A nyúl neonatális FC receptor klónozása, jellemzése = Cloning and characterisation of the rabbit neonatal FC receptor

Az emlősök szervezetében található immunglobulinok közül legjelentősebb az IgG, mely a fertőzésekkel szemben biztosít védelmet. A neonatális Fc receptor (FcRn) dimer molekula, nehéz lánca az MHC-I osztályú fehérjékhez tartozik. A molekula részt vesz az anyai IgG utódoknak történő átadásában, a szérum IgG szintjének fenntartásában, és az immunválaszban –fagocitózisban és antigén prezentációban. A főemlősökhöz hasonlóan nyúlban az anyai immun transzport folyamatok a magzati korban történnek a szikzacskón keresztül. Bár a nyúlban a transzport folyamatát már vizsgálták, a receptor molekulát még nem klónozták és jellemezték. Hogy pontosan megértsük a nyúlban, mint modellállatban az FcRn működését RACE PCR-el klónoztuk a nyúl FcRn nehéz láncát és jellemeztük. Megállapítottuk, hogy nagy hasonlóságot mutat az ismert emlős ortológokhoz. Részletesen vizsgáltuk a gén expresszióját a placentában, az amnionban és a szikzacskóban. A receptor megjelenik a placenta kapilláris endotél sejtjeiben és a szikzacskó endoderm sejtek apikális régiójában. RNS szinten kimutattuk jelenlétét monocitákban és makrofágokban is. Megállapítottuk, hogy a nyúl FcRn pH függő módon működik. A fenti vizsgálatok igazolják, hogy az általunk klónozott és jellemzett receptor a nyúl FcRn. | IgG is the most important immunoglobulin in mammals. IgG protects against infections. The neonatal Fc receptor (FcRn) is a dimer its heavy chain is similar in structure to MHC class I proteins. Multiple functions have been shown for neonatal Fc receptor, as it mediates maternal transfer of IgG to offsprings, responsible for the maintenance of serum IgG level and plays an important role in immune respons-phagocytosis and antigen presentation. Like primates, rabbit maternal IgG transport occurs during the fetal life and mediated by the FcRn through the yolk sac. Although, there were several studies in analyzing IgG transport in rabbit yolk sac, the rabbit FcRn has not been cloned and analyzed so far. In order to have a better understanding in this valuable model, first we cloned the rabbit FcRn using RACE PCR and found that the coding region of the rabbit FcRn alpha-chain shows high similarity to the other mammalian orthologues. We also characterized the rabbit FcRn by detecting its expression in monocytes, macrophages, fetal placenta, yolk sac and amnion, though this last one presented less FcRn expression, by RT-PCR. We detected this receptor in the endothelial cells of the placental capillaries as well as in the apical region of endoderm cells in the yolk sac. We found that the rabbit FcRn, binds IgG in a pH dependent manner, like its other mammalian FcRns. These results confirm that the rabbit Fc receptor in the rabbit yolk sac is a bona fide FcRn receptor

Fejlődés specifikus gének expressziós mintázatának összehasonlítása pluripotens nyúl embrionális és epiblaszt őssejtekben. = Comparison of developmental stage specific gene expression patterns in pluripotent rabbit embryonic and epiblast derived stem cell lines .

Transzgénikus nyulak kiváló modell állatként szolgálnak az öröklött humán betegségek tanulmányozására. Jelenleg nem áll rendelkezésre olyan pluripotens embrionális őssejt vonal, ami lehetővé tenné célzott genetikai módosítások végrehajtását nyúlban. Ivarsejt kimérákat mind a mai napig nem sikerült előállítani ezek felhasználásával. A Nanog és az Oct4 transzkripciós faktorok a legfontosabb gének, amik elősegítik a pluripotencia megtartását egér és humán őssejtekben, azonban nyúl esetében nagyon keveset tudtunk ezen gének expressziójáról. Sikerült feltérképeznünk a nyúl őssejt specifikus markerek expresszióját a nyúl embriók fejlődése során, valamint embrionális és epiblaszt őssejtekben. Az őssejtek passzálásának előrehaladtával az epiblaszt specifikus Oct4, és Nanog szint csökkenni kezdett, a trofoblaszt (Cdx2) és hipoblaszt (Gata4, Gata6) sejtekre jellemző markerek expressziója viszont továbbra is kimutatható maradt. Ezekben sejttenyészetekben az őssejt kolóniák nagy része differenciálódni kezdett, valószínűleg a sejttenyészetben megtalálható hipoblaszt és trofoblaszt eredetű sejtekből származó faktorok hatásának eredményeként. A munka folytatásaként szeretnénk megismerni a nyúl Ins, Wnt, FGF4 és LIF jelátvitelben résztvevő elemeket, kiegészítve a regulációban fontos szerepet játszó őssejt specifikus nyúl mikroRNS-ek szerepének megismerésével. Azt reméljük, hogy ezen jelátviteli rendszerek megismerése segíthet valóban pluripotens nyúl ES sejtvonalak létrehozásában. | Laboratory rabbits have long been used in biomedical research. Recently, they have been used as experimental models for human diseases. Rabbit ES cells would be invaluable for the study of testing stem cell therapies for human applications. Although many attempts have been made to derive real pluripotent ES cell lines from rabbits, none has been successful. We analyzed the expression pattern of embryonic stem cell specific markers in rabbit embryos and epiblast cells. The expression pattern of Oct4, Nanog transcriptions factors during the rabbit embryonic development was not known. Epiblast specific Oct4 and Nanog, trophoblast specific Cdx2 and hypoblast specific Gata4 and Gata6 were examined in blastocysts, attached embryos and epiblast like cells. The Oct4 and Nanog in rabbit ES-like cells expressed at high level reflecting their pluripotent state, but reduced passage by passage. We could recognize Cdx2 expression which might be related to cells derived from remaining trophoblast cells. We could also detect low level of Gata4 and Gata6 expressing colonies correlated with some differentiated and hypoblast derived cells. After few passages the most of the epiblast derived colonies begin to differentiate, most likely as a result of the effect of remaining hypoblast and trophoblast cells derived factors. As a continuation of our work, we want to investigate the rabbit Ins, Wnt, FGF4 and LIF signaling elements in addition with stem cells specific rabbit microRNAs

Characterization of the Rabbit Neonatal Fc Receptor (FcRn) and Analyzing the Immunophenotype of the Transgenic Rabbits That Overexpresses FcRn

The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes an active role in phagocytosis, and delivers antigen for presentation. We have previously shown that overexpression of FcRn in transgenic mice significantly improves the humoral immune response. Because rabbits are an important source of polyclonal and monoclonal antibodies, adaptation of our FcRn overexpression technology in this species would bring significant advantages. We cloned the full length cDNA of the rabbit FcRn alpha-chain and found that it is similar to its orthologous analyzed so far. The rabbit FcRn - IgG contact residues are highly conserved, and based on this we predicted pH dependent interaction, which we confirmed by analyzing the pH dependent binding of FcRn to rabbit IgG using yolk sac lysates of rabbit fetuses by Western blot. Using immunohistochemistry, we detected strong FcRn staining in the endodermal cells of the rabbit yolk sac membrane, while the placental trophoblast cells and amnion showed no FcRn staining. Then, using BAC transgenesis we generated transgenic rabbits carrying and overexpressing a 110 kb rabbit genomic fragment encoding the FcRn. These transgenic rabbits – having one extra copy of the FcRn when hemizygous and two extra copies when homozygous - showed improved IgG protection and an augmented humoral immune response when immunized with a variety of different antigens. Our results in these transgenic rabbits demonstrate an increased immune response, similar to what we described in mice, indicating that FcRn overexpression brings significant advantages for the production of polyclonal and monoclonal antibodies

Impact of cycling cells and cell cycle regulation on Hydra regeneration

Hydra tissues are made from three distinct populations of stem cells that continuously cycle and pause in G2 instead of G1. To characterize the role of cell proliferation after mid-gastric bisection, we have (i) used flow cytometry and classical markers to monitor cell cycle modulations, (ii) quantified the transcriptomic regulations of 202 genes associated with cell proliferation during head and foot regeneration, and (iii) compared the impact of anti-proliferative treatments on regeneration efficiency. We confirm two previously reported events: an early mitotic wave in head-regenerating tips, when few cell cycle genes are up-regulated, and an early-late wave of proliferation on the second day, preceded by the up-regulation of 17 cell cycle genes. These regulations appear more intense after mid-gastric bisection than after decapitation, suggesting a position-dependent regulation of cell proliferation during head regeneration. Hydroxyurea, which blocks S-phase progression, delays head regeneration when applied before but not after bisection. This result is consistent with the fact that the Hydra central region is enriched in G2-paused adult stem cells, poised to divide upon injury, thus forming a necessary constitutive pro-blastema. However a prolonged exposure to hydroxyurea does not block regeneration as cells can differentiate apical structures without traversing S-phase, and also escape in few days the hydroxyureainduced S-phase blockade. Thus Hydra head regeneration, which is a fast event, is highly plastic, relying on large stocks of adult stem cells paused in G2 at amputation time, which immediately divide to proliferate and/or differentiate apical structures even when S-phase is blocked

Reduced IgG catabolism in Tg rabbits that carry two extra copies of the rabbit FcRn.

<p>We have analyzed the half-life of rabbit IgG in Tg (homozygous; +/+) and wt rabbits from days 2–13 after injecting OVA-specific rabbit IgG i.v. into these animals. <b>A–B.</b> Our analysis showed that the Tg rabbits have increased serum persistence of rabbit IgG as the beta phase half-lives of the IgG were 7.1±0.5 days (mean ± SEM) as compared to their controls which showed 5.3±0.3 days. <b>C.</b> This difference may be even greater as non-immunized Tg rabbits have higher total IgG levels as compared to their controls. Values shown are the mean ± SEM. (*, <i>P</i><0.05). The experiment was repeated two times with similar results.</p

Augmented humoral immune response in transgenic rabbits.

<p><b>A–B</b>. Tg+/− and wt rabbits (4 and 8 animals, respectively) were immunized with OVA. After the booster immunization the OVA-specific IgG titers were nearly double in Tg rabbits as compared with the wt animals. We found that the total IgG levels rose steadily after immunization and reached peak levels on day 49 in Tg and wt animals. <b>C–D</b>. Tg+/− and wt rabbits (5 and 5, respectively) were immunized with TNP-BSA. The mean TNP-specific IgG level was higher in Tg rabbits as compared to their wt controls at the peak of the immune response, on day 49, one week after the second booster immunization with an almost doubled level of total IgG as compared to their wt controls. <b>E–F</b>. The same rabbits which had been immunized with TNP-BSA, were immunized with a conserved influenza hemagglutinin epitope (HA2) conjugated to KLH. The mean of the HA2-specific IgG levels was double at the peak of the immune response, on day 56, two weeks after the second booster immunization, with an almost doubled level of total IgG as compared to their wt controls. Values shown are the means ± SEM. (*, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.).</p

Detection of the rabbit FcRn in Western blot.

<p>This Western blot shows that the chicken antibody strongly and specifically reacted with the soluble recombinant bovine FcRn which was used for immunization (its estimated molecular weight is 30 kDa), a ∼40-kDa band that is consistent with the known molecular weight of the bovine FcRn α-chain in the protein extract from a bovine FcRn stably transfected cell line (B4) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028869#pone.0028869-Kacskovics4" target="_blank">[46]</a> which strongly expresses the functional form of the bovine FcRn and a ∼40-kDa band that is consistent with the calculated molecular weight of the rabbit FcRn α-chain in the protein extract from the rabbit yolk sac from 24 dpc fetuses.</p

Rabbit embryos start to express FcRn by 6 dpc, close to the implantation time.

<p>Rabbit FcRn α-chain expression was analysed in rabbit blastocysts and embryos at different time points by PCR. L-ladder, 1–3.5 dpc rabbit blastocyst, 2–4.5 dpc rabbit blastocyst, 3–6 dpc rabbit embryo, 4–9 dpc rabbit embryo, 5–13.5 dpc rabbit embryo, 6 - negative control (DNA omitted).</p

Cytoplasmic domains of the FcRn sequences most likely reflect their phylogenetic position.

<p>Marsupials (possum, opossum, and wallaby) have relatively short cytoplasmic domains composed of 27–28 amino acid residues. The next phylogenetic step resulted in clades Atlantogenata and Boreoeutheria. The only (predicted) sequence we found belonging to Atlantogenata (elephant) shows a 7–8 amino acid longer cytoplasmic domain as compared to Marsupials. Boreoeutheria is composed of the sister taxa Laurasiatheria and Euarchontoglires. Species belong to Euarchontoglires analyzed so far (human, chimp, gorilla, orangutan, gibbon, rhesus, marmoset, lemur, rabbit, pika, squirrel, hamster, rat and mouse) preserved these extra amino acids of the FcRn C-terminal with the exception of the guinea pig (based on its predicted amino acid sequence). Rabbit lost five amino acids in a more N-terminal (or middle) part of the cytoplasmic domain. As pika (Ochotona), another Lagomorphs, possesses these residues, the five amino acid deletion is thus specific of either rabbit or Leporidae family. Animals belong to the Laurasiatheria clade (bovine, sheep, pig, horse, bat, dog and panda) lost 10 amino acids of their FcRn C-terminals. Phylogenetic tree was created based on Prasad et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028869#pone.0028869-Prasad1" target="_blank">[45]</a> where some branch lengths were optimized for clarity and space.</p