Uloga inaktivisane vakcine protiv gripa u regulaciji autoimunskih procesa u eksperimentalnom autoimunskom encefalomijelitisu

Influenza is a contagious respiratory illness caused by influenza viruses. Influenza infection per se has seldom been associated with various organ-specific and systemic autoimmune diseases but neurological autoimmune phenomena have been reported following influenza vaccine. Multiple sclerosis (MS) is the most common chronic neurological disease of an autoimmune nature that has been viewed in conjunction with vaccination. Experimental autoimmune encephalomyelitis (EAE) is the most extensively studied mouse model of MS and myelin oligodendrocyte glycoprotein (MOG) induced EAE more closely resembles MS than other EAE variants. Since the debate about vaccine safety in patients with MS is still wide open, the objective of our study was to shed more light on and supply additional information about the influenza vaccination effects on regulation of EAE through monitoring of clinical signs, serum antibody titers by using ELISA, expression of MHC (Major Histocompatibility Complex) class I and II molecules in brain tissue by immunohistochemical detection, and its effect on cognitive functions (spatial learning and memory measured by Morris Water Maze test). C57BL/6 mice were first vaccinated with influenza split virion inactivated vaccine and three days later EAE was induced with MOG35-55 peptide. The overall data presented in this study indicate that influenza vaccine had no effect on the production of anti-MOG autoantibodies and the development of clinical signs. Although the unbalanced immune system is evident in patients with MS, immune defense against common viral and bacterial infections appears to be preserved. In this study anti-influenza antibody titers in healthy vaccinated mice and in MOG induced EAE-vaccinated mice, four weeks after vaccination with influenza vaccine have indicated preserved long-lasting antibody response in MOG induced EAE mice. High expression of MHC II and mild-to-low expression of MHC I was detected in mice with EAE...Influenca je infektivna respiratorna bolest uzrokovana virusima influence. Iako je sama infekcija virusom influence retko bila povezivana sa različitim organ-specifičnim i sistemskim autoimunskim bolestima, neurološke autoimunske manifestacije posle primene influenca vakcine su bile dokumentovane tokom vremena. Multipla skleroza (MS) je hronična, u osnovi autoimunska neurološka bolest koja se najčešće dovodi u vezu sa vakcinacijom. Eksperimentalni autoimunski encefalomijelitis (EAE) je najintenzivnije izučavan mišji model MS, a mijelin oligodendrocitnim glikoproteinom (MOG) indukovan EAE je mnogo sličniji MS kod ljudi nego druge varijante EAE. S obzirom da je rasprava o dobrobiti vakcinacije bolesnika sa MS još uvek otvorena cilj naše studije je bio davanje doprinosa u rasvetljavanju uloge vakcine protiv gripa i njenog efekta na regulaciju EAE kroz praćenje kliničkih znakova, titra serumskih antitela ELISA testom, ekspresije molekula glavnog histokompatibilnog kompleksa (MHC) I i II klase na presecima moždanog tkiva imunohistohemijskim metodama i efekat na kognitivne funkcije (prostorno učenje i pamćenje određivano Morisovim testom vodenog lavirinta; MWM). C57BL/6 miševi su prvo vakcinisani split virion inaktivisanom influenca vakcinom i tri dana kasnije je indukovan EAE sa MOG35-55 peptidom. Ukupno dobijeni rezultati predstavljeni u ovoj disertaciji ukazuju da influenca vakcina nije uticala na produkciju anti- MOG antitela i na razvoj kliničkih znakova EAE. I pored toga što je poremećaj na nivou (auto)imunskog odgovora kod MS bolesnika evidentan, imunski odgovor na većinu uobičajenih virusnih i bakterijskih infekcija izgleda da je očuvan. Rezultati ove studije su pokazali da je titar anti- influenca antitela kod zdravih vakcinisanih miševa i kod onih kod kojih je nakon vakcinacije indukovan EAE, četiri nedelje nakon vakcinacije, bio identičan, što ukazuje na očuvanost humoralnog imunskog odgovora i kod miševa kod kojih je indukovan EAE..

Role of inactivated influenza vaccine in regulation of autoimmune processes in experimental autoimmune encephalomyelitis

Инфлуенца је инфективна респираторна болест узрокована вирусима инфлуенце. Иако је сама инфекција вирусом инфлуенце ретко била повезивана са различитим орган-специфичним и системским аутоимунским болестима, неуролошке аутоимунске манифестације после примене инфлуенца вакцине су биле документоване током времена. Мултипла склероза (МС) је хронична, у основи аутоимунска неуролошка болест која се најчешће доводи у везу са вакцинацијом. Експериментални аутоимунски енцефаломијелитис (EAE) је најинтензивније изучаван мишји модел МС, а мијелин олигодендроцитним гликопротеином (MOG) индукован EAE је много сличнији МС код људи него друге варијанте ЕАЕ. С обзиром да је расправа о добробити вакцинације болесника са MС још увек отворена циљ наше студије је био давање доприноса у расветљавању улоге вакцине против грипа и њеног ефекта на регулацију ЕАЕ кроз праћење клиничких знакова, титра серумских антитела ELISA тестом, експресије молекула главног хистокомпатибилног комплекса (MHC) I и II класе на пресецима можданог ткива имунохистохемијским методама и ефекат на когнитивне функције (просторно учење и памћење одређивано Морисовим тестом воденог лавиринта; MWM). C57BL/6 мишеви су прво вакцинисани split virion инактивисаном инфлуенца вакцином и три дана касније је индукован ЕАЕ са MOG35-55 пептидом. Укупно добијени резултати представљени у овој дисертацији указују да инфлуенца вакцина није утицала на продукцију анти- MOG антитела и на развој клиничких знакова ЕАЕ. И поред тога што је поремећај на нивоу (ауто)имунског одговора код МС болесника евидентан, имунски одговор на већину уобичајених вирусних и бактеријских инфекција изгледа да је очуван. Резултати ове студије су показали да је титар анти- инфлуенца антитела код здравих вакцинисаних мишева и код оних код којих је након вакцинације индукован ЕАЕ, четири недеље након вакцинације, био идентичан, што указује на очуваност хуморалног имунског одговора и код мишева код којих је индукован ЕАЕ...Influenza is a contagious respiratory illness caused by influenza viruses. Influenza infection per se has seldom been associated with various organ-specific and systemic autoimmune diseases but neurological autoimmune phenomena have been reported following influenza vaccine. Multiple sclerosis (MS) is the most common chronic neurological disease of an autoimmune nature that has been viewed in conjunction with vaccination. Experimental autoimmune encephalomyelitis (EAE) is the most extensively studied mouse model of MS and myelin oligodendrocyte glycoprotein (MOG) induced EAE more closely resembles MS than other EAE variants. Since the debate about vaccine safety in patients with MS is still wide open, the objective of our study was to shed more light on and supply additional information about the influenza vaccination effects on regulation of EAE through monitoring of clinical signs, serum antibody titers by using ELISA, expression of MHC (Major Histocompatibility Complex) class I and II molecules in brain tissue by immunohistochemical detection, and its effect on cognitive functions (spatial learning and memory measured by Morris Water Maze test). C57BL/6 mice were first vaccinated with influenza split virion inactivated vaccine and three days later EAE was induced with MOG35-55 peptide. The overall data presented in this study indicate that influenza vaccine had no effect on the production of anti-MOG autoantibodies and the development of clinical signs. Although the unbalanced immune system is evident in patients with MS, immune defense against common viral and bacterial infections appears to be preserved. In this study anti-influenza antibody titers in healthy vaccinated mice and in MOG induced EAE-vaccinated mice, four weeks after vaccination with influenza vaccine have indicated preserved long-lasting antibody response in MOG induced EAE mice. High expression of MHC II and mild-to-low expression of MHC I was detected in mice with EAE..

Neutrophil Death in Myeloproliferative Neoplasms: Shedding More Light on Neutrophils as a Pathogenic Link to Chronic Inflammation

Neutrophils are an essential component of the innate immune response, but their prolonged activation can lead to chronic inflammation. Consequently, neutrophil homeostasis is tightly regulated through balance between granulopoiesis and clearance of dying cells. The bone marrow is both a site of neutrophil production and the place they return to and die. Myeloproliferative neoplasms (MPN) are clonal hematopoietic disorders characterized by the mutations in three types of molecular markers, with emphasis on Janus kinase 2 gene mutation (JAK2V617F). The MPN bone marrow stem cell niche is a site of chronic inflammation, with commonly increased cells of myeloid lineage, including neutrophils. The MPN neutrophils are characterized by the upregulation of JAK target genes. Additionally, MPN neutrophils display malignant nature, they are in a state of activation, and with deregulated apoptotic machinery. In other words, neutrophils deserve to be placed in the midst of major events in MPN. Our crucial interest in this review is better understanding of how neutrophils die in MPN mirrored by defects in apoptosis and to what possible extent they can contribute to MPN pathophysiology. We tend to expect that reduced neutrophil apoptosis will establish a pathogenic link to chronic inflammation in MPN

Extracellular Hemoglobin: Modulation of Cellular Functions and Pathophysiological Effects

Hemoglobin is essential for maintaining cellular bioenergetic homeostasis through its ability to bind and transport oxygen to the tissues. Besides its ability to transport oxygen, hemoglobin within erythrocytes plays an important role in cellular signaling and modulation of the inflammatory response either directly by binding gas molecules (NO, CO, and CO2) or indirectly by acting as their source. Once hemoglobin reaches the extracellular environment, it acquires several secondary functions affecting surrounding cells and tissues. By modulating the cell functions, this macromolecule becomes involved in the etiology and pathophysiology of various diseases. The up-to-date results disclose the impact of extracellular hemoglobin on (i) redox status, (ii) inflammatory state of cells, (iii) proliferation and chemotaxis, (iv) mitochondrial dynamic, (v) chemoresistance and (vi) differentiation. This review pays special attention to applied biomedical research and the use of non-vertebrate and vertebrate extracellular hemoglobin as a promising candidate for hemoglobin-based oxygen carriers, as well as cell culture medium additive. Although recent experimental settings have some limitations, they provide additional insight into the modulatory activity of extracellular hemoglobin in various cellular microenvironments, such as stem or tumor cells niches

Podklasa teških lanaca i nivo ekspresije sijalinske kiseline na njima određuju hromatografsku raspodelu humanih monoklonskih imunoglobulina G

Anion exchange chromatography is a widely accepted method for purification of immunoglobulins. In this work, we used human monoclonal immunoglobulin G (IgG) with structure and solubility of normal human IgG as a model for studying chromatographic behavior of particular molecular forms of IgG. Human sera with monoclonal IgG were fractionated on a strong anion exchanger, Q Sepharose Fast Flow. With 20 mM Tris pH 7.5 as a start buffer, 42% of human monoclonal IgG passed through column, and 58% of them remained adsorbed. Bound monoclonal IgG were eluted from the exchanger by linear increasing of concentration of NaCl from 0 to 0.5 M. The chromatographic distribution of human monoclonal IgG correlated with their electrophoretic mobilities in agarose gels, and it was dependent on ã heavy chain isotype. Light chain type, as well as serum concentration of monoclonal IgG did not influence their chromatographic behavior. The level of heavy chain sialic acid expression, but not of galactose and N-acetylglucosamine, significantly determined chromatographic distribution of serum monoclonal IgG. In addition to the information on the chromatographic behavior of human monoclonal IgG, we believe that the presented data could provide useful information about the possible use of Q Sepharose Fast Flow matrix for the isolation of specific molecular forms of human IgG.Zbog velike molekulske heterogenosti humanih imunoglobulina G (IgG) (4 izotipa, 20 alotipova i preko 600 mogućih glikoformi) ne postoji jedinstven protokol za izolovanje svih molekulskih formi IgG. Jonoizmenjivačka hromatografija je široko prihvaćen metod za izolovanje i prečišćavanje humanih IgG, pre svega zbog činjenice da se separacija odvija u blagim uslovima, što pored efikasnog prečišćavanja omogućava da struktura i funkcija IgG kao antitela ostane očuvana. Za izolovanje humanih IgG mogu se koristiti i slabi i jaki anjonski izmenjivači. Međutim, koji će od brojnih matriksa koji danas postoje na tržištu biti odgovarajući za izolovanje specifičnih molekulskih formi humanih IgG, može se znati samo nakon testiranja. U ovom radu, humani monoklonski IgG iz seruma bolesnika sa monoklonskim gamapatijama su izolovani preparativnom hromatografijom na Q Sepharose Fast Flow anjonskom izmenjivačkom matriksu. Kada je kao startni pufer korišćen 20 mM Tris-HCl, pH 7,5, 42% analiziranih monoklonskih IgG nije se vezivalo za matriks i bilo je moguće izolovati ih u čistoj formi. Preostalih 58% monoklonskih IgG, različitim intenzitetom vezivali su se za matriks, i sa njega su eluirani 0,09-0,43 M Tris, pH 7,5/NaCl. Hromatografska raspodela monoklonskih IgG je bila u korelaciji sa njihovom elektroforetskom pokretljivosti u gelu agaroze i podklasom γ teškog lanca, a nije bila određena tipom lakog lanca i serumskom koncen- tracijom monoklonskog IgG. Nivo ekspresije sijalinske kiseline na teškim lancima je značajno uticao na hromatografsku raspodelu humanih monoklonskih IgG, dok je nivo ekspresije druga dva terminalna šećera, galaktoze i N-acetilglukozamina, bio bez uticaja. Smatramo da prikazani rezultati mogu biti od koristiti u kreiranju protokola za izolovanje humanih monoklonskih IgG jer pored toga što daju informacije o njihovom hromatografskom ponašanju daju i informaciju o mogućnosti korišćenja Q Sepharose Fast Flow anjonskog matriksa za izolovanje pojedinih molekulskih formi humanih IgG

Putative Role of Neutrophil Extracellular Trap Formation in Chronic Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are hematologic malignancies characterized by gene mutations that promote myeloproliferation and resistance to apoptosis via constitutively active signaling pathways, with Janus kinase 2-signal transducers and the activators of transcription (JAK-STAT) axis as a core part. Chronic inflammation has been described as a pivot for the development and advancement of MPNs from early stage cancer to pronounced bone marrow fibrosis, but there are still unresolved questions regarding this issue. The MPN neutrophils are characterized by upregulation of JAK target genes, they are in a state of activation and with deregulated apoptotic machinery. Deregulated neutrophil apoptotic cell death supports inflammation and steers them towards secondary necrosis or neutrophil extracellular trap (NET) formation, a trigger of inflammation both ways. NETs in proinflammatory bone marrow microenvironment induce hematopoietic precursor proliferation, which has an impact on hematopoietic disorders. In MPNs, neutrophils are primed for NET formation, and even though it seems obvious for NETs to intervene in the disease progression by supporting inflammation, no reliable data are available. We discuss in this review the potential pathophysiological relevance of NET formation in MPNs, with the intention of contributing to a better understanding of how neutrophils and neutrophil clonality can orchestrate the evolution of a pathological microenvironment in MPNs

Morphological changes in lymph nodes and spleen upon EAE induction in C57BL/6 mic

Myelin oligodendrocyte glycoprotein (MOG) is a protein widely used in the induction of experimental autoimmune encephalomyelitis (EAE) for studying human multiple sclerosis (MS). In C57BL/6 female mice aged eight weeks, we administered subcutaneously MOG35-55 peptide in CFA (complete Freund's adjuvant) along with pertussis vaccine injected intraperitoneally. We observed the sign of flaccid tail as early as thirteen days post-immunization in five of twelve animals. Hematoxylin and eosin staining of paraffin-embedded sections of lymph nodes and spleen revealed the presence of germinal centers in the immunized animals. In the control group of animals, lymphoid follicles without germinal centers were observed. Immunohistochemical staining of spleen sections revealed an expression of MHC II molecules in the EAE-induced group. We would like to point out that even though the clinical signs are mild, the morphological changes are apparent in the lymph nodes and spleen of MOG35-55-immunized mice

Raman spectral analysis of the brainstem and responses of neuroglia and cytokines in whole-body gamma-irradiated rats after administration of aminothiol-based radioprotector GL2011

The search for an effective and non-toxic radioprotector is ongoing. We tested a novel, natural aminothiol-based radioprotector, GL2011, that was applied 30 min, 3 h or 6 h after the exposure of male albino Wistar rats to a 6.7 Gy mild dose of gamma radiation. The molecular signatures of radioprotection were investigated with Raman microspectroscopy of brainstem tissue samples. Morphological changes and activation of astrocytes and microglia were assessed by immunohistochemistry. Global markers of neuroinflammation were followed by ELISA to monitor blood plasma levels of proinflammatory (IL-6 and TNF-α) and anti-inflammatory (IL-10) cytokines. A thirty-day follow-up determined survival of unprotected animals 37.5%. A survival increase was observed after radioprotection (75%, irrespective of the time of application). Raman spectra revealed a slightly deleterious effect of radiation on nucleic acids in surviving animals that was mitigated with the radioprotector, as GL2011 preserved the morphology of both astrocytes and microglia, with reduced microglial infiltration. Cytokine assessment revealed an immunomodulatory effect of the novel radioprotector. The overall results point out the positive effects of a single dose of GL2011 applied at different times. The molecular and cellular changes in the brainstem indicate that the radioprotector applied after radiation conferred better protection, which underlines its translation to cure radiation accidents

Insight into the Biological Activity of Hennosides-Glucosides Isolated from Lawsonia inermis (henna): Could They Be Regarded as Active Constituents Instead

Henna is the current name of the dye prepared from the dry leaf powder of Lawsonia inermis (Lythraceae). Several studies have focused on the chemistry and pharmacology of the henna dyeing active compound, lawsone, obtained from the main constituents of leaves, hennosides, during the processing of plant material. However, knowledge regarding the biological activity of hennosides is largely lacking. In this paper, the redox activity of three hennoside isomers is reported. The pro-oxidative activity was confirmed by their ability to induce mild lysis of erythrocytes and to increase the level of methemoglobin at the concentration gt = 500 mu g/mL. The antioxidant activity of hennosides (concentration gt = 100 mu g/mL) was determined by FRAP and ABTS assays. At concentration of 500 mu g/mL, antioxidant activity of hennoside isomers was equivalent to 0.46 +/- 0.08, 0.62 +/- 0.28 and 0.35 +/- 0.03 mM FeSO4 x 7H(2)O, and 0.15 +/- 0.01, 0.30 +/- 0.01 and 0.09 +/- 0.01 mM Trolox. Hennosides at 100 mu g/mL concentration did not influence viability of human breast cancer cell lines MDA231 and MCF-7 and primary human peripheral blood and periodontal ligament-mesenchymal stem cells, but produced a modest increase in concentration of antioxidants in the cell culture supernatants. The evidenced antioxidant and pro-oxidant activities indicate their potential to act as redox balance regulator, which opens up the possibility of using hennosides in commercial phytomedicines