Karakteristike tkivno specifičnih makrofaga pre i posle aktivacije in vitro

Macrophages derived from different tissues: bone marrow, spleen, peritoneal cavity and alveolus, were examined from the aspects of their morphology and functional characteristics expression of Fc receptors (FcR), phagocytic activity towards yeast particles and nonspecific esterase (NSE) content¹ before and after in vitro activation. Twenty four-hour-adherent cells were isolated with the aim of analyzing the characteristics of resident tissue macrophages. Following cultivation in vitro 8-day-adherent cells were used to investigate the influence of macrophage activation on their morphology and function. Morphological analysis of cell smears, performed in respect to cell size, showed significant enlargement, especially in the population of alveolar cells cultured for 8 days and activated with colony-stimulating factors (CSFs) and lymphokines. It was also demonstrated that 24-hour- and 8-dayadherent macrophages derived from different tissues exhibited similar properties. All these cells were more than 90% FcR-positive (FcR+) NSE-positive (NSE+) and had phagocytic properties. However, within the population of alveolar macrophages there were some NSE+ cells lacking FcR and phagocytic activity, even after in vitro activation. These results confirmed that the properties of alveolar macrophages differing from those of macrophages from other tissues were dependent on their microenvironment.Morfološke i funkcionalne karakteristike makrofaga izolovanih iz različitih organa: kostne srži, slezine, peritonealne šupljine i alveola ispitivane su pre i posle aktivacije u in vitro uslovima. Da bi se ispitale karakteristike makrofaga koji se nalaze u ispitivanim tkivima analizirane su adherentne ćelije dobijene nakon inkubacije od 24 časa. Uticaj aktivacije makrofaga na njihovu morfologiju i funkciju (ekspresiju Fc receptora, sposobnost fagocitoze čestica kvasca i sadržaj enzima nespecifične esteraze) ispitivan je nakon kultivisanja adherentnih ćelija u toku 8 dana. Morfološkom analizom utvrđeno je značajno povećanje veličine alveolarnih makrofaga kultivisanih tokom 8 dana u prisustvu faktora koji stimulišu rast kolonija i limfokina. Pokazano je da adherentne ćelije iz različitih tkiva izolovane nakon 24 sata i 8 dana imaju slične funkcionalne karakteristike. Više od 90% tih ćelija je eksprimiralo Fc receptore, imalo sposobnost fagocitoze i sadržavalo nespecifičnu esterazu. Međutim, u populaciji alveolarnih makrofaga, pre kao i nakon in vitro aktivacije, utvđeno je prisustvo ćelija koje su sadržavale nespecifičnu esterazu u citoplazmi, ali nisu eksprimirale Fc receptore, niti su imale sposobnost fagocitoze. Ovi rezultati potvrđuju da su karakteristike alveolarnih makrofaga u odnosu na makrofage iz drugih tkiva zavisne od njihovog mikrookruženja

Proliferacija naivnih i aktiviranih T limfocita u prisustvu tkivno specifičnih makrofaga

In this study the antigen-presenting ability of tissue specific macrophages isolated from bone marrow, spleen, peritoneal cavity and lungs was analyzed. Murine macrophages were isolated by a one-step adherence procedure (for 24 hours) and pretreated with mytomycin C. The antigen-presenting ability of the macrophages was tested in T cell proliferation assays. The ability of macrophages to support antigenspecific proliferation of T lymphoblasts was investigated when sheep red blood cell (SRBC)-specific T blasts were stimulated in vitro by antigen in the presence of different numbers of tissue specific macrophages. On the other hand, the abilities of macrophages to induce proliferation of naïve T cells were analyzed in allogeneic and syngeneic mixed leukocyte reactions (MLRs). It was demonstrated that tissue specific macrophages supported antigen specific proliferation of T lymphoblasts in vitro. They also induced the activation of allogeneic and syngeneic T cells. Increasing the number of macrophages co-cultured with T cells, led to a certain inhibitory effect on T cell proliferation.U ovom radu je ispitivana sposobnost mišjih makrofaga izolovanih iz kostne srži, slezine, peritonealne šupljine i alveola da prezentuju antigen i indukuju proliferaciju T limfocita. Makrofazi su izolovani adherencijom tokom 24-časovne kulture i pretretirani mitomicinom C. Da bi se ispitala sposobnost makrofaga da indukuju proliferaciju limfoblasta, T limfociti specifični za ovčje eritrocite izolovani iz limfnih čvorova imunizovanih miševa u in vitro uslovima, su restimulisani antigenom u prisustvu tkivnih makrofaga izolovanih iz različitih tkiva. Pored toga, sposobnost makrofaga da indukuju proliferaciju in vivo naivnih T limfocita ispitivana je u mešanoj kulturi tkivnih makrofaga i alogenih ili singenih limfocita. Dokazano je da tkivni makrofazi izolovani iz kostne srži, slezine, peritonealne šupljine i alveola mogu da potpomognu antigen-specifičnu proliferaciju T limfoblasta u in vitro uslovima, kao i da aktiviraju naivne alogene i singene T limfocite. Povećanje broja makrofaga dovelo je do smanjenja T limfocitne proliferacije

Monoklonsko antitelo 26 napravljeno na tetanus toksoidu reaguje i sa tetanus toksinom i β2-glikoproteinom I - karakteristike vezivanja in vitro i moguća primena

A murine monoclonal IgG1 antibody, marked as MAb26, specific for tetanus toxoid has been immunochemically characterized. By performing enzyme-linked immunosorbent assays (ELISAs) and western blot analyses, it was demonstrated that MAb26 reacted with tetanus toxoid, tetanus toxin and β2-glycoprotein I (β2GPI). According to the results, MAb26 recognized the sequential epitope on the tetanus heavy chain. The affinity constant, calculated from Scatchard plots of MAb26 binding to tetanus toxoid, was 1.145×108 M-1 and the measurement of the relative affinity of MAb26 by ELISA using thiocyanate elution showed a significantly higher affinity of MAb26 to the toxoid (p = 0.0012) in comparison to the toxin. Additionally, the reactivity of MAb26 toward the toxoid forms increased when the tetanus toxin was detoxified using 8 mM and higher formaldehyde concentrations. The similarity of the tetanus toxoid to several sera proteins, either at the level of its conformation (IL-1α) or at the level of peptide sequences (â2GPI, laminin) favors its role in autoimmunity by the mechanism of molecular mimicry. As the induction of an autoimmune disease is dependent on the breakdown of tolerance, which could be the result of an overt hyperstimulation, the control of the presence and concentration of self-reactive epitopes in vaccine preparations is a prerequisite. In this study, it was shown that MAb26 can: 1) discriminate between the tetanus toxin and different toxoid forms, which makes it a good candidate for antibody control during vaccine preparation; 2) due to its cross-reactivity with β2GPI, it could provide information on the presence of a potentially dangerous sequential epitope expressed at the protein surface.Ovaj rad opisuje imunohemijsku karakterizaciju mišjeg IgG1 monoklonskog antitela označenog kao MAt26. Enzimskim imunosorbentnim testom (ELISA) i Western blot analizom je pokazano da MAt26 reaguje sa tetanus toksoidom, tetanus toksinom i β2-glikoproteinom I (β2GPI). Prema našim rezultatima, MAt26 prepoznaje sekvencioni epitop na teškom lancu molekula tetanusa. Konstanta afiniteta MAt26 za tetanus toksoid, izračunata na osnovu Skačardovog dijagrama, je 1,145×108 M-1. Na osnovu elucije tiocijanatom, korišćene za određivanje relativnog afiniteta MAt26 za tetanus toksin i tetanus toksoid, postupkom baziranim na ELISA-i, pokazan je znatno veći (p = 0,0012) afinitet MAt26 ka toksoidnoj formi. Takođe, reaktivnost MAt26 ka toksoidnoj formi rasla je sa porastom koncentracije formaldehida, počevši od 8 mM, korišćenog u procesu detoksifikacije. Sličnost tetanus toksoida sa različitim serumskim proteinima na nivou konformacije i/ili peptidnih sekvencija (β2GPI, laminin) ukazuje na njegovu potencijalnu ulogu u indukciji autoimunosti mehanizmom molekulske mimikrije. Budući da nastanak autoimunske bolesti podrazumeva narušavanje tolerancije, na primer, prekomernom stimulacijom imunskog sistema, kontrola prisustva i koncentracije sebi sličnih epitopa se nameće kao neophodna. U ovom radu je pokazano da: 1) MAt26 može da pravi razliku između tetanus toksina i različitih toksoidnih formi što ga čini potencijalno dobrim antitelom koje bi se koristilo u kontroli tokom proizvodnje vakcina; 2) zahvaljujući unakrsnoj reaktivnosti sa β2GPI, MAt26 može da pruži informacije o prisustvu potencijalno opasnih epitopa na površini proteina

The Possible Role of Natural Idiotopes in Immune Memory

In this paper we report on the generation of Abs possessing specificities similar to those of Abs used in immunization, and on the generation of Id and anti-Id specificities in the sera of mice immunized with commensal bacterial antigens

Network connectivity is shown to change in C57BL/6 mice during a continuing immune response subsequent to tetanus toxoid hyperimmunization

We have already demonstrated (Stojanovic et al., 2009) a connection between tetanus toxoid (TTd) hyperimmunization and the induction of anti-phospholipid syndrome (APS) in BALB/c mice. Here we show that C57BL/6 mice subjected to an identical procedure do not exhibit any like pathology attributable to anti-phospholipid antibodies; we explain that this absence results from idiotypic connectivity. Six groups of C57BL/6 mice were hyperimmunized with TTd in aluminum hydroxide or glycerol, with or without pretreatments. Pretreated mice had been injected with polyclonal or nonspecific immune stimulators, such as complete Freund's adjuvant (CFA) or glycerol. The epitope specificity of induced antibodies was tested by indirect ELISA using a tetanus toxoid immunogen and these autoantigens: phospholipids, gangliosides, laminin. Idiotypic connectivity was tested by competitive ELISA and gauged from the degree to which the interaction of idiotypic/anti-idiotypic complementary antibodies was inhibited in the presence of immunized sera antibodies. Higher idiotypic connectivity was noted amongst pretreated mice. There was a positive correlation between higher connectivity and autoantibody levels that acted to favor the participation of natural autoantibodies in the inhibitory process. We conclude that idiotypic connectivity plays a protective role in immunization-induced autoimmunity

Supplementary data for the article: Mihailovic, J.; Inic-Kanada, A.; Smiljanic, K.; Stein, E.; Barisani-Asenbauer, T.; Cirkovic Velickovic, T. Lysine Acetylation of Major Chlamydia Trachomatis Antigens. EuPA Open Proteomics 2016, 10, 63–69. https://doi.org/10.1016/j.euprot.2016.01.007

Supplementary material for: [https://doi.org/10.1016/j.euprot.2016.01.007]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/295

Effects of water-filtered infrared A and visible light (wIRA/VIS) radiation on heat- and stress-responsive proteins in the retina and cornea of guinea pigs

Water-filtered infrared A and visible light (wIRA/VIS), shown to reduce chlamydial infections in vitro and in vivo, might represent an innovative therapeutic approach against trachoma, a neglected tropical disease caused by ocular infection with the bacterium C. trachomatis. In this in vivo study, we assessed the impact of wIRA radiation in combination with VIS (wavelength range 595–1400 nm, intensity 2100 W/m2) on the retina and cornea in a guinea pig animal model of inclusion conjunctivitis. We investigated the effects 19 days after wIRA/VIS irradiation by comparing a single and double wIRA/VIS treatment with a sham control. By immunolabeling and western blot analyses of critical heat- and stress-responsive proteins, we could not detect wIRA/VIS-induced changes in their expression pattern. Also, immunolabeling of specific retinal marker proteins revealed no changes in their expression pattern caused by the treatment. Our preclinical study suggests wIRA/VIS as a promising and safe therapeutic tool to treat ocular chlamydial infections

Effects of iota-carrageenan on ocular Chlamydia trachomatis infection in vitro and in vivo

Ocular chlamydial infections with the ocular serovars A, B, Ba, and C of Chlamydia trachomatis represent the world's leading cause of infectious blindness. Carrageenans are naturally occurring, sulfated polysaccharides generally considered safe for food and topical applications. Carrageenans can inhibit infection caused by a variety of viruses and bacteria. To investigate whether iota-carrageenan (I-C) isolated from the red alga Chondrus crispus could prevent ocular chlamydial infection, we assessed if targeted treatment of the conjunctival mucosa with I-C affects chlamydial attachment, entry, and replication in the host cell. Immortalized human conjunctival epithelial cells were treated with I-C prior to C. trachomatis infection and analyzed by flow cytometry and immunofluorescence microscopy. In vivo effects were evaluated in an ocular guinea pig inclusion conjunctivitis model. Ocular pathology was graded daily, and chlamydial clearance was investigated. Our study showed that I-C reduces the infectivity of C. trachomatis in vitro. In vivo results showed a slight reduced ocular pathology and significantly less shedding of infectious elementary bodies by infected animals. Our results indicate that I-C could be a promising agent to reduce the transmission of ocular chlamydial infection and opens perspectives to develop prophylactic approaches to block C. trachomatis entry into the host cell

Supplementary data for the article: Mihailovic, J.; Inic-Kanada, A.; Smiljanic, K.; Stein, E.; Barisani-Asenbauer, T.; Cirkovic Velickovic, T. Lysine Acetylation of Major Chlamydia Trachomatis Antigens. EuPA Open Proteomics 2016, 10, 63–69. https://doi.org/10.1016/j.euprot.2016.01.007

Supplementary material for: [https://doi.org/10.1016/j.euprot.2016.01.007]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/295

Mišje monoklonsko antitelo specifično za kapa lanac humanih imunoglobulina i mogućnosti njegove primene

Monoclonal antibodies (MoAb) are usually produced as the secretion products of cloned B lymphoblastoid cell lines. Advantages of MoAbs are that they could be selected to have desired specificity and produced in relatively large quantities of consistent quality and char acteristics. Interest in the production of re agents specific for human kappa (K) chain can be explained by the fact that many pathological conditions are accompanied by frequency of immunoglobulins with K light chain. The aim of this study was production of stable MoAb-se creating hybridoma with ability to secrete imunoglobulin specific for human K chain, immunochemical characterization of this MoAb and its possible application. Murine monoclonal antibody, as signed as MoAt 44, produced by hybridoma technology is specific for human kappa chain and fully immunochemically characterized. In this paper, we reported that MoAb 44 demonstrated excellent properties in most immunochemical techniques (immunoblot, dot-blot, immunofluorescence, double and radial immunodiffusion, immunoelectrophoresis...), which highly recommend this MoAb for the application as a tool for re search and immunodiagnostics.Monoklonska antitela (MoAt) su sekretorni proizvodi kloniranih B ćelijskih limfoblastoidnih linija. Od poliklonskih antitela ih razlikuje: definisana specifičnost vezivanja, homogenost i mogućnost proizvodnje u velikoj količini. Budući da su brojna patološka stanja povezana sa sekrecijom imunoglobulina u čijem sastavu se nalazi kapa (k) tip lakog lanca, proizvodnja specifičnih monoklonskih antitela našla bi svoje mesto u njihovoj dijagnostici. Stoga je cilj našeg rada bio dobijanje stabilnog mišjeg hibridomskog klona koji bi sekretovao MoAt specifično za humani k lanac, imunohemijska karakterizacija tog MoAt i ispitivanje mogućnosti njegove primene. Mišje monoklonsko antitelo, označeno kao MoAt 44, proizvedeno hibridomskom tehnologijom, specifično je za k lanac humanih imunoglobulina i u potpunosti imunohemijski okarakterisano. MoAt 44 može da se koristi u većini imunohemijskih tehnika (imunoblot-u, dot-blot-u, imunofluorescenci, dvostrukoj i radijalnoj imunodifuziji, imunoelektroforezi...), što omogućava njegovu primenu u naučno-istraživačkom radu i imunodijagnostici