolj imunogen od S proteina. Potrdili smo tudi, da je genski elektroprenos ustrezna metoda za vnašanje DNA-cepiva v tarčno tkivo. At the end of December 2019, an outbreak of atypical pneumonia of unknown origin was reported from the Wuhan, China. Genomic analysis has shown that it is a new representative of coronviruses, which has been named SARS-CoV-2. In less than a year after the outbreak, the virus caused a pandemic due to its high infectivity. The development of an effective vaccine was necessary for its control. To date, four SARS-CoV-2 vaccines based on mRNA and adenoviral vectors have been approved. A lot of attention has also been focused on the development of DNA-vaccines against SARS-CoV-2. The effectiveness of the DNA-vaccine depends on the method of introduction into the target tissue. One of the methods is electroporation, which increases the permeability of cell membranes and facilitates the entry od DNA into the cell cytoplasm. The transfection of DNA into a target cell by electroporation is called gene electrotransfer. The aim of the master's thesis was to compare the immune response after gene electrotransfer of the DNA-vaccine against the SARS-CoV-2 virus and cytokine IL-12 in the muscle and skin tissue. The immune response was evaluated in samples taken on the 13th and 28th day after vaccination. Commercially available ELISA tests were used to determine the presence and concentrations of IFNγ (non-specific immunity), as well as the titer of anti-N and anti-S IgG antibodies (specific humoral immunity). The tetramer test was used to determine portion of CD8+N+ and CD8+S+ T cells (specific cellular immunity). We have demonstrated that gene electrotransfer of DNA-vaccine elicits both non-specific and specific immunity against N and S protein. We found that the activation of the immune response is more intense after gene electrotransfer into the muscle and that in our case the N protein is more immunogenic than the S protein. We have also confirmed that gene electrotransfer is suitable method for introducing a DNA-vaccine into the target tissue.

NOD2 is an innate immune receptor that constitutes an important target for the development of small molecule immunopotentiators with great potential to be used as vaccine adjuvants. We report here the results of an in vivo study of the adjuvant properties of a desmuramylpeptide NOD2 agonist SG29 and its lipidated analogs featuring an adamantyl moiety or a stearoyl group. These compounds have been synthesized, incorporated into liposomes, and evaluated for their in vivo adjuvant activity. The characterization of liposome formulations of examined compounds revealed that their size increased in comparison to that of empty liposomes. The introduction of a stearoyl or an adamantane lipophilic anchor into the structure of SG29, to produce SG115 and ZSB63, respectively, substantially improved the in vivo adjuvant activity. Of note, the attachment of the stearoyl moiety produced a Th2-biased immune response, while the incorporation of the adamantyl moiety greatly enhanced the production of total IgG but mostly augmented the production of IgG2a antibodies, which indicated a shift toward a Th1 immune response. The identified bona fide capacity of ZSB63 to initiate a cellular immune response thus highlights its untapped potential as an alternative vaccine adjuvant. Nasl. z nasl. zaslona. Opis vira z dne 9. 12. 2022. Bibliografija: str. 8-9. Abstract. ARRS ARRS ARRS ARRS Croatian Science Foundation

Nucleotide-binding oligomerization domain 1 (NOD1) receptor and Toll-like receptor 4 (TLR4) belong to the family of pattern recognition receptors. Interactions between these receptors profoundly shape the innate immune responses. We previously demonstrated that co-stimulation of peripheral blood mononuclear cells (PBMCs) with D-glutamyl-meso-diaminopimelic acid (iE-DAP)-based NOD1 agonists and lipopolysaccharide (LPS), a TLR4 agonist, synergistically increased the cytokine production. Herein, we postulate that stimulation of NOD1 alone or a combined stimulation of NOD1 and TLR4 could also strengthen PBMC-mediated cytotoxicity against cancer cells. Initially, an in-house library of iE-DAP analogs was screened for NOD1 agonist activity to establish their potency in HEK-Blue NOD1 cells. Next, we showed that our most potent NOD1 agonist SZZ-38 markedly enhanced the LPS-induced cytokine secretion from PBMCs, in addition to PBMC- and natural killer (NK) cell-mediated killing of K562 cancer cells. Activation marker analysis revealed that the frequencies of CD69+, CD107a+, and IFN-γ+ NK cells are significantly upregulated following NOD1/TLR4 co-stimulation. Of note, SZZ-38 also enhanced the IFN-γ-induced PBMC cytotoxicity. Overall, our findings provide further insight into how co-engagement of two pathways boosts the non-specific immune response and attest to the importance of such interplay between NOD1 and TLR4. Nasl. z nasl. zaslona. Opis vira z dne 8. 8. 2022. Objavljeno 22. julija 2022. Št. članka: 920928. Abstract. Bibliografija: str. 11-12. ARRS, program ARRS, program ARRS, projekt

Pri hujšem poteku koronavirusne bolezni 2019 so nivoji vnetnih dejavnikov, kot sta interlevkin 6 (IL-6) in serumski amiloid A (SAA), izrazito povišani. Vlogo pri regulaciji teh dejavnikov bi lahko igrala tudi proti njim usmerjena avtoprotitelesa. Pri raziskovalnem delu znotraj magistrske naloge smo zato želeli določili vrednosti avtoprotiteles proti IL-6 in proti SAA v serumskih vzorcih zdravih krvodajalcev in bolnikov s koronavirusno boleznijo 2019 ter te vrednosti preučiti. Za določanje protiteles proti IL-6 smo postavili in ovrednotili novo hišno metodo ELISA. Določili smo potrebne parametre za ustrezno natančnost metode, specifičnost metode pa smo dokazali z inhibicijo v tekoči in v trdni fazi. V skupini krvodajalcev smo opazili, da spol in starost posameznika ne vplivata na vrednosti protiteles proti IL-6 in proti SAA. Glede na izmerjene nivoje protiteles proti SAA ni bilo razlik med skupinama, bolniki pa so imeli v primerjavi s krvodajalci signifikantno višje vrednosti protiteles proti IL 6 (p = 0,0018). Višja raven protiteles proti IL 6 pri bolnikih nakazuje na to, da ta protitelesa morda tvorijo komplekse z IL-6, ki bi lahko podaljšala čas zadrževanja IL-6 v krvnem obtoku. Preverjali smo tudi prisotnost korelacij med preiskovanimi dejavniki in opazili signifikantno korelacijo med protitelesi proti IL-6 in protitelesi proti SAA v obeh skupinah. V skupini krvodajalcev smo opazili tudi korelacijo med IL 6 in protitelesi proti SAA. Da bi ugotovili, kako se vrednosti preiskovanih protiteles spreminjajo tekom bolezni, smo nekaj izbranih bolnikov spremljali longitudinalno. Vrednosti preiskovanih protiteles so pri večjem deležu pregledanih bolnikov v času hospitalizacije upadle. Pri protitelesih proti SAA smo preverili tudi nevtralizacijsko sposobnost s funkcijskimi testi na celicah. Uporabljeni serumi so SAA nespecifično inhibirali, pri izolirani frakciji protiteles IgG pa nevtralizacijske sposobnosti nismo opazili. Levels of inflammatory factors such as interleukin 6 (IL-6) and serum amyloid A (SAA) are distinctly elevated in severe cases of coronavirus disease 2019. A role in regulation of such factors could be attributed to autoantibodies against them. The aim of our research was to determine the values of autoantibodies against IL-6 and against SAA in sera samples of healthy blood donors and coronavirus disease 2019 patients and to analyze these values. To determine the values of antibodies against IL-6 we set up and validated a new in-house ELISA. We determined the needed parameters for the adequate precision of the method and verified the specificity by fluid phase and solid phase inhibition. In the healthy blood donors’ group, we noticed that gender and age of an individual don’t influence the value of the antibodies against IL-6 and SAA. There was no significant difference in the levels of antibodies against SAA between the two groups, but the patients had significantly higher values of antibodies against IL-6 compared to blood donors (p = 0,0018). The higher levels of those antibodies among patients suggest that the antibodies might form complexes with IL-6, that could prolong their half-life in the circulation. We also checked for correlations among the examined factors and observed the significant correlation between antibodies against IL-6 and against SAA in both groups. We also noticed a significant correlation between IL-6 and antibodies against SAA. To see how the values of the examined antibodies change during the disease we chose the samples of some of the patients for longitudinal observation. The levels of the examined antibodies mostly decreased during the time of hospitalization. We also checked the neutralizing ability of the antibodies against SAA with functional tests on cells. The used sera have non-specifically inhibited SAA, but there was no neutralizing ability to be observed with isolated IgG fractions.

Preobilica plastičnih ostankov v okolju je vedno večja grožnja vsem organizmom v ekosistemu. Še posebej problematični so mikro- in nanoplastični delci, ki so pogosto skriti našim očem. Magistrsko nalogo smo si zastavili kot pregled raziskav o imunskem odzivu nevretenčarjev na izpostavitev plastičnim delcem. V člankih, objavljenih v podatkovni zbirki ScienceDirect, smo iskali ključne podatke o vrsti modelnega organizma, lastnostih uporabljenih plastičnih delcev, dodanih kemikalijah, koncentraciji plastičnih delcev in času izpostavitve. Pri analizi smo ločili odzive organizma na splošne ter imunske, ki so bili merjeni v hemolimfi ali direktno na hemocitih. V nadaljno analizo smo vključili le slednje. Ločeno smo analizirali celične imunske parametre in ekspresijo genov, povezanih z imunostjo. Med najbolj pogosto spremenjenimi parametri so bili: zmanjšana fagocitotska aktivnost, viabilnost ter gostota vseh hemocitov in stabilnost lizosomalne membrane, medtem ko so se vrednosti koncentracije reaktivnih kisikovih zvrsti v hemocitih, genotoksičnosti (poškodbe DNA) in aktivnosti lizocimov povečale. Med najbolj proučevanimi geni pa se je nivo izražanja najbolj očitno spremenil pri genih za lizocim, mitilin B in katalazo. Najpogosteje so izražanje spremenili geni, ki so povezani z apoptozo, delovanjem antimikrobnih peptidov ter funkcije prepoznavanja in signalizacije. Zaradi večinske uporabe PS plastičnih delcev, smo posebej analizirali imunski odziv na izpostavitev organizmov polistirenu. Analiza ni pokazala večjih odstopanj od zaključkov analize izpostavitve vsem možnim tipom plastičnih delcev. Zaključujemo, da imunski odziv organizmov ni odvisen od tipa in ostalih lastnosti plastičnih delcev. Prav tako imunski odziv organizma ni odvisen od koncentracije plastičnih delcev, saj analiza ni pokazala možnih korelacij. Zagotovo lahko trdimo, da ima izpostavitev organizmov plastičnim delcem vpliv in sproži imunski odziv. V prihodnjih študijah bi se bilo smotrno osredotočiti še na vpliv izpostavitve organizmov nanoplastiki, saj je na tem področju še veliko neznanega. The abundance of plastic debris in the environment is a growing threat to all organisms. Particularly problematic are micro- and nanoplastic particles. With this master's thesis we wanted to review studies on the immune response upon exposure of invertebrates to plastic particles. The articles, published in database ScienceDirect, were searched for key information on the type of model organism, the properties of the particles used, the chemicals added, the concentration of the particles and the exposure time. The analysis distinguished between general and immune responses, which were measured in haemolymph or directly on haemocytes. Only the latter were included in the further analysis. Cellular immune parameters and expression of immunity-related genes were analysed separately. The most frequently changed parameters were: decreased phagocytotic activity, viability, density of total haemocytes and LMS, while the concentration of ROS in haemocytes, genotoxicity and lysozyme activity increased. Among the most studied genes, changes in expression levels were observed for the lysozyme, mytillin B and catalase genes. The most commonly altered expression is in genes related to apoptosis, antimicrobial peptide action and recognition and signalling functions. Due to the predominant use of PS plastic particles, the immune response to exposure to polystyrene was analysed separately. The analysis showed no significant deviations from the conclusions of the exposure analysis for all possible types of plastic particles. We conclude that the immune response of the organisms is independent of the type and other properties of the plastic particles. The immune response is also independent of the concentration of particles, as the analysis showed no possible correlation. However it can certainly be argued that exposure of organisms to plastic particles triggers an immune response. Future studies should also focus on the impact of exposure of organisms to nanoplastics, as much is still unknown in this area.

Konjska astma je pogosta kronična bolezen dihal, ki pomembno vpliva na zdravje in dobrobit konj. Trenutne strategije zdravljenja konjske astme temeljijo na lajšanju simptomov in ne zdravijo bolezni. Lahko imajo tudi znatne stranske učinke. Cilj naše raziskave je bil oceniti lokalne in sistemske učinke intrabronhialne administracije avtolognih mezenhimskih matičnih celic, pridobljenih iz maščobnega tkiva (Adipose derived mesenchymal stem cells, AD-MSC) na potek konjske astme. Konje smo naključno razdelili v dve skupini in jih zdravili z AD-MSC ali deksametazonom. Za ugotavljanje terapevtskih učinkov smo natančno opredelili klinično izražanje bolezni pri posameznem konju in opravili meritve pljučnih funkcij in endoskopski pregled dihalnih poti. Ob endoskopiji smo opravili tudi bronhoalveolarno lavažo, izpirke pa citološko pregledali in izmerili vrednosti izražanja sporočilne RNK določenih vnetnih citokinov in koncentracijo njihovih beljakovin. Po končani obravnavi smo konje spremljali še eno leto in primerjali dolgoročno stabilnost kliničnih znakov med obema skupinama. Kljub temu, da zdravljenje z AD-MSC ni povzročilo takšnega izboljšanja kliničnih znakov astme kot zdravljenje kontrolne skupine, smo zaznali znatno izboljšanje pri izražanju in koncentraciji posameznih vnetnih spremenljivk. V primerjavi s kontrolnim zdravljenjem, pa je imelo zdravljenje z AD-MSC pozitiven dolgoročni učinek na stabilnost klinične slike. Na podlagi rezultatov te raziskave zaključujemo, da ima intrabronhialno zdravljenje z AD-MSC omejen kratkoročen učinek in pozitiven dolgoročen učinek na simptome kronične astme pri konjih. Equine asthma is a common chronic disease of the respiratory tract that significantly affects horses' health and well-being. Current therapeutic strategies are based on symptomatic treatment and do not alter the disease's long-term progression, and their side effects are worrying. Our study aimed to evaluate the local and systemic effects of intrabronchial administration of autologous adipose-derived mesenchymal stem cells (AD-MSC) on severe equine asthma. Horses were randomly divided into two groups and treated with AD-MSC or dexamethasone. To determine the therapeutic effects of intrabronchial treatment with AD-MSC, we precisely defined clinical signs, and performed lung function tests and endoscopic examination of the airways in horses included in the study. Bronchoalveolar lavage was performed, and the samples were cytologically examined, and the mRNA expression values of specific inflammatory cytokines and the concentration of their proteins were measured. After treatment, the horses were monitored for another year, and the long-term stability of clinical signs between the two groups was compared. Although non-inferiority of AD-MSC treatment was not confirmed, improvement in certain inflammatory variables was observed after cell therapy. However, intrabronchial treatment with AD-MSC had a positive long-term effect on clinical stability of horses with severe asthma. Results of the study indicate that intrabronchial treatment with AD-MSC has a limited short-term anti-inflammatory effect and a positive long-term clinical effect in asthmatic horses.

Alongside physiochemical properties (PCP), it has been suggested that the protein corona of nanoparticles (NPs) plays a crucial role in the response of immune cells to NPs. However, due to the great variety of NPs, target cells, and exposure protocols, there is still no clear relationship between PCP, protein corona composition, and the immunotoxicity of NPs. In this study, we correlated PCP and the protein corona composition of NPs to the THP-1 macrophage response, focusing on selected toxicological endpoints: cell viability, reactive oxygen species (ROS), and cytokine secretion. We analyzed seven commonly used engineered NPs (SiO2, silver, and TiO2) and magnetic NPs. We show that with the exception of silver NPs, all of the tested TiO2 types and SiO2 exhibited moderate toxicities and a transient inflammatory response that was observed as an increase in ROS, IL-8, and/or IL-1β cytokine secretion. We observed a strong correlation between the size of the NPs in media and IL-1β secretion. The induction of IL-1β secretion was completely blunted in NLR family pyrin domain containing 3 (NLRP3) knockout THP-1 cells, indicating activation of the inflammasome. The correlations analysis also implicated the association of specific NP corona proteins with the induction of cytokine secretion. This study provides new insights toward a better understanding of the relationships between PCP, protein corona, and the inflammatory response of macrophages for different engineered NPs, to which we are exposed on a daily basis.

MicroRNAs (miRNAs) are members of the non-coding regulatory RNA family that play pivotal roles in physiological and pathological conditions, including immune response. They are particularly interesting as promising therapeutic targets, prognostic and diagnostic markers due to their easy detection in body fluids and stability. There is accumulating evidence that different miRNAs provide disease-specific signatures in liquid samples of distinct kidney injuries. Using experimental models and human samples, there have been numerous suggestions that immune-related miRNAs are also important contributors to the development of different kidney diseases as well as important markers for monitoring response after kidney transplantation. However, there are limited data for understanding their function in the molecular pathways of allograft pathologies. In our review, we focused on microRNAs that are related to different aspects of immune response after kidney transplantation.

Klopni meningoencefalitis (KME) je virusna bolezen centralnega živčnega sistema (CŽS), ki jo povzroča virus klopnega encefalitisa (KMEV). Najbolj učinkovita zaščita pred KME je cepivo, vendar nekateri posamezniki zbolijo kljub cepljenju. Cepljeni bolniki s KME so v primerjavi z necepljenimi starejši, imajo enofazni potek bolezni in razvijejo hujše bolezenske znake. Zaradi vse več dokazov o vpletenosti imunskega sistema v patološke manifestacije KME, smo z doktorsko nalogo želeli raziskati, ali se funkcionalnost Toll-u podobnih receptorjev (TLR) 3, 7 in 8 ter aktivacija imunskega sistema pri bolnikih s KME, ki zbolijo kljub cepljenju proti bolezni, razlikuje od necepljenih bolnikov s KME. Z genotipizacijo enonukleotidnih polimorfizmov (SNP), povezanih s funkcionalnostjo TLR, smo ugotovili, da je frekvenca divjega alela za TLR8 pri cepljenih bolnicah s KME višja kot pri necepljenih bolnicah, zato bi lahko funkcionalni TLR8 predstavljal dejavnik tveganja za pojav KME kljub cepljenju. S stimulacijo mononuklearnih celic iz periferne krvi (PBMC) cepljenih in necepljenih bolnikov s KMEV in vitro smo poskusili poustvariti dogajanje v začetni fazi patogeneze KME. Ugotovili smo, da se cepljeni in necepljeni bolniki s KME ne razlikujejo po izražanju površinskih aktivacijskih molekul na dendritičnih celicah, smo pa pri cepljenih bolnikih s KME v zgodnjih časovnih točkah in vitro simulacije izmerili statistično značilno nižje koncentracije citokinov IFN-α, IL-12p70 in IL-15, kar bi lahko omogočilo obsežnejše virusno razmnoževanje in vodilo v šibkejšo aktivacijo drugih imunskih celic ter nižji protitelesni odziv. Po drugi strani pa smo v pozni točki in vitro stimulacije izmerili višjo koncentracijo IL-6, ki bi lahko ojačala vnetni odziv in prispevala k porušenju krvno-možganske pregrade. V kliničnih vzorcih cepljenih bolnikov s KME, odvzetih v nevrološki fazi bolezni, smo izmerili sistemsko povišane koncentracije vnetnih citokinov. Večina vnetnih citokinov, povezanih s prirojenim in Th1 celičnim odzivom, je bila koncentrirana v likvorju in je pozitivno korelirala z imunskimi celicami, kar kaže na lokalizacijo Th1 in vnetnih odzivov v CŽS. Cepljeni bolniki s KME so imeli še posebej izrazito povišan VEGF-A, ki bi lahko povzročil obsežnejše porušenje krvno-možganske pregrade in omogočil vstop imunskih celic v CŽS ter tako prispeval k oblikovanju intratekalnega vnetnega žarišča in k težjemu poteku bolezni. Obenem so imeli cepljeni bolniki s KME še dva meseca po pojavu nevroloških simptomov sistemsko povišane vrednosti IL-8/CXCL8 in GROα/CXCL1, kar kaže na dolgotrajno aktivacijo imunskega sistema. Obsežnejši in dlje časa trajajoč vnetni odziv pri cepljenih bolnikih s KME nakazuje na vpletenost imunskega odziva v pojav in potek bolezni pri tej skupini bolnikov s KME, vendar so za podrobnejše razumevanje mehanizma, ki leži za pojavom bolezni kljub cepljenju, potrebne še dodatne raziskave. Tick-borne meningoencephalitis (TBE) is a viral disease of the central nervous system (CNS) caused by tick-borne encephalitis virus (TBEV). The most effective protection against TBE is vaccination, however, despite highly immunogenic vaccine, vaccine breakthrough (VBT) infections still occur. VBT TBE patients are usually older, develop a monophasic disease and more severe symptoms compared to unvaccinated TBE patients. In contrast to unvaccinated TBE patients, VBT TBE patients are older and more often develop monophasic disease with more severe clinical symptoms. With growing evidence on pathological involvement of immune system in TBE, we aimed to investigate the functionality of Toll-like receptors (TLRs) 3, 7 and 8 and the activation of the immune system in VBT TBE patients. Genotyping of single nucleotide polymorphisms (SNPs) associated with TLR functionality showed that the frequency of the wild allele for TLR8 was higher in vaccinated females with TBE than in unvaccinated females with TBE, therefore functional TLR8 receptor could represent a risk factor for VBT TBE in women. To recreate the events in the initial phase of TBE pathogenesis, we stimulated peripheral blood mononuclear cells (PBMC) from VBT TBE and unvaccinated TBE patients with TBEV in vitro. Vaccinated and unvaccinated TBE patients did not differ in the expression of activation molecules dendritic cells after stimulation with TBEV in vitro, however, significantly lower concentrations of IFN-α, IL-12p70 and IL-15 were measured in supernatants of stimulated PBMCs from VBT patients in early time points. This could lead to more extensive viral replication and impair the activation of other immune cells. On the other hand, an observed increased IL-6 expression in VBT patients in late stages of in vitro stimulation, could support the extensive inflammatory responses and contribute to the blood-brain barrier disruption. Additionally, in acute phase of the disease, VBT TBE patients had systemically elevated inflammatory cytokines, with majority of them concentrated in cerebrospinal fluid and positively correlating with immune cell counts, indicating a localized Th1 and inflammatory responses in the central nervous system. Markedly elevated VEGF-A could lead to an extensive disruption of the blood-brain barrier, supporting immune cells entry and the formation of an intrathecal inflammatory focus, therefore contributing to a more severe disease. Also, VBT TBE patients had systemically upregulated levels of IL-8/CXCL8 and GROα/CXCL1 up to two months after the onset of neurological symptoms, indicating long-term systemic immune activation, which could explain the need for longer hospitalization of VBT TBE patients. An observed more extensive and prolonged inflammatory response in VBT TBE patients suggests an involvement of the immune response in the onset and development of the disease. However, additional studies are needed to better understand the mechanism behind vaccination breakthrough infections.

We compared the changes of selected immune parameters of Porcellio scaber to different stressors. The animals were either fed for two weeks with Au nanoparticles (NPs), CeO$_2$ NPs, or Au ions or body-injected with Au NPs, CeO$_2$ NPs, or lipopolysaccharide endotoxin. Contrary to expectations, the feeding experiment showed that both NPs caused a significant increase in the total haemocyte count (THC). In contrast, the ion-positive control resulted in a significantly decreased THC. Additionally, changes in phenoloxidase (PO)-like activity, haemocyte viability, and nitric oxide (NO) levels seemed to depend on the stressor. Injection experiments also showed stressor-dependant changes in measured parameters, such as CeO$_2$ NPs and lipopolysaccharide endotoxin (LPS), caused more significant responses than Au NPs. These results show that feeding and injection of NPs caused an immune response and that the response differed significantly, depending on the exposure route. We did not expect the response to ingested NPs, due to the low exposure concentrations (100 µg/g dry weight food) and a firm gut epithelia, along with a lack of phagocytosis in the digestive system, which would theoretically prevent NPs from crossing the biological barrier. It remains a challenge for future research to reveal what the physiological and ecological significance is for the organism to sense and respond, via the immune system, to ingested foreign material.

V zadnjem stoletju so cepiva najučinkovitejši način preprečevanja smrti zaradi nalezljivih bolezni. Napredek v znanosti je ključno gonilo razvoja cepiv, zato so raziskave na področju cepiv zelo pomembne. Raziskovanje različnih pristopov načrtovanja novih cepiv je pomembno orodje za izboljšanje njihovih lastnosti. Cepiva ločimo na živa in neživa. Razlika med prvo in drugo skupino je v tem, da prva vsebuje oslabljene patogene, ki se lahko v telesu razmnožujejo, druga pa vsebuje mrtve patogene ali njihove komponente. V doktorskem delu smo se osredotočili na peptide, ki tvorijo obvite vijačnice (ang. coiled coils, CC-peptide) kot nosilce, s katerimi bi združili imunološko aktivne komponente v učinkovito ne-živo cepivo. Zaradi svoje modularne narave, so nam CC-peptidi služili kot dobri gradniki za načrtovanje nanocepiva, ki omogoča hkratno dostavo antigena in adjuvansa v tarčno celico, kar je za učinkovitost cepiva zelo pomembno. Načrtovali smo več parov stabilnih in med seboj ortogonalnih CC-peptidov. Pri tem smo upoštevali dobro definirana pravila umeščanja posamezne aminokisline znotraj CC-peptida. Z energijskega vidika je najpomembnejši položaj tisti, ki tvori hidrofobno sredico, elektrostatske interakcije med nasprotno nabitimi aminokislinskimi ostanki pa so drugi pomemben doprinos k stabilizaciji obvite vijačnice ter ključne za selektivnost parjenja. Sintetične peptide smo, po določitvi njihovih lastnosti in vitro, smo jih uporabili kot dimerizacijske domene za sestavljanje cepljenih proteinov. Na ta način smo se prepričali, ali načrtovani peptidi ohranijo svoje lastnosti v fuziji z večjimi domenami (proteini), kar je bilo ključno za nadaljnje eksperimente. Peptidna para z najustreznejšimi lastnostmi smo uporabili kot sestavna dela nanoogrodja, kjer smo načrtovane peptide izrazili v fuziji s fluorescenčnimi proteini ter s celičnimi dostavnimi domenami. Fluorescenčni protein nam je omogočal spremljanje celične dostave s pomočjo konfokalne mikroskopije ali pretočne citometrije. Ker je za cepivo poleg izbire ustreznega antigena in adjuvansa pomembna tudi tarčna dostava v celice, smo preizkusili več dostavnih domen. Po izbiri najučinkovitejše dostavne domene, smo nanoogrodje uporabili kot nanocepivo s katerim smo tarčno dostavili antigen ter adjuvans (kratek flagelin) v modelno antigen predstavitveno celico. Dokazali smo, da s CC-interakcijami posredovana dostava imunsko aktivnih molekul izboljša vnetni odziv. In the last century, vaccines have been the most effective way to prevent deaths caused by infectious diseases. Advances in science are a key driver of vaccine development, so research in the field of vaccines is very important. Exploring different approaches to designing new vaccines is important for improvement of vaccine efficacy. Vaccines are separated in two types: live-attenuated and inactivated. The difference between the two is that the first contains attenuated pathogens that can reproduce in our body, while non-living ones contain dead pathogens or their components. In this doctoral dissertation, we focused on peptides that form coiled coils (CC-peptides) as carriers with which to combine immunologically active components into an effective inactivated vaccine. Due to their modular nature, CC-peptides have served as good building blocks for the design of a nanovaccine that allows simultaneous delivery of antigen and adjuvant to the target cell, which is important for vaccine effectiveness. We designed several sets of stable and orthogonal CC-peptides. Well-defined rules for individual position within a CC peptide were followed. Most important positions are those that form the hydrophobic core. Electrostatic interactions between oppositely charged amino acid residues are another important contribution to the stabilization of the coiled helix and crucial for selectivity. After determining the properties of synthetic peptides in vitro, they were used as dimerization domains for the assembly of split proteins. Using split proteins method, we made sure the designed peptides retained their properties in fusion with larger domains (proteins), which was crucial for further experiments. The peptide pairs with most suitable properties were used as components of the nanoscaffold, where the designed peptides were expressed in fusion with fluorescent proteins and cell delivery domains. The fluorescent protein added to the nanostructure allowed us to monitor cell delivery by confocal microscopy or flow cytometry. In addition to selecting the appropriate antigen and adjuvant, target delivery into cells is also important for vaccine efficacy. We thus focused on several delivery domains. After selecting the most efficient delivery domain, the nano framework was used as a nanovaccine to target the antigen and adjuvant (short flagellin) to the model antigen-presenting cell. We have shown that CC-mediated delivery of immunologically active molecules improves the inflammatory response.

The success of vaccination with subunit vaccines often relies on the careful choice of adjuvants. There is great interest in developing new adjuvants that can elicit a cellular immune response. Here, we address this challenge by taking advantage of the synergistic cross-talk between two pattern recognition receptors: nucleotide-binding oligomerization-domain- containing protein 2 (NOD2) and Toll-like receptor 7 (TLR7). We designed two conjugated NOD2/TLR7 agonists, which showed potent immunostimulatory activities in human primary peripheral blood mononuclear cells and murine bone-marrow-derived dendritic cells. One of these, 4, also generated a strong antigen-specific immune response in vivo, with a Th1-polarized profile. Importantly, our study shows that novel NOD2/TLR7 agonists elicit sophisticated and fine-tuned immune responses that are inaccessible to individual NOD2 and TLR7 agonists.

Bolezen COVID-19 je posledica okužbe z virusom SARS-CoV-2 pri ljudeh. Bolezen poteka raznoliko: od poteka bolezni brez kliničnih znakov, do kritične oblike, ki lahko privede do smrti. Glavni klinični znaki pri COVID-19 so vročina, kašelj in utrujenost, neredko pa se pojavlja tudi oteženo dihanje in tesnoba v prsih. V magistrski nalogi smo preiskovali morebitno vzročno povezavo med kliničnim potekom COVID-19 in specifičnim imunskim odzivom gostitelja na posamezne virusne beljakovine (N, S1, S2 in RBD). Dodatno nas je zanimalo tudi, ali potek bolezni (blažji, zmeren, resen ali kritičen) vpliva na koncentracijo protiteles proti posameznim virusnim beljakovinam in/ali na dinamiko protitelesnega imunskega odziva. V raziskavo smo vključili 79 bolnikov s COVID-19. Ugotovili smo, da ima v prvem vzorcu, ki je bil v povprečju odvzet v dveh tednih po dokazu okužbe s SARS-CoV-2, 64,6 % bolnikov protitelesa razreda IgG, 79,7 % bolnikov protitelesa razreda IgA in 60,8 % bolnikov protitelesa razreda IgM. Prav tako smo ugotovili, da se vsa protitelesa pojavijo že zelo zgodaj po okužbi in da najverjetneje jakost protitelesnega odziva vpliva na težo poteka bolezni. Ugotovili smo, da koncentracija protiteles proti RBD in N lahko vpliva na potek bolezni. Pri večini protiteles, glede na posamezne virusne epitope, nismo ugotovili razlike v dinamiki. Pričakovano dinamiko protiteles smo dokazali v primeru protiteles razreda IgA, usmerjenih proti virusni beljakovini N. Dodatno smo dokazali, da imata na potek bolezni in klinično sliko poleg jakosti imunskega odziva velik vpliv tudi spol in starost. SARS-CoV-2 virus is the causative agent of COVID-19. The disease progresses in a variety of ways. It varies from infection without clinical symptoms, to severe disease that can lead to death. The main clinical symptoms of COVID-19 are fever, cough, and fatigue, as well as shortness of breath and chest tightness. The aim of our study was to investigate the possible causal relationship between clinical manifestation of COVID-19 and the specific immune response to individual viral proteins (N, S1, S2 in RBD). In addition, we were interested in whether the course of the disease (mild, moderate, serious, or critical) is affected by the concentration of antibodies against individual viral proteins or the dynamic antibody immune response. The study included 79 patients with COVID-19. We found that in the first sample, which was on average obtained 2 weeks after a confirmed infection with SARS-CoV-2, 64.6% of patients had IgG antibodies, 79.7% of patients had IgA antibodies, and 60.8% had IgM antibodies against SASR-CoV-2. We also found that all antibodies appear soon after infection and that most likely the strength of the antibody response directly affects the severity of the disease. We found that the concentration of antibodies against RBD and N can affect the course of the disease. No differences in dynamics were found for most viral epitopes in most antibodies. The expected antibody dynamic was observed in the case of IgA antibodies specific against the N protein. Furthermore, we have shown that in addition to a strong immune response, gender and age also greatly impact the course of the disease and its clinical manifestation.

Microglia are resident macrophages in the central nervous system that are involved in immune responses driven by Toll-like receptors (TLRs). Microglia-mediated inflammation can lead to central nervous system disorders, and more than one TLR might be involved in these pathological processes. The cysteine peptidase cathepsin X has been recognized as a pathogenic factor for inflammation-induced neurodegeneration. Here, we hypothesized that simultaneous TLR3 and TLR4 activation induces synergized microglia responses and that these phenotype changes affect cathepsin X expression and activity. Murine microglia BV2 cells and primary murine microglia were exposed to the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)) and the TLR4 ligand lipopolysaccharide (LPS), individually and simultaneously. TLR3 and TLR4 co-activation resulted in increased inflammatory responses compared to individual TLR activation, where poly(I:C) and LPS induced distinct patterns of proinflammatory factors together with different patterns of cathepsin X expression and activity. TLR co-activation decreased intracellular cathepsin X activity and increased cathepsin X localization at the plasma membrane with concomitant increased extracellular cathepsin X protein levels and activity. Inhibition of cathepsin X in BV2 cells by AMS36, cathepsin X inhibitor, significantly reduced the poly(I:C)- and LPS-induced production of proinflammatory cytokines as well as apoptosis. Additionally, inhibiting the TLR3 and TLR4 common signaling pathway, PI3K, with LY294002 reduced the inflammatory responses of the poly(I:C)- and LPS-activated microglia and recovered cathepsin X activity. We here provide evidence that microglial cathepsin X strengthens microglia activation and leads to subsequent inflammation-induced neurodegeneration. As such, cathepsin X represents a therapeutic target for treating neurodegenerative diseases related to excess inflammation.

Nepravilnosti v odzivu limfocitov B na dražljaje tekom zorenja ali aktivacije vodijo do avtoimunskih obolenj, limfomov in levkemij. B-celični odziv je natančno reguliran proces, v katerem imajo ključno vlogo premreženje receptorja BCR, T-celično posredovani signali, stimulacija receptorjev TLR ter prisotnost citokinov in drugih ligandov za membranske receptorje, kot so na primer prostaglandini, v mikrookolju limfocitov B. Identifikacija novih molekul, ki sodelujejo v kompleksni signalni mreži limfocitov B, bo pripomogla k boljšemu razumevanju B-celičnega odziva in izboru obetavnih tarč za razvoj ciljanih terapij. V okviru doktorskega dela smo kot zanimive tarče za modulacijo apoptoze in proliferacije celic limfoma B predstavili serinske proteaze ter prostaglandinski receptor EP4. V prvem delu smo testirali nabor inhibitorjev serinskih proteaz, sintetiziranih na Fakulteti za farmacijo, na zmožnost indukcije apoptoze na človeških in mišjih linijah limfoma B. Ugotovili smo, da so spojine, zasnovane na azafenilalaninskem skeletu, citotoksične za celice limfoma B in povzročajo apoptotično celično smrt. Te spojine so povzročile biokemijske in morfološke značilnosti apoptoze: aktivacijo kaspaz, izgubo mitohondrijskega potenciala, razpad celičnega jedra in fragmentacijo DNA. Večina inhibitorjev je bila selektivnih za trombin, s konstantami inhibicije v nanomolarnem območju, vendar so močno inhibirali vsaj še eno dodatno serinsko proteazo (tripsin, kimotripsin in/ali faktor koagulacije X). V drugem delu doktorskega dela smo se osredotočili na prostaglandinski receptor EP4, ki v limfocitih B posreduje inhibitorne učinke endogenega liganda PGE2. Iz naših rezultatov lahko sklepamo, da agonist receptorja EP4 modulira učinke posredovane preko receptorjev BCR in TLR4. Stimulacija receptorjev BCR in TLR4 namreč poveča ekspresijo EP4, stimulacija receptorja EP4 pa nato kot pozitivna povratna zanka pospešuje učinke specifične vezave antigena na receptor BCR in kot negativna povratna zanka zavira nespecifično aktivacijo preko poliklonalnega aktivatorja LPS. Vsaj del teh učinkov je posredovan preko inhibicije aktivacije transkripcijskega faktorja NFκB. Nizke koncentracije ireverzibilnega inhibitorja serinskih proteaz TPCK prav tako zavirajo aktivacijo NFκB, ne da bi signifikantno vplivale na proliferacijo celic WEHI-231.Kombinacija razmeroma netoksičnih koncentracij TPCK in agonista EP4 (PGE1-OH) pa je za celice usodna, saj povzroči popolno smrt celic. Predvidevamo, da je za ta učinek ključna sinergistična inhibicija aktivacije NFκB. Many diseases of the immune system such as autoimmune diseases, lymphomas and leukemias are caused by defects in the process of B cell maturation and activation. Various signals contribute to the tightly regulated B cell response, among which are crosslinking of BCR, T-cell help, TLR stimulation and the presence of cytokines and other ligands for membrane receptors, such as prostaglandins, in the B cell microenvironment. The identification of novel molecules that participate in the complex B cell signaling network will contribute to a better understanding of B cell responses and give rise to novel molecular targets for drug discovery and development. In the present work we present two novel molecular targets for the modulation of B cell responses: serine proteases and the prostaglandin receptor EP4. In the first part of our research we tested if a series of inhibitors of serine proteases synthesized at the Faculty of Pharmacy were able to induce apoptotic cell death in human and murine B lymphoma cell lines. We have shown that compounds built on an azaphenylalanine scaffold are severely cytotoxic for B-cell lymphoma cells and they provoke biochemical and morphological changes typical of apoptosis, such as DEVDase activation, loss of mitochondrial membrane potential, nuclear degradation and genomic DNA fragmentation. Most of the inhibitors proved to be selective for thrombin, with inhibition constants (Ki) in the nanomolar range. However, they could also inhibit at least one additional serine protease (trypsin, chymotrypsin and/or coagulation factor X) with Ki values in the nanomolar or low micromolar range. These serine protease inhibitors constitute novel apoptosis inducing compounds in B-cell lymphoma cells. In the second part of our work we focused on the prostaglandin receptor EP4, which is the principal molecule conveying the growth-suppressive effect of prostaglandin E2. Our results suggest that an agonist of the EP4 receptor modulates the effects mediated by the receptors BCR and TLR4. We have shown that both receptors up-regulate the expression of EP4, which stimulation can in turn act as a positive feedback loop increasing the inhibitory effect of BCR crosslinking and as a negative feedback loop suppressing the proliferatory effect of TLR4 signaling. In WEHI-231 cells we demonstrated that this effect is at least in part mediated by the inhibition of activation of the transcription factor NFκB. Non-cytotoxic concentrations of the irreversible inhibitor of serine proteases TPCK were also able to inhibit NFκB activation. However, the combination of low concentrations of TPCK and an EP4 agonist (PGE1-OH) proved to be lethal for WEHI-231 cells, causing cell death. We suggest that this effect is caused by a synergistic inhibition of NFκB activation.

Highlights • Immune parameters are slightly changed upon exposure to plastic fibres or crumb rubber. • Chlorpyrifos caused significant changes in Porcellio scaber immune parameters. • Microplastics decreased the bioavailability of chlorpyrifos for P. scaber. • Mixtures of plastic fibres and chlorpyrifos resulted in greater response in haemocyte count. Microplastics and agrochemicals are common pollutants in terrestrial ecosystems. Their interaction during coexistence in soils may influence their fate and adverse effects on terrestrial organisms. The aim of this study was to investigate how the exposure to two types of microplastics; polyester fibres, and crumb rubber; induce changes in immune parameters of Porcellio scaber and if the co-exposure of microplastics affects the response induced by the organophosphate pesticide chlorpyrifos. A number of immune parameters, such as total haemocyte count, differential haemocyte count, and phenoloxidase-like activity were assessed. In addition, the acetylcholinesterase (AChE) activity in the haemolymph was evaluated as a measure of the bioavailability of chlorpyrifos. After three weeks of exposure, the most noticeable changes in the measured immune parameters and also a significantly reduced AChE activity were seen in chlorpyrifos-exposed animals. Both types of microplastic at environmentally relevant concentrations caused only slight changes in immune parameters which were not dependent on the type of microplastic, although the two types differed significantly in terms of the chemical complexity of the additives. Mixtures of chlorpyrifos and microplastics induced changes that differed from individual exposures. For example, alterations in some measured parameters suggested a reduced bioavailability of chlorpyrifos (AChE activity, haemocyte viability) caused by both types of microplastics exposure, but the increase of haemocyte count was promoted by the presence of fibres implying their joint action. In conclusion, this study suggests that immune processes in P. scaber are slightly changed upon exposure to both types of microplastics and microplastics can significantly modulate the effects of other co-exposed chemicals. Further research is needed on the short-term and long-term joint effects of microplastics and agrochemicals on the immunity of soil invertebrates.

NOD1 and NOD2 are pattern recognition receptors that have important roles in innate immune responses. Although their overactivation has been linked to a number of diseases, NOD2 in particular remains a virtually unexploited target in this respect, with only one structural class of antagonist reported. To gain insight into the structure-activity relationships of NOD2 antagonists, a series of novel analogs was designed and synthesized, and then screened for antagonist activity versus NOD2, and counter-screened versus NOD1. Compounds 32 and 38 were identified as potent and moderately selective NOD2 antagonists, and 33 and 42 as dual NOD1/NOD2 antagonists, with balanced activities against both targets in the low micromolar range. These data enable in-depth exploration of their structure-activity relationships and provide deeper understanding of the structural features required for NOD2 antagonism. Članek: št. 112089. Bibliografija: str. 14-15. Abstract. ARRS COST action CA15135 “Multi-target para- digm for innovative ligand identification in the drug discovery proces

V raziskavi smo želeli ugotoviti pojavnost okužb dihal povzročenih z bakterijo M. pneumoniae pri slovenskih bolnikih v obdobju desetih let, spoznati kateri genotipi so bili v tem obdobju navzoči ter proučiti, če obstajajo med posameznimi genotipi razlike v patogenosti in vitro. Pregledali smo podatke iz strokovno zdravstvene dejavnosti za dokaz okužb z M. pneumoniae in jih analizirali. Shranjene izolate M. pneumoniae smo uporabili za genotipizacijo bakterije na podlagi gena za beljakovino P1 s pirosekvenciranjem in dodatno še z metodo MLVA. Pet genotipov smo uporabili za okužbo celic A549 in merili hitrost razmnoževanja mikoplazem, citotoksični učinek na gostiteljske celice ter tvorbo provnetnih citokinov IL-1β, IL-6, IL-8, TNF-α in IFN-γ. Ugotovili smo, da smo v obdobju med 2006 in 2015 v Sloveniji imeli dve epidemiji (2010/11 in 2014/15). V prvi epidemiji je prevladoval P1 tip 2, medtem ko v drugi P1 tip 1. Skupaj smo odkrili devet genotipov MLVA od katerih so bili trije (MLVA-3,5,6,2 MLVA-3,6,6,2 in MLVA-4,5,7,2) navzoči stalno in predstavljali 95,2 % vseh izolatov. Med petimi izbranimi genotipi MLVA nismo opazili razlik v hitrosti razmnoževanja ali citotoksičnosti. Celice so se na okužbo z vsemi testiranimi genotipi, razen MLVA-3,5,6,2, odzvale enako: z visoko tvorbo IL-6 in IL-8, manjšim porastom IL-1β ter brez odziva s TNF-α ali IFN-γ. Za genotip MLVA-3,5,6,2 smo opazili zavrto tvorbo IL-6 glede na negativno kontrolo ter statistično značilno manjšo tvorbo IL-8 glede na ostale genotipe. Vse skupaj nakazuje ne morebiten vpliv genetske različice bakterije na odziv gostiteljskih celic. The aims of this study were to assess the epidemiology of M. pneumoniae respiratory tract infections in Slovenian patients during a 10-year period, to recognize which genotypes were present in the study population and to investigate if different genotypes show diverse in vitro pathogenicity potentials. We analysed the results from routine diagnostics for the detection of M. pneumoniae and used stored M. pneumoniae isolates for genotyping (P1 typing and MLVA). Five genotypes were used for in vitro infection of A549 cells in order to determine bacterial growth, cytotoxicity and pro-inflammatory cytokine (IL-1β, IL-6, IL-8, TNF-α and IFN-γ) production. The results showed that in the period from 2006 to 2015 two distinct epidemics occurred (2010/11 and 2014/15) and that the dominant P1 type shifted from P1 type 2 to P1 type 1 just prior to the second epidemic. All together we detected nine MLVA genotypes from which three (MLVA-3,5,6,2 MLVA-3,6,6,2 and MLVA-4,5,7,2) were present during the whole study period and represented 95.2 % of isolates. We found no differences in bacterial growth dynamics or cytotoxicity for five selected MLVA genotypes. The host cells responded to all genotypes except MLVA-3,5,6,2 by producing IL-1β, IL-6 and IL-8 but not TNF-α or IFN-γ. For the genotype MLVA-3,5,6,2 we observed an inhibition of IL-6 production and more than half less IL-8 response. These results may indicate a possible impact of bacterial genotype on the immune response of host cells.

Manipulacija črevesne mikrobiote s koristnimi mikroorganizmi predstavlja obetavno alternativo in podporo za zdravljenje črevesnega vnetja in motenj v delovanju črevesne epitelne bariere. Probiotiki lahko namreč stimulirajo tako pridobljeni kot tudi naravni imunski odziv. Namen raziskave je bil zato pojasniti signalne poti, ki jih v črevesnih epitelnih celicah sprožijo probiotični bakterijski sevi Lactobacillus gasseri K7 (K7), Lactobacillus fermentum L930BB (L930BB), Bifidobacterium animalis subsp. animalis IM386 (IM386) in Lactobacillus plantarum WCFS1 (WCFS1). Izhodišče raziskave je bila in vivo študija, v kateri so na miših s povzročenim kolitisom proučevali zaščitni vpliv sevov L930BB in IM386. Rezultati analize transkriptoma mišjega kolona so pokazali spremembe v izražanju genov, ki so povezani z aktivacijo proti-apoptostskih poti preko fosfatidil inozitol 3-kinaze (PI3K)/Akt in aktivacijo poti, ki vodijo v regulacijo aktinskega citoskeleta in tesnih stikov preko protein-kinaze C (PKC). Tako reorganizacija aktinskega citoskeleta, kot tudi zmanjšana apoptoza, prispevata k obnavljanju monosloja črevesnih epitelnih celic. V in vitro poskusih smo dokazali, da so aktivirane signalne poti posledica signalizacije preko receptorja naravne imunosti, Toll-u podobnega receptorja 2 (TLR2). Z uporabo pretočne citometrije smo potrdili, da so probiotični sevi in ligand za TLR2 sposobni zmanjšati s citokini povzročeno celično smrt. S pregledovanjem črevesne epitelne celične linije Caco-2 pod konfokalnim mikroskopom pa smo opazili, da probiotični sevi, ob simulaciji vnetnih razmer s H2O2, zmanjšajo internalizacijo proteina tesnih stikov ZO-1 in tako stabilizirajo medcelične povezave. V stanju vnetja se je ob dodatku probiotičnih sevov, zmanjšala tudi permeabilnost celičnega monosloja, kar dodatno dokazuje zaščitni vpliv izbranih sevov in njihovo zmožnost podpore vzdrževanja črevesne epitelne bariere. Manipulation of the intestinal microbiota with beneficial microorganisms represents a promising alternative and support to the treatment of intestinal inflammation and disorders in intestinal epithelial barrier function. Probiotics can stimulate both the adaptive and innate immune responses. The aim of the study was therefore to clarify the signalling pathways in intestinal epithelial cells triggered by probiotic bacterial strains Lactobacillus gasseri K7 (K7), Lactobacillus fermentum L930BB (L930BB), Bifidobacterium animalis subsp. animalis IM386 (IM386) and Lactobacillus plantarum WCFS1 (WCFS1). The starting point of the research was an in vivo study in which the protective effect of strains L930BB and IM386 was examined in a mouse model with induced colitis. Results of mice colon transcriptome analysis revealed changes in gene expression, involved in activation of anti-apoptotic pathways through phosphatidylinositol 3-kinase (PI3K)/Akt and activation of pathways that lead to regulation of actin cytoskeleton and tight junctions through protein kinase C (PKC). Reorganisation of actin cytoskeleton and decreased apoptosis are both helpful in intestinal epithelial cell monolayer reconstitution. We proved in in vitro experiments that activated signalling pathways are a consequence of signalization through the innate immune receptor Toll-like receptor 2 (TLR2). With the use of flow cytometry we confirmed that probiotic strains and TLR2 ligand are able to reduce cytokine induced cell death. By examining the intestinal epithelial cell line Caco-2 under the confocal microscope, we observed that probiotic strains, in addition to the simulation of inflammatory conditions with H2O2, reduce the internalization of the tight junction protein ZO-1 and thus stabilize intercellular connections. In inflammatory conditions, the addition of probiotic strains also reduced the permeability of the cell monolayer, further proving the protective effect of selected strains and their ability to support the maintenance of the intestinal epithelial barrier.

Dendritične celice se zaradi svoje osrednje vloge pri prepoznavanju in predstavitvi antigenov ter aktivaciji imunskega odziva v vedno večjem obsegu uporabljajo za pripravo celičnih cepiv pri raziskavah zdravljenja raka. Za aktivacijo tumorsko specifičnih citotoksičnih CD8+ limfocitov T sta bistvenega pomena popolna stopnja zrelosti in visoka migracijska sposobnost dendritičnih celic v sekundarne limfatične organe. Postopek ex vivo priprave dendritičnih celic zajema diferenciacijo in zorenje v gojišču, ki mora zagotavljati optimalno okolje. Želeli smo raziskati potencialno uporabo lizata alogenskih trombocitov kot dodatka brezserumskemu gojišču za diferenciacijo, zorenje in gojenje človeških dendritičnih celic, pri čemer smo se osredotočili na karakterizacijo ključnih funkcijskih lastnosti. Iz zgoščene levkocitne plasti periferne krvi smo s pomočjo imunomagnetne selekcije izolirali CD14+ monocite in jih diferencirali v nezrele dendritične celice v gojišču RPMI 1640 z dodanimi 10 % lizata človeških alogenskih trombocitov. Kot referenčno gojišče smo uporabili brezserumski medij CellGenix® z 1 % človeškega seruma AB. Z uporabo treh kombinacij agonistov Tollu podobnih receptorjev in vnetnih citokinov smo pridobili zrele dendritične celice z različno sposobnostjo migracije in stimulacije alogenskih limfocitov T. Dodatek lizata trombocitov je omogočil diferenciacijo in nastanek zrelih dendritičnih celic z visoko sposobnostjo migracije v smeri kemokina CCL19 ter sposobnostjo alo-stimulacije CD4+ limfocitov T. Izmerjene visoke koncentracije interferona-?, ki so ga izločale celice T po stimulaciji z dendritičnimi celicami, so potrdile polarizacijo naivnih CD4+ limfocitov T v celice T pomagalke tipa 1. Dendritične celice, ki smo jih gojili v prisotnosti lizata trombocitov, so izkazovale zmanjšano sposobnost za alo-stimulacijo CD8+ limfocitov T v primerjavi s celicami iz referenčnega gojišča. Na aktivacijo z dendritičnimi celicami iz gojišča z lizatom trombocitov so se CD8+ limfociti T odzvali z znižanim izločanjem interferona-? in oslabljenim znotrajceličnim izražanjem grancima B v primerjavi z uporabo dendritičnih celic iz brezserumskega medija, kar kaže na znižano citotoksično aktivnost. Ugotovili smo, da je prisotnost lizata alogenskih trombocitov izboljšala kemotaktično migracijsko sposobnost, a je hkrati delovala inhibitorno na alo-stimulacijsko funkcijo dendritičnih celic. Rezultati našega raziskovalnega dela bi lahko predstavljali izhodišče za proučevanje uporabe lizata trombocitov za pripravo dendritičnih celic, usmerjenih v druga aktivacijska stanja, na primer z imunosupresivnimi lastnostmi. Because of their central role in recognition and presentation of antigens as well as in activation of immune response, dendritic cells are increasingly being used for cell vaccine preparation in research for cancer treatment. To induce tumour-specific immune response of cytotoxic CD8+ T cells, the complete maturation status and high migratory capacity of dendritic cells to the secondary lymphoid organs are required. Ex vivo generation of dendritic cells includes differentiation and maturation in the cell culture medium that must provide an appropriate environment. The aim of this study was to investigate the potential use of allogeneic platelet lysate as a serum-free medium supplement for differentiation, maturation, and cultivation of human dendritic cells. We focused on the characterisation of key functional properties. Using immunomagnetic selection we isolated CD14+ monocytes from the buffy coat and differentiated them into immature dendritic cells in RPMI 1640 cell culture medium supplemented with 10% of human platelet lysate. As a reference, we used serum-free CellGenix® medium supplemented with 1% of human AB serum. By culturing dendritic cells in the presence of three different maturation cocktails consisting of Toll-like receptor agonists and inflammatory cytokines, we obtained dendritic cells with different migratory capacity and the capacity to induce allogeneic T-cell responses. The addition of platelet lysate enabled the differentiation and maturation of dendritic cells with a high capacity of migration towards chemokine CCL19 and a potential to stimulate allogeneic CD4+ T cells. High interferon-? concentrations, measured after T-cell activation with dendritic cells, confirmed the capacity of dendritic cells to induce type 1 polarisation of na?ve CD4+ T cells. Dendritic cells generated in the presence of platelet lysate displayed lower capacity to stimulate allogeneic CD8+ T cells in comparison to dendritic cells from the reference medium. After stimulation with dendritic cells generated in the presence of platelet lysate, CD8+ T cells displayed reduced cytotoxic activity with lower interferon-? production and decreased intracellular granzyme B expression. The presence of allogeneic platelet lysate improved the chemotactic migratory potential, but displayed inhibitory effect on the allo-stimulatory function of dendritic cells. The results of our study could open the possibility of investigating the use of platelet lysate for generation of dendritic cells focused on other activation stages, for example, with immunosupressive properties.

Izhodišča. O dejavnikih, ki določajo, kakšna bosta potek in izid klopnega meningoencefalitisa (KME), o (dolgo)trajnih posledicah prebolele bolezni (postencefalitisni sindrom) in o mehanizmih, ki so povezani z neugodnim potekom in izidom bolezni, je malo znanega. Namen. Namen raziskave je opredeliti klinične značilnosti akutne bolezni, ugotoviti, kateri klinični in/ali laboratorijski parametri so povezani s težjim potekom bolezni in/ali neugodnim izidom, oceniti (dolgo)trajne posledice, ki jih pušča prebolela bolezen, in osvetliti osnovne imunske značilnosti KME. Metode dela. V raziskavo smo vključili 717 odraslih bolnikov, ki so bili v obdobju od 1. 1. 2007 do 31. 12. 2012 na Kliniki za infekcijske bolezni in vročinska stanja zdravljeni zaradi KME. Pri podskupini 420 bolnikov, ki smo jih povabili na kontrolni pregled leta 2014, 2–7 let po prebolelem KME, smo ocenili dolgoročni izid bolezni. V raziskavo smo vključili tudi kontrolno skupino oseb, ki so se z bolniki ujemale glede na starost in v preteklosti niso prebolele KME. Imunske odzive smo v obdobju akutne bolezni ocenili v serumu in možganski tekočini, 2 meseca in 2–7 let po preboleli bolezni pa le v serumu. Določili smo koncentracije 24 citokinov/kemokinov, ki zastopajo prirojene ter pridobljene Th1-, Th17- ter B-celične odzive. Rezultati. Multipla regresija je pokazala, da so s težjim potekom KME značilno povezani višja starost bolnikov (razmerje obetov (RO) 1,26, 95 % interval zaupanja (IZ) 1,11–1,44 P = 0,001), predhodno cepljenje proti KME (RO 14,23, 95 % IZ 1,72–117,87 P = 0,014), nizka koncentracija protiteles IgG proti virusu KME v serumu (RO 0,85, 95 % IZ 0,75–0,96 P = 0,009), večja koncentracija levkocitov v krvi (RO 1,45, 95 % IZ 1,13–1,85 P = 0,004) ter večja koncentracija C-reaktivnega proteina v serumu (ocenjeni koeficient 1,20, 95 % IZ 0,48–1,91 P = 0,001). Pogostost postencefalitisnega sindroma se zmanjšuje s časom in se stabilizira 12 mesecev po preboleli bolezni, ko ima postencefalitisni sindrom tretjina bolnikov kasneje se zmanjšuje le še njegova teža. Neugoden izid 6 mesecev po preboleli bolezni je povezan s koncentracijo levkocitov v možganski tekočini (RO 1,43, 95 % IZ 1,07–1,93, P = 0,017), po 12 mesecih z izidom bolezni po 6 mesecih (RO 497,89, 95 % IZ 39,42–6289,16, P < 0,001) in 2–7 let po preboleli bolezni z izidom bolezni po 12 mesecih (RO 38,56, 95 % IZ 8,40–177,14, P < 0,001). Bolniki so 2&#8210 7 let po prebolelem KME navedli večje število nespecifičnih simptomov v primerjavi s kontrolnimi preiskovanci, ki niso preboleli KME, prisotnost vsakega od ponujenih simptomov pa bolj pogosto in bolj stalno. Slabše so ocenili tudi kakovost telesnega in duševnega zdravja, vendar so bile razlike med skupinama majhne in niso bile statistično značilne. Koncentracije velike večine citokinov/kemokinov v serumu in možganski tekočini se v času akutne bolezni značilno razlikujejo: koncentracije citokinov/kemokinov prirojenega in Th1-pridobljenega imunskega odziva so večje v možganski tekočini, tiste, povezane s pridobljenim Th17- in B-celičnim imunskim odzivom, pa v serumu. Večina predstavnikov prirojenega in pridobljenega Th1-imunskega odziva je povezana s težo akutne bolezni, z neugodnim izidom bolezni pa nismo dokazali statistično značilnih povezav. Sklep. Raziskava, temelječa na velikem številu dobro definiranih odraslih bolnikov s KME, je omogočila določitev dejavnikov, ki so povezani s težkim potekom in neugodnim izidom KME, ter oceno pogostosti in kliničnih značilnosti postencefalitisnega sindroma. Omogočila je tudi vpogled v imunske značilnosti akutne bolezni ter v vnetne parametre, povezane s težko akutno boleznijo in neugodnim izidom. Background. Information on parameters associated with the severity of tick-borne encephalitis (TBE), on the long-term outcome (post-encephalitic syndrome), and on the mechanisms responsible for the unfavorable clinical course and outcome of TBE are limited. Aims. The aims of the study were to describe the clinical characteristics of acute TBE, to determine the clinical and/or laboratory parameters associated with the severity of acute illness and/or unfavorable outcome, to assess the frequency and severity of post-encephalitic syndrome at different time points after TBE, and to highlight the basic immune characteristics of TBE. Methods. Seven hundred seventeen adult patients, diagnosed with TBE and hospitalized at the Department of Infectious Diseases, University Medical Centre Ljubljana, in the period from 1. 1. 2007 to 31. 12. 2012, qualified for the study. In a subgroup of 420 patients who participated in a follow-up visit in 2014, i.e. 2–7 years after TBE, we assessed the long-term outcome of the disease. The study also included control group of subjects of the same age (± 5 years). Immune responses were evaluated in serum and cerebrospinal fluid in the period of acute illness, and in the serum 2 months and 2–7 years after the onset of illness. The concentrations of 24 cytokines /chmokines that represent innate and adaptive Th1, Th17 and B cell responses were determined. Results. Multivariate regression showed that patient age (odds ratio (OR) 1.26, 95% confidence interval (CI) 1.11–1.44 P = 0.001), previous vaccination against TBE (OR 14.23, 95% CI 1.72–117.87 P = 0.014), low level of specific TBE virus serum IgG antibodies (OR 0.85, 95% CI 0.75–0.96 P = 0.009), blood leukocyte count (OR 1.45, 95% CI 1.13–1.85 P = 0.004), and serum C-reactive protein level (estimated coefficient 1.20, 95% CI 0.48–1.91 P = 0.001) were associated with severe acute illness. The frequency of post-encephalitic syndrome diminished over time and stabilized 12 months after the acute illness when it was present in one-third of patients whereas the severity of post-encephalitis syndrome continued to decline. Unfavourable outcome at 6 months was associated with cerebrospinal fluid leukocyte count (OR 1.43, 95% CI 1.07–1.93, P = 0.017), at 12 months with the disease outcome at 6 months (OR 497.89, 95% CI 39.42–6289.16, P < 0.001), and at the final visit with disease outcome at 12 months (OR 38.56, 95% CI 8.40–177.14, P < 0.001). Unspecific symptoms 2–7 years after TBE were more frequent and more constant in patients than in the control group. Similarly, patients have poorly assessed their physical and mental health, but the differences between the groups were small and were not statistically significant. The concentrations of most cytokines/chemokines in serum and cerebrospinal fluid were significantly different during the time of acute illness: cytokines and chemokines associated with innate immune responses and Th1 adaptive immune responses were significantly higher in cerebrospinal fluid, whereas mediators associated with Th17 and B cell immune responses were generally higher in serum. The majority of studied cytokines/chemokines of innate and adaptive Th1 immune responses were associated with the severity of acute illness, while statistically significant correlations with the unfavorable outcome of the disease have not been found. Conclusion. A study based on a large number of well-defined adult patients with TBE enabled the identification of parametrs associated with the severity of acute illness and/or unfavourable (long-term) outcome of TBE, and the assessment of the frequency and severity of postencephalitic syndrome. It also provided an insight into the immune characteristics of acute illness and inflammatory parameters associated with severe acute illness and an unfavorable outcome of TBE.

The biphasic course of tick-borne encephalitis (TBE) is well described, but information on the monophasic course is limited. We assessed and compared the clinical presentation, laboratory findings, and immune responses in 705 adult TBE patients: 283 with monophasic and 422 with biphasic course. Patients with the monophasic course were significantly (p ≤ 0.002) older (57 vs. 50 years), more often vaccinated against TBE (7.4% vs. 0.9%), more often had comorbidities (52% vs. 37%), and were more often treated in the intensive care unit (12.4% vs. 5.2%). Multivariate logistic regression found strong association between the monophasic TBE course and previous TBE vaccination (OR = 18.45), presence of underlying illness (OR = 1.85), duration of neurologic involvement before cerebrospinal fluid (CSF) examination (OR = 1.39), and patients’ age (OR = 1.02). Furthermore, patients with monophasic TBE had higher CSF levels of immune mediators associated with innate and adaptive (Th1 and B-cell) immune responses, and they had more pronounced disruption of the blood–brain barrier. However, the long-term outcome 2–7 years after TBE was comparable. In summary, the monophasic course is a frequent and distinct presentation of TBE that is associated with more difficult disease course and higher levels of inflammatory mediators in CSF than the biphasic course, however, the long-term outcome is similar.

Probiotiki, ki so trenutno na tržišču, so omejeni le na nekaj vrst mikroorganizmov. Med njimi prevladujejo predstavniki rodov laktobacilov in bifidobakterij. Znano pa je, da se v našem prebavnem traktu nahaja veliko več rodov bakterij, ki jih znanstveniki še preučujejo v upanju, da postanejo probiotiki nove generacije. Za razliko od konvencionalnih probiotikov, ki imajo zgodovino varne uporabe v prehrani ljudi, je pri slednjih potrebno poleg učinkovitosti, dokazati tudi varnost. Nobena od obravnavanih vrst namreč še nima statusa QPS. Med temi obetavnimi bodočimi probiotiki izpostavljajo predvsem bakterijske vrste Feacalibacterim prausnitzii, Akkermansia muciniphila in Eubacterium hallii. Predstavnice omenjenih bakterijskih vrst imajo pozitiven učinek predvsem na telesno težo in na imunski odziv. Način delovanja, ki privede do teh učinkov, pa je pri različnih vrstah in celo sevih lahko različen. Potrebno pa je izpostaviti tudi konvencionalne probiotike, spremenjene s pomočjo genskega inženiringa. Slednji predstavljajo velik potencial v prihodnosti, saj bi na ta način v ge-nom lahko uvedli željene spremembe in dosegli večjo odpornost proti fiziološkim pogo-jem. Probiotiki, ki so danes v uporabi, so namreč v primerjavi z novimi, precej bolj od-porni na biotehnološke procese pridelave in niso tako občutljivi na atmosferski kisik, kot prej omenjene bakterije, ki so striktni anaerobi. Omenjena lastnost namreč zelo otežuje samo formulacijo probiotičnega izdelka. Probiotics currently available on the market, are limited only on a few different species of microorganisms. Among them the well known members of genera Lactobacillus and Bifidobacterium are prevailing. But there is a lot of different genera of bacteria in our gut, that could potentionally serve as next-generation probiotics. Unlike the conventional probiotics, that have a history of safe use in human nutrition, next-generation probiotics have to be proven to be safe, not only effective. Neither one of the potential next-generation probiotics species has a QPS status. Among the most promising are species Feacalibacterim prausnitzii, Akkermansia muciniphila and Eubacterium hallii. The representatives of aforementioned species can have a positive effect on body weight and immune response. The mechanisms on which these effects rely on, however, are different among species and even strains. It is worth to mention also genetically engineered conventional probiotics. With this approach, we could introduce new traits in the genomes of bacteria and potentionally produce microorganisms, that are more resistant to different physical conditions, such as resistance to oxygen. Most candidates of the next-generation probiotics are obligate anaerobes, unlike the commercial probiotics used today. This trait makes the formulation of probiotic products more difficult.

Probiotiki so živi mikroorganzimi, ki ob prejemu v zadostni količini nudijo pozitivne vplive na zdravje gostitelja. Preko interakcije z gostiteljevim gastrointestinalnim traktom so sposobni vplivati na njegov prirojeni in pridobljeni immunski odziv. Probiotki vplivajo na sposobnost fagocitoze makrofagov in drugih celic, vplivajo na število in učinkovitost celic NK, spodbujajo produkcijo citokinov in protiteles IgA, preko celic DC vplivajo na celice Treg, ter uravnavajo ravnovesje med Th1 in Th2 celičnimi sub-populacijami. Delovanje probiotikov je sevno specifično. L. reuteri, je na primer zaviral produkcijo citokina IL-8, medtem ko jo je sev L. plantarum 299v spodbujal. Nekateri probiotiki imajo sposobnost uravnavati imunski odziv, glede na zdravstveno stanje gostitelja. L. rhamnosus GG je pri zdravih posameznikih stimuliral fagocitozno aktivnost ter jo inhibiral pri preobčutljivih na mleko. Najbolj pogosto uporabljeni probiotiki so G+ mlečnokislinske bakterije iz rodu Lactobacillus in Bifidobacterium, vendar tudi G- bakterije, na primer E. coli Nissle 1917, veliko obetajo, saj kažejo večje imunostimulatorne učinke verjetno zaradi različnih PAMP na njihovi površini. Probiotiki s svojimi imunomodulatornimi lastnostmi kažejo velik potencial za uporabo pri zdravljenju različnih bolezni (diareja, ulcerozni kolitis, preobčutljivosti, alergije, atopični dermatitis, avtoimunske bolezni idr.), uporabni pa bi bili tudi kot adjuvanti oralnim cepivom. Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. Through interaction with the host gastrointestinal tract, they are able to influence its innate and adaptive immune response. Probiotics affect the ability of phagocytosis of macrophages and other cells, affect the number and efficiency of NK cells, stimulate the production of cytokines and IgA antibodies, affect the Treg cells via DC cells, and balance Th1 and Th2 sub-populations. Probiotics are strain specific. L. reuteri, for example, inhibited the production of cytokine IL-8, while the strain L. plantarum 299v stimulated it. Some probiotics have the ability to control the immune response, depending on the health status of the host. L. rhamnosus GG stimulated phagocytosis in healthy individuals and inhibited it in milk-hypersensitive patients. The most commonly used probiotics are G + lactic acid bacteria from the genus Lactobacillus and Bifidobacterium, but also G-bacteria, for example E. coli Nissle 1917, are very promising, as they show greater immunostimulatory effects probably due to different PAMPs on their surface. Probiotics, with their immunomodulatory properties, show great potential for use in the treatment of various diseases (diarrhea, ulcerative colitis, hypersensitivity, allergies, atopic dermatitis, autoimmune diseases, etc.) and they would also be useful as adjuvants to oral vaccines.

Toll-u podoben receptor 4 (TLR4) že vrsto let povezujejo s pojavom sepse, povzročene z vdorom Gram negativnih bakterij v človeško kri. Bolezen se v veliki meri pojavlja pri hospitaliziranih bolnikih, smrtnost zanjo pa je izredno visoka. Pri tem se poraja dosti vprašanj o načinu ter ustreznosti zdravljenja. Vse to je razlog za veliko povpraševanje po zdravilih za preprečevanje ali zdravljenje sepse, a kljub številnim raziskavam na tem področju, na tržišču še ni registriranega specifičnega zdravila. Antagonisti TLR4 predstavljajo velik potencial pri iskanju novih zdravil pri zdravljenju sepse. Toll-u podobni receptorji 4 igrajo pomembno vlogo v prirojenem imunskem sistemu, hkrati pa v manjši meri vplivajo tudi na pridobljeni imunski odziv posameznika. Gre za najbolj poznan in opisan transmembranski receptor iz skupine TLR. Značilno prepoznava molekulske vzorce povezane s patogeni, najpomembnejše pa je njegovo prepoznavanje lipopolisaharida, ki je glavna sestavina celične membrane Gram negativnih bakterij. V okviru magistrske naloge smo načrtovali sintezno pot za derivate spojine z dokazanim antagonističnim delovanjem na TLR4, ki je bila odkrita na Fakulteti za farmacijo v Ljubljani s pomočjo virtualnega rešetanja knjižnice spojin. Preizkusili smo več različnih sinteznih poti za pripravo gvanidinskih derivatov. Za najuspešnejšo pa se je izkazala tristopenjska sinteza, ki smo jo uporabili pri sintezi štirih končnih spojin. Sintetiziranim spojinam smo določili topnost v celičnem mediju, citotoksičnost na celični liniji HEK 293 ter ovrednotili antagonistično delovanje na celicah s selektivno izraženim TLR4. Izmed testiranih končnih spojin je ena izkazovala precej obetajoče antagonistično delovanje z določeno vrednostjo srednje inhibitorne koncentracije 27 μM. Za dodatne informacije o odnosu med strukturo in delovanjem spojin pa bi bilo potrebno sintetizirati še nekaj več derivatov. For many years Toll-like receptor 4 has been associated with the occurrence of sepsis, caused by intrusion of Gram negative bacteria into the human blood. The disease largely occurs in hospitalized patients and its mortality rate is extremely high, therefore it raises a lot of questions about methods and adequacy of the treatment. This is the reason for the high demand for medicinal products with the purpose of treatment or prevention of sepsis. However, despite numerous research in this area, a specific product has not been registered on the market. TLR4 antagonists present high potencial for the development of novel agents against sepsis. These receptors play an important role in the innate immune system and to a lesser extent also affect the adaptive immune response of an individual. TLR4 is well-known and described transmembrane receptor in the TLR group. It typically recognizes molecular patterns associated with pathogens, however, the most important is the recognition of lipopolysaccharide, which is a major component of the cell membrane of Gram negative bacteria. The aim of our master's thesis was to design the synthetic pathway for derivatives of a hit compound with proven antagonism of TLR4, which was discovered at the Faculty of Pharmacy in Ljubljana by virtual screening of the compound library. We examined several synthetic routes and finally discovered the three-stage synthesis of guanidine derivatives. Four final compounds were synthesized and evaluated. We have determined the solubility of compounds in the cell medium, the cytotoxicity in HEK 293 cell line and evaluated the antagonist activity of final compounds on cells which selectively express TLR4. Half maximal inhibitory concentration (IC50) of 27 μM has been determined for one of the final compounds, which shows promising antagonistic activity. However, it would be necessary to synthesize more derivates to obtain additional informations about structure-activity relationship.

When nanoparticles enter the body, their interactions with cells are almost unavoidable. Unintended nanoparticle interaction with immune cells may elicit a molecular response that can have toxic effects and lead to greater susceptibility to infectious diseases, autoimmune disorders, and cancer development. As evidenced by several studies, nanoparticle interactions with biological systems can stimulate inflammatory or allergic reactions and activate the complement system. Nanoparticles can also stimulate immune response by acting as adjuvants or as haptens. Immunosuppressive effects have also been reported. This article gives a brief review of in vitro and in vivo research evidencing stimulatory or suppressive effects of nanoparticles on the immune system of mammals. In order to ensure safe use of nanosized particles, future research should focus on how their physical and chemical properties influence their behaviour in the biological environment, as they not only greatly affect nanoparticle-immune system interactions but can also interfere with experimental assays., Ko nanodelci vstopijo v organizem, pridejo v kontakt s celicami imunskega sistema. Nezaželene interakcije nanodelcev z imunskim sistemom lahko sprožijo molekularni odziv, ki lahko pripelje do toksičnih učinkov in povečane dovzetnosti organizma za okužbe, avtoimunska obolenja ter razvoj raka. Dosedanje raziskave so pokazale, da nanodelci lahko sprožijo vnetne in alergijske reakcije, lahko pa tudi aktivirajo sistem komplementa. Nanodelci lahko delujejo kot adjuvansi ali kot hapteni. Obstajajo pa tudi poročila, ki kažejo na sposobnost nanodelcev, da zavrejo imunski odziv. V članku bomo povzeli ugotovitve dosedanjih raziskav in vitro ter in vivo, ki so bile narejene na področju proučevanja vplivov nanodelcev na stimulacijo ali supresijo imunskega sistema sesalcev. Za zagotovitev varne uporabe nanodelcev moramo razumeti kako fizikalno-kemijske lastnosti nanodelcev vplivajo na njihovo obnašanje v biološkem okolju. Lastnosti nanodelcev moramo upoštevati tudi ob izvajanju poskusov, da se izognemo lažnim rezultatom zaradi potencialne interference nanodelcev z dejavniki v eksperimentalnem okolju. Čeprav je bilo do sedaj narejenih že več nanotoksikoloških raziskav, je vpliv nanodelcev na imunski sistem še vedno slabo razumljen. Sposobnost nanodelcev za modulacijo imunskega odziva narekuje potrebo po nadaljnjih raziskavah interakcij nanodelcev z imunskim sistemom.

Iz vzorcev kozjega mleka in fecesa smo uspešno izolirali 16 bakterijskih vzorcev in jih karakterizirali s pomočjo klasičnih in sodobnih mikrobioloških metod. Izmed izoliranih in karakteriziranih 16 bakterij jih 14 pripada rodu Lactobacillus ter 2 rodu Lactococcus. Dokazali smo pozitiven vpliv bakterij v in vitro celičnem modelu intestinalnega trakta. Rezultati so pokazali dobro stopnjo vezave na intestinalni epitelij, sorazmerno nizko citotoksičnost ter spodbujanje nespecifičnega imunskega odziva. S tem smo dokazali tudi zadostnost naših sevov za selekcijske kriterije. So funkcionalni, predvsem pa ne patogeni. Zanimivi za nadaljnje raziskave so predvsem vzorci M5K1 (Lactobacillus Brevis), F1K1K2 (Lactobacillus Plantarum) ter F1K1K1 (Lactococcus Rafinolactis), ki so najbolj izstopali po učinkovitosti. Navkljub dejstvu, da so vsi izolirani sevi pripadniki mlečnokislinskih bakterij je opazna vrstna specifičnost posameznih izoliranih sevov, ki se med seboj močno razlikujejo glede biološke aktivnosti. Prav tako prihaja do razhajanj znotraj posamezne vrste. Naše delo predstavlja v svetu edinstven eksperimentalni model za tovrstne analize. We isolated and characterized successfully 16 bacterial samples from goat milk and feces with classical and modern practices. Among the isolated bacteria, 14 belong to the genus of Lactobacillus and 2 belong to the genus of Lactococcus. We proved the beneficial influence of bacteria in a in vitro intestinal cell model. The results showed a good degree of attachment to the intestinal epithelium, low citotoxicity and stimulation of non-specific immune responses. With this we also showed that these bacteria fulfill the criteria for probiotic selection. They are functional and non-pathogenic. The isolates M5K1 (Lactobacillus Brevis), F1K1K2 (Lactobacillus Plantarum) and F1K1K1 (Lactococcus Rafinolactis) are especially interesting for further research due to their efficiency. We observed strong specifities among different genus groups concerning their bio-activity. Our work presents a unique experimental approach for this kind of analysis.