Promene u stepenu galaktozilacije IgG kunića tokom dugotrajne imunizacije goveđim serumskim albuminima

The galactose content of isolated IgG was analyzed before immunization and at intervals during prolonged immunization over 16 months. It was found that IgGs from non-immunized and immunized animals differed in galactose content and that the galactose content of serum IgG varied during the course of the immune response. These results suggest possible intraclonal variations or the emergence of subclones of cells secreting IgG that is differently galactosylated relative to the IgG of non-immunized animals. Since isotypic polymorphism of IgG (subclasses), which determines the effector potential(s) of IgG antibodies, does not exist in rabbits, glycosylation may be what defines functional subsets of isotypically uniform antibodies produced during an immune response in these animals.Ugljeni hidrati su strukturni element imunoglobulina G (IgG), bitan za konformaciju i efektorne funkcije molekula. Ugljenohidratni sadržaj čine oligosaharidni lanci (glikani) vezani kovalentno za konzervirani Asn 297 u svakom CH2 domenu Fc regiona IgG molekula. Sadržaj pojedinih šećera koji ulaze u sastav glikana nije isti u svim molekulima IgG. Imunoglobulini G su populacija različitih glikoformi čiji sadržaj varira pod fiziološkim, a naročito pod raznim patološkim uslovima. Pretpostavlja se da su te varijacije odraz promena u distribuciji specifičnih antitela IgG klase tokom tekućeg često hroničnog, imunog odgovora na neki antigen. O dinamici tih promena se međutim, malo zna, a posebno o pojavi glikoformi tokom imunog odgovora koje bi mogle imati patogenetski značaj. U ovom radu su analizirane promene u glikozilaciji serumskih IgG tokom imunog odgovora kunića dugotrajno imunizovanih goveđim serumskim albuminima. Rezultati su ukazali da je taj odgovor praćen permanentnim promenama u sadržaju terminalne galaktoze. S obzirom da su molekuli IgG kunića, za razliku od drugih sisara, izotipski uniformni, tj. nemaju podklase (kojima je determinisana brojnost efektornih funkcija IgG), razlike u galaktozilaciji bi mogle determinisati pojavu funkcionalnih subsetova IgG antitela stvorenih tokom imunog odgovora kod ove vrste

Imunokompleksi u krvnom serumu teladi u neonatalnom periodu i kod odraslih goveda

In this work immune complexes (IC) in the sera of healthy calves from birth to four months of age, heifers and cows were investigated. Polyethylen glycol (PEG) precipitation assay was applied after previous standardization. Optical density (OD) of redissolved precipitates, formed after adding PEG to serum samples, was a measure of IC level. The upper-limit values for normal IC levels were established using isolated cattle IgG in different amounts and aggregated by heat as an IC model in vitro, in the same assay. In newborn calves before colostrum intake, OD of 0.047±0.024 was registered in redissolved PEG precipitates, far below the values obtained for native monomeric IgG. Therefore, these values can not be considered to refer to immune complexes. After colostrum intake, OD values for normal IC levels were higher, with significant interindividual differences: in calves at 6 24 and 48 hours after birth the mean OD values of solubilized PEG precipitates were 0.069±0.025, 0.148±0.079, and 0.136±0.062 respectively. In the sera of calves 10 days after birth mean OD value was slightly lower 0.089±0.053, whereas in the serum of 1, 2, 3 and 4 month old calves normal levels of IC rose to 0.165±0.067, 0.157±0.080, 0,231±0,124 and 0.261±0.092 respectively. OD values for IC levels for adult animals were found to be 0,456±0,150 for heifers, and 0,183±0,031 for cows. Together, the obtained data may be included in a reference values system for normal levels of IC at different ages, in relation to which the IC levels under pathological conditions could be estimated.U radu je ispitivan nivo imunokompleksa (IC) u krvnom serumu teladi od rođenja do četiri meseca starosti, kao i kod junica i krava. Za analize je posle predhodne standardizacije korišćen polietilenglikol precipitacioni test (direktan PEG test). Meru nivoa IC predstavljale su vrednosti optičke gustine (OD) rastvorenog precipitata dobijenog nakon taloženja imunokompleksa iz seruma PEG-om. Gornja granica normalnih vrednosti za nivo IC utvrđena je prema vrednostima dobijenim za izolovane i toplotom agregirane kravlje IgG, u istom testu, gde su takvi IgG korišćeni kao modeli IC in vitro. U grupi novorođene teladi pre uzimanja kolostruma vrednosti OD su bile ekstremno niske (OD 0,047±0.024) i nisu mogle biti pripisane imunokompleksima. Vrednosti OD za normalan nivo IC u serumu teladi nakon uzimanja kolostruma su bile više: za telad 6, 24 i 48 sati nakon rođenja taj nivo je iznosio 0,069±0,025, 0,148±0,079 i 0,136±0,062 respektivno. U odnosu na ove vrednosti, nivo IC u serumu teladi 10 dana od rođenja je bio nešto niži i iznosio 0,089±0,053, da bi se, zatim, kontinuirano povećavao kod teladi uzrasta od 1 do 4 meseca i iznosio 0,165±0,067 za telad od 1-og meseca, 0,157±0,080 za telad od 2 meseca, 0,231±0,124 za telad od 3 meseca i 0,261±0,092 za telad od 4 meseca starosti. Nivo imunokompleksa u serumu odraslih goveda iznosio je kod junica 0,456±0,150 i krava 0,183±0,031. Dobijeni rezultati mogu biti uključeni u kontrolni sistem vrednosti za normalan nivo IC u raznim starosnim grupama goveda, koji se može koristiti pri određivanju nivoa IC PEG testom pod patološkim uslovima kod teladi junica i krava

Elektroforetske i imunoelektroforetske karakteristike IgG kao konstituenata peg precipitabilnih imunokompleksa seruma kod teladi do četvrtog meseca starosti

Preruminant calves encounter numerous antigens, and formation of immune complexes is necessary for antigens elimination. The capability of immunologically immature calves to form immune complexes has not yet been studied in detail. For immune complexes studies, selective precipitation by PEG was performed, in combination with agarose gel electrophoresis and immunoelectrophoresis, with an aim to determine some properties of IgG, as constituents of immune complexes. In our previous work it was shown that the level of PEG precipitable immune complexes increased in the period from birth to 48-hours of life, decreased at day 10, increased in one month old animals, and after that, stayed unchanged until 4th month of age. Electrophoretic and immunoelectrophoretic analysis showed that until one month of age, calves' sera and PEG precipitates contained only one part of IgG molecules which corresponded to fast, anionic γ globulins. Although at the age of one month, preruminant calves' sera contained all molecular forms of IgG molecules present in the sera of adult cattle, only one part of serum IgG (fast, anionic IgG) was precipitated by PEG. In older calves, all molecular forms of serum IgG had the capacity to form PEG precipitable immune complexes. Presented data, as well as the results of our previous work, can be used as parameters with reference to physicochemical and immunochemical characteristics of IgG immune complexes of preruminant calves under pathological conditions.Telad od rođenja dolazi u kontakt sa velikim brojem antigena za čije je efikasno uklanjanje neophodno formiranje imunokompleksa. Sposobnost ovih, još uvek imunološki nezrelih, životinja da formiraju iminokomplekse, kao i sastav njihovih konstituenata, nisu detaljno ispitivani. U ovom radu smo imunokomplekse iz seruma teladi izolovali precipitacijom polietilen glikolom (PEGom), a osobine IgG, kao potencijalnih konstituenata imunokompleksa, analizirali metodama elektroforeze i imunoelektroforeze. U našem prethodnom radu dokazali smo da se nivo PEG precipitabilnih imunokompleksa menja sa uzrastom teladi. On raste od rođenja do 48. sata, opada desetog dana, ponovo raste u prvom mesecu, nakon čega ostaje nepromenjen do četvrtog meseca starosti. Elekroforetske i imunoelektroforetske analize su ukazale da se u serumu i PEG precipitatima seruma teladi do mesec dana starosti nalazi samo jedna molekulska frakcija IgG koja odgovara brzim, anjonskim IgG. Iako se u serumima teladi već u prvom mesecu starosti nalaze sve molekulske forme IgG koje se nalaze i u serumu odraslih krava, samo jedan njihov deo (brzi, anjonski γ globulini) ima sposobnost da formira imune komplekse. Kod starijih teladi, kao i kod odraslih krava, sve molekulske forme serumskih IgG mogu da formiraju imune komplekse. Ovi rezultati, zajedno sa rezultatima našeg predhodnog rada, se mogu koristiti za dobijanje referentnog sistema vrednosti nivoa i sastava imunokompleksa kod zdravih teladi, a koji bi se mogao koristiti za određivanje nivoa i sastava imunokompleksa kod teladi u patološkim uslovima

Hipogalaktozilacija imunoglobulina G (IgG) gingivalne tečnosti i pljuvačke u obolelih od parodontopatije

Changed glycosylation of immunoglobulin G (IgG), above all, the expression of thermal galactose, influence to numerous functions of those immunoglobulin and correlate with the inflammatory level in a number of diseases. Aim: This work analyses the distribution of IgG subclasses and the content of thermal galactose in them, in saliva and gingival fluid of the patients with periodontal disease and different gum inflammatory level. Materials and methods: It was used saliva and gingival fluid of 30 adults with clinical picture of periodontal disease and 20 persons with healthy periodontium. The qualification of IgG was done by “dot-blot” procedure and the, and thermal galactose was determined by lectin immunoblot procedure. Results: The division of IgG subclasses in both fluids was different in the patients with periodontal disease and in control samples. In saliva and gingival fluid of the diseased quantitatively dominated IgG2 subclasses, independently from periodontal status. In IgG of both fluids, thermal galactose was exprimated at the healthy periodontium persons (control) and with the person with initial periodontal disease, while at the person with increased periodontal disease the expression of this saccharide wasn’t registered in neither of fluids. Conclusion: The results showed that there is a shift towards hypogalactosyled IgG glikoforms during the process of gum inflammation at the periodontal disease patients.Promenjena glikozilacija imunoglobulina G (IgG), pre svega ekspresija terminalne galaktoze, utiče na brojne funkcije ovih imunoglobulina i korelira sa stepenom zapaljenja u mnogim bolestima. Cilj rada: U ovom radu analizirana je distribucija IgG podklasa i sadržaj terminalne galaktoze u njima, u pljuvačci i gingivalnoj tečnosti bolesnika sa parodontopatijom različitog stepena inflamacije gingive. Materijal i metod: Kao materijal u ispitivanjima korišćena je gingivalna tečnost i pljuvačka 30 odraslih osoba sa kliničkom dijagnozom parodontopatija i 20 osoba sa zdravim parodoncijumom. Kvantifikacija IgG urađena je “dot-blot” postupkom, a određivanje terminalne galaktoze lektinskim imunoblot postupkom. Rezultati: Raspodela IgG podklasa u obe tečnosti se razlikovala kod parodontopatija i u kontrolnim uzorcima. I u pljuvačci i u gingivalnoj tečnosti obolelih, kvantitativno je dominirala IgG2 podklasa, nezavisno od parodontalnog statusa. U IgG obe oralne tečnosti terminalna galaktoza je bila eksprimirana kod osoba sa zdravim parodoncijumom (kontrola) i kod osoba sa početnom (inicijalnom) parodontopatijom, dok kod osoba sa uznapredovalom parodontopatijom ekspresija ovog šećera nije registrovana ni u jednoj od ove dve tečnosti. Zaključak: Rezultati ovih istraživanja ukazuju da postoji pomeranje prema hipogalaktozilovanim IgG glikoformama tokom procesa inflamacije gingive u obolelih od parodontopatije

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

Imunski sistem uretre muškaraca

Sluznica muške uretre je inicijalno mesto infekcije brojnim patogenima koji su prenosivi seksualnim putem. U njenim donjim segmentima ce odvijaju složene aktivnosti vezane za kontrolu infekcije i sprečavanje njenog širenja prema gornjim segmentima muškog genitalnog trakta. Sluznica urogenitalnog trakta i kod muškaraca i kod žena sagledava ce kao integralni deo svekolikog mukoznog imunskog sistema, ali i kao imunološki samosvojni induktivni prostor sa specifičnim mehanizmima ugrađivanja i procesiranja antigena i pokretanja sekretornih imunskih odgovora. U velikoj meri neispitan, imunoglobulinski sistem muške uretre je predmet naših istraživanja i povod za ovaj osvrt na novija saznanja o mukoznom imunitetu, jednom od uzbudljivih polja savremenih istraživanja u imunobiologiji

Uloga citokina u ortodontskom pomeranju zuba

One of the most important breakthroughs in the understanding of bone biology was the identification of the role of cytokines in bone remodelling including the alveolar bone exposed to the effect of mechanical forces during orthodontic treatment. Since bone remodelling is associated, in its early phase, with inflammation of the surrounding tissue, the hypothesis has been suggested on the role of proinflammatory cytokines in the process of bone remodelling, primarily IL-1β, IL-6 and TNF- α. These cytokines function as response mediators in the acute phase of inflammation, as well as in the processes of metabolism, and stimulation of resorption and inhibition of bone formation. Mostly uninvestigated, the dynamics of concurrent changes of these three cytokines during the early phase of orthodontic teeth movement in children and adults was the subject of our investigation presented in this article on the current knowledge on the role of cytokines in this process.Jedan od najznačajnijih prodora u razumevanju bioloških osobina kosti jeste rasvetljavanje uloge citokina u preoblikovanju kosti (remodeliranje, remodeling), uključujući i alveolarnu kost zuba, izloženog delovanju mehaničke sile tokom ortodontskog lečenja. S obzirom na to da je remodeliranje kosti, koje pokreću ortodontske sile, u svojoj ranoj fazi povezano sa zapaljenjem okolnog tkiva, izneta je pretpostavka o ulozi proinflamatornih citokina u procesu remodeliranja kosti, pre svega IL-1β, IL-6 i TNF-α. Ovi citokini posreduju u reakcijama akutne faze zapaljenja, kao i u procesima metabolizma, stimulacije resorpcije i inhibicije stvaranja kosti. U velikoj meri neispitana, dinamika promena ova tri citokina istovremeno tokom rane faze ortodontskog pomeranja zuba kod dece i odraslih jeste predmet naših istraživanja i povod za ovaj osvrt na postojeća saznanja o ulozi citokina u tom procesu

Acquired von Willebrand syndrome in multiple myeloma

Acquired von Willebrand syndrome (AvWS) is an uncommon complication of multiple myeloma (MM), and it is believed to be connected with paraprotein. The aim of this study was to determine the incidence of AvWS in patients with MM, and estimate the role of paraprotein in its occurrence. The study included 40 patients with MM. The plasma level of paraprotein, platelet adhesion on glass pearls, plasma von Willebrand factor antigen concentration, and ristocetin-induced platelet aggregation (RIPA) were measured initially. Absence of RIPA was found in six patients with MM (15%); however, all six of them had normal levels of von Willebrand factor antigen. Paraprotein was isolated from the serum of these patients. Platelet aggregation was measured in six healthy donors before and after addition of the isolated paraprotein. RIPA was significantly decreased in healthy donors in the presence of paraprotein (P lt 0.001). The same test was repeated with added human immunoglobulins for intravenous use without any change in RIPA. A significant negative correlation between plasma paraprotein level and RIPA was found (P lt 0.001). These investigations have shown that paraprotein is associated with AvWS in patients with MM

Matične ćelije zubne pulpe i njihov potencijalni značaj u regenerativnoj medicini

To date, three types of dental stem cells have been isolated: Dental Pulp Stem Cells (DPSC), Stem Cells From Human Exfoliated Deciduous Teeth (SHED) and Immature Dental Pulp Stem Cells (IDPC). These dental stem cells are considered as mesenchymal stem cells. They reside within the perivascular niche of dental pulp. They are highly proliferative, clonogenic, multipotent and are similar to mesenchymal Bone Marrow Stem Cells (BMSC). Also, they have high plasticity and can be easy isolated. The expressions of the alkaline phosphatase gene, dentin matrix protein 1 and dentinsialophosphoprotein are verified in these cells. Analyses of gene expression patterns indicated several genes which encode extracellular matrix components, cell adhesion molecules, growth factors and transcription regulators, cell signaling, cell communication or cell metabolism. In both conditions, in vivo and in vitro, these cells have the ability to differentiate into odontoblasts, chondrocytes, osteoblasts, adipocytes, neurons, melanocytes, smooth and skeletal muscles and endothelial cells. In vivo, after implantation, they have shown potential to differentiate into dentin but also into tissues like bone, adipose or neural tissue. In general, DPSCs are considered to have antiinflammatory and immunomodulatory abilities. After being grafted into allogenic tissues these cells are ableto induce immunological tolerance. Immunosuppressive effect is shown through the ability to inhibit proliferation of T lymphocytes. Dental pulp stem cells open new perspectives in therapeutic use not only in dentin regeneration, periodontal tissues and skeletoarticular, tissues of craniofacial region but also in treatment of neurotrauma, autoimmune diseases, myocardial infarction, muscular dystrophy and connective tissue damages.Iz zubne pulpe su do danas izolovane tri populacije matičnih ćelija koje su označene kao matične ćelije zubne pulpe (engl. Dental Pulp Stem Cells, DPSC), matične ćelije iz eksfoliranih mlečnih zuba (engl. Stem Cells From Human Exfoliated Decidual Teeth, SHED) i nezrele matične ćelije zubne pulpe (engl. Immature Dental Pulp Stem Cells, IDPC). Sve matične ćelije zubne pulpe su ektomezenhimalnog porekla i lokalizovane su u perivaskularnoj niši. One se lako i efikasno izoluju, visoko su proliferativne, klonogene, multipotentne, ispoljavaju visok stepen plasticiteta i i slične su mezenhimalnim matičnim ćelijama koštane srži (BMSC). U njima je pokazana visoka ekspresija gena alkalne fosfataze, proteina 1 matriksa dentina i dentin-sijalofosfoproteina. Takođe, istaknuta je važnost u ovim ćelijama ekspresije više gena koji kodiraju sintezu komponenti ekstracelularnog matriksa, molekula ćelijske adhezije, faktora rasta, transkripcionih faktora, gena prenosa ćelijskih signala, ćelijske komunikacije i metabolizma. U uslovima in vitro ili in vivo ove ćelije mogu da se diferenciraju, s određenim međusobnim razlikama, u pravcu odontoblasta, hondrocita, osteoblasta, adipocita, neurona/glije, glatkih i skeletnih mišićnih ćelija, endotelnih ćelija i melanocita. U uslovima in vivo, nakon implantacije, pokazuju različit potencijal za formiranje dentina, ali i koštanog, masnog i nervnog tkiva. Generalno se smatra da DPSC imaju anti-inflamatorno dejstvo i ispoljavaju imunom-odulatorni efekat. Takođe, dovode do imunološke tolerancije ukoliko se implantiraju u alogena tkiva. Sposobnost inhibicije proliferacije T limfocita ukazuje na njihovo imunosupresivno dejstvo. Matične ćelije zubne pulpe otvorile su nove perspektive u terapijskoj primeni ovih ćelija ne samo u regeneraciji dentina, tkiva periodoncijuma i koštano-zglobnog tkiva kraniofacijalne regije, već i u lečenju neurotraume, autoimunskih oboljenja, infarkta miokarda, mišićne distrofije i oštećenja vezivnog tkiva

Interaction between fibronectin fragments and immunoglobulin G in gingival crevicular fluid of patients with periodontal disease

Introduction Fibronectin (FN) can interact with immunoglobulin G (IgG) molecules affecting the process of physiological elimination and causing abnormal deposition of immune complexes. The aim of the study was to analyze interaction between FN fragments and IgG molecules with different glycosylation profiles in gingival crevicular fluid (GCF) of patients with periodontal disease and healthy controls. Material and Methods The study included 30 patients with moderate and advanced periodontitis and 22 healthy subjects. IgG and FN content in GCF were measured as well as the presence of FN and galactose expression on IgG molecules. Results IgG content in GCF was five times higher in patients with moderate (p<0.01) and eight time higher in patients with advanced periodontitis (p<0.001) compared to healthy subjects. Also, hypogalactosylated forms of IgG were found in higher concentration in GCF of patients with advanced periodontitis compared to moderate periodontitis and healthy subjects (p<0.05). FN fragments of molecular mass 48 - 53 kDa were the most commonly found fragments in all three groups. Furthermore, in patients with advanced periodontitis, fibronectin fragments were attached to IgG molecules. Conclusion IgG and FN fragments form complexes in GCF in patients with periodontal disease and healthy subjects