Thesis (doctoral)--University of Turku.

No description available.

The aim of this study was to identify common messenger molecules used in both the immune and the neuroendocrine systems in birds, and to shed light on a cell type within the bursa of Fabricius that has historically been postulated as a potential neuroendocrine-immune link, the bursal secretory dendritic-like cells (BSDC). An immunocytochemical approach was used to identify neuroendocrine cell populations in the thymus, pituitary and bursa of Fabricius in the chicken. Molecular confirmation of the neuroendocrine cell marker, chromogranin A (CgA) in the thymus tissue of the chicken was reported. Previously the serine protease inhibitor, ovoinhibitor, was localized in bursal follicles, specifically the cortico-medullary border region. The presence of ovoinhibitor was identified and confirmed in the chicken pituitary by this study. Continued focus on the neuroendocrine-immune interactions in chicken immune tissue narrowed the study around the BSDC population. The BSDC are a component of the stromal, non-lymphoid cellular environment of the bursa of Fabricius and are thought to play a role in B-cell maturation and differentiation. They are located mainly along the cortico-medullary border of the bursal follicles in the same area as the majority of the ovoinhibitor-positive cell population. During attempts to isolate the BSDC population by flow cytometry and laser capture microdissection, a cell culture method was developed that enriched the BSDC population by 10-fold. This enriched population was used to evaluate protein product secretion following lipopolysaccharide (LPS) challenge and compared to in vivo challenge with live Salmonella. For the first time, up-regulation of the pro-inflammatory cytokine IL-12 was documented in the chicken following in vivo challenge. In addition, the gene expression of serine protease inhibitors was markedly decreased in the adherent cell population following LPS stimulation. As a result of this research a novel method for the enrichment of an adherent population, including the BSDC, was developed, providing a valuable tool for the analysis of this population during immune stimulation.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1963. / Typescript. Vita. Includes reprints (included in pagination) of four journal articles from Endocrinology and Journal of immunology, by Aspinall et. al. Includes bibliographical references.

The bursa of Fabricius is a central lymphoid organ essential for B cell development and generation of humoral immune responses in birds. The bursa is connected dorsally to the cloaca and continually “samples” the intestinal fluid phase for the presence of antigen. It is comprised of folds or plicae, which are seeded with individual follicles, where antigen processing and presentation occurs for B cell development as well as generation of antibody responses. The plicae are separated from the bursal lumen by interfollicular epithelium (IFE) and specific regions of follicle-associated epithelium (FAE). The FAE is comprised of M cell-like cells, which are specialized for transcytosis of antigen from the bursal lumen into underlying follicles. The uptake of particulate and soluble antigen in the bursa of Fabricius has been previously demonstrated, but how particle size affects their internalization within bursal FAE and the transport of particulate antigen into deeper follicles has not been explored. It has been shown that nanoparticles (NPs) ≤40 nm are most efficiently internalized by the epithelium of the murine intestine and vaginal tract. Therefore, we examined the uptake of 0.04 - 2 μm fluorescent polystyrene NPs in bursa at 1 hour or 6 hours after bursal administration to determine whether bursal epithelium is similarly constrained. Using immunofluorescence microscopy (IFM) and spectrofluorophotometry we found that NP uptake is inversely correlated with NP size. NPs ≤40 nm are most efficiently internalized by the bursal epithelium and bursal follicles, while NPs ≥500 nm are not effectively taken up by the bursal epithelium within 6 hours of administration. Moreover, once the size-limited capabilities of the bursal epithelium were established, we also found that bursal priming of chickens with 20 nm NPs conjugated to IMP-1, a protective antigen of an important avian pathogen Eimeria maxima, induces IMP-1-specific serum IgG following sub-cutandous boosting. We induced similar IMP-1-specific serum antibody responses in chickens primed bursally and sub-cutaneously boosted as those primed and boosted sub-cutaneously. Whether this route of immunization is able to elicit long-term protection must be investigated. A number of infectious diseases, including Infectious Bursal Disease Virus (IBDV), which directly targets the bursa of young birds and prevents the development of the immune system and causes mortality, are prevalent in the poultry industry. While vaccines exist for many of these diseases they confer only partial or incomplete protection; therefore, alternative vaccine strategies must be investigated. In addition, the bursa is an ideal in vivo model for the investigation of endocytic mechanisms for uptake of particulate antigen. Therefore, further characterizing the mechanisms of NP uptake at mucosal surfaces and their immunogenicity will be important for the development of NP-based mucosal vaccines for agriculturally relevant species such as poultry.

The chicken bursa of Fabricius is a primary lymphoid tissue important for B-cell development. Our long-term goal is to understand the role of bursal microenvironment in an early B-cell differentiation event initiating repertoire development through immunoglobulin gene-conversion in the chick embryo. We hypothesize that early bursal B-cell differentiation is guided by signals through cytokine receptors. Our theory is based on previous evidence for expression of the receptor tyrosine kinase superfamily members and interleukin receptors in unseparated populations of bursal B-cells and bursal tissue. Knowledge of the expressed genes that are responsible for B-cell differentiation is a prerequisite for understanding the bursal microenvironment’s function. This project uses transcriptomic analysis to examine gene expression across an early B-cell differentiation event. RNA-seq was performed with total RNA isolated from developing B-cells at embryonic day (ED) 16 and ED 19 (n=3). Approximately 90 million high quality clean reads where obtained from the cDNA libraries. The analysis revealed differentially expressed genes involved in Wnt signaling pathway, Jak-STAT pathway, metabolic pathways, tyrosine metabolism, Toll-like receptor signaling pathway, MAPK signaling pathway, and cellhesion molecules. The transcripts for surface receptors, signal transduction and transcription factors identified in this study represent gene candidates for controlling B-cell differentiation in response to bursal microenvironmental factors.

In the present investigation, microarray analysis was used to identify potential TCDD gene targets. Three microarray experiments were performed to study the effect of TCDD in an established chicken B-cell line (DT40), in a chicken macrophage cell line (HD11), and in the bursa of Fabricius from embryos exposed in ovo at 6 days of incubation. From the DT40 microarray analyses, clones with sequence similarity to the apoptotic genes caspase 8 and caspase 9, and the transcription factor NFΜB, among others, were identified. Real-time quantitative polymerase chain reaction (RT-PCR) revealed that TCDD elicits aryl hydrocarbon receptor (AhR)-mediated apoptosis in the avian DT40 pre-B-cell line through activation of caspases 9 and 3 (see chapter III). During the course of the HD11 microarray analyses, a consistent down-regulation of the matrix metalloprotease MMP-2 was observed. This finding was the basis for the hypothesis that TCDD has an effect on the gene expression of the MMP-2 and MMP-9 in macrophages. Then, gene expression analysis and functional zymography showed that TCDD impairs the MMP-2 and MMP-9 response to LPS stimulation in HD11 chicken macrophages (see chapter V). The microarray analyses of the embryonic bursa of Fabricius provided the basis to further study of the effect of TCDD in the chicken embryo. The shifted genes were classified according to their function. The down-regulated genes included: precursor of matrix metalloprotease-inhibitor, histone acyl-transferase 1, homeobox protein CUX-2, Death Associated Protein Kinase, and UDPglucosyl transferase, among others. The up-regulated genes included: phosphoinositidespecific phospholipase, acyl Co-A oxidase, and protein effector of Cdc42, among others. Together, these microarray analyses produced a database of genes of interest that will provide sufficient hypotheses to inspire multiple investigations aimed at confirming and refining the gene expression alterations as a consequence of TCDD exposure.

<p>Инфективна болест бурзе (Гамборо болест) је контагиозна вирусна болест младих пилића. Узрочник је РНК вирус који припада фамилији Birnaviridae. Испољава тропизам према Фабрицијусовој бурзи доводећи до оштећења лимфоцита и атрофије. Болест се контролише вакцинацијом родитељских јединки и пријемчивих пилића. Употреба атенуираних вакцина може довести до смањења неких технолошких параметара. Циљ овог рада је био да се докаже које од примењених атенуираних вакцина, интермедијарних Гумбокал, Гумбокал D78 и интермедијарне-плус Гумбокал 228Е, индукује најбољи развитак имнунолошког одговора и доводе до најмањих оштећења у ткиву Фабрицијусове бурзе и слезене. Пратили смо да ли делују имуносупресивно на вакцину против Њукастл болести и да ли утичу на смањење телесне масе бројлерских пилића. Основне огледне групе су вакцинисане четрнаестог дана старости против Гамборо болести, а затим су од основних огледних група оформљене подгрупе које су вакцинисане против Њукастл болести у различитим временским периодима: О1-1, О2-2, О3-1 после седам дана, О1-2, О2-2, О3-2 после четрнаест дана, О1-3, О2-3, О3-3 после двадесетједан дан од вакцинације против Гамборo болести .Од првог до четрдесетдругог дана експеримента сваких седам дана је вађена крв пилићима, мерена њихова телесна маса и узорковане бурзе и слезене. Крвни серуми су испитивани на висину титра антитела ЕЛИСА тестом за Гамборо болест, а методом инхибиције хемагутинације за Њукастл болест. Највишу висину титра на Гамборо болест је показала огледна група О1, затим група О2 и најнижи О3. У огледним подгрупама О3-1, О3-2 и О3-3 се испољио имуносупресивни ефекат на имунолошки одговор према Њукастл болести. Фабрицијусовим бурзама је одређивана релативна маса и бурзални индекс да би се установило да ли је дошло до атрофије након примене вакцина. Најнижи бурзални индекс је установљен у огледној групи О3, затим у О2 и у О1. Таквим редоследом су устновљене и патохистолошке промене у бурзи. Добијени резултати указују да употреба интермедијане-плус вакцине индукује најбољи имунолошки одговор, али и најнижу релативну масу и вредност бурзалног индекса, као и нижу телесну масу у односу на инермедијарне вакцине. Примењене вакцине нису утицале на релативну масу слезене и нису довеле до појаве патохистолошких промена у њој.</p> / <p>Infektivna bolest burze (Gamboro bolest) je kontagiozna virusna bolest mladih pilića. Uzročnik je RNK virus koji pripada familiji Birnaviridae. Ispoljava tropizam prema Fabricijusovoj burzi dovodeći do oštećenja limfocita i atrofije. Bolest se kontroliše vakcinacijom roditeljskih jedinki i prijemčivih pilića. Upotreba atenuiranih vakcina može dovesti do smanjenja nekih tehnoloških parametara. Cilj ovog rada je bio da se dokaže koje od primenjenih atenuiranih vakcina, intermedijarnih Gumbokal, Gumbokal D78 i intermedijarne-plus Gumbokal 228E, indukuje najbolji razvitak imnunološkog odgovora i dovode do najmanjih oštećenja u tkivu Fabricijusove burze i slezene. Pratili smo da li deluju imunosupresivno na vakcinu protiv NJukastl bolesti i da li utiču na smanjenje telesne mase brojlerskih pilića. Osnovne ogledne grupe su vakcinisane četrnaestog dana starosti protiv Gamboro bolesti, a zatim su od osnovnih oglednih grupa oformljene podgrupe koje su vakcinisane protiv NJukastl bolesti u različitim vremenskim periodima: O1-1, O2-2, O3-1 posle sedam dana, O1-2, O2-2, O3-2 posle četrnaest dana, O1-3, O2-3, O3-3 posle dvadesetjedan dan od vakcinacije protiv Gamboro bolesti .Od prvog do četrdesetdrugog dana eksperimenta svakih sedam dana je vađena krv pilićima, merena njihova telesna masa i uzorkovane burze i slezene. Krvni serumi su ispitivani na visinu titra antitela ELISA testom za Gamboro bolest, a metodom inhibicije hemagutinacije za NJukastl bolest. Najvišu visinu titra na Gamboro bolest je pokazala ogledna grupa O1, zatim grupa O2 i najniži O3. U oglednim podgrupama O3-1, O3-2 i O3-3 se ispoljio imunosupresivni efekat na imunološki odgovor prema NJukastl bolesti. Fabricijusovim burzama je određivana relativna masa i burzalni indeks da bi se ustanovilo da li je došlo do atrofije nakon primene vakcina. Najniži burzalni indeks je ustanovljen u oglednoj grupi O3, zatim u O2 i u O1. Takvim redosledom su ustnovljene i patohistološke promene u burzi. Dobijeni rezultati ukazuju da upotreba intermedijane-plus vakcine indukuje najbolji imunološki odgovor, ali i najnižu relativnu masu i vrednost burzalnog indeksa, kao i nižu telesnu masu u odnosu na inermedijarne vakcine. Primenjene vakcine nisu uticale na relativnu masu slezene i nisu dovele do pojave patohistoloških promena u njoj.</p> / <p>Infectious bursal disease (Gumboro disease) is a contagious viral disease of young chickens. The causative agent is an RNA virus that belongs to the family Birnaviridae. It exhibits tropism toward bursa of Fabricius causing damage to cells and atrophy. The disease is controlled by vaccination of susceptible broiler breeders and chickens. The use of attenuated vaccines may lead to the reduction of certain production parameters. The aim of this study was to prove which of the applied attenuated vaccines, intermediate Gumbokal, Gumbokal D78 and intermediate-plus Gumbokal 228E, induces the best immunological response and leads to fewest damages to burza of Fabricijus tissue and spleen. We tracked whether it influences on immunosuppressive vaccine against Newcastle disease and if it leads to reduce of body mass of broiler chickens. Basic experimental groups were vaccinated on the fourteenth day of age against Gamboro diseases, and sub-groups were formed of the basic experimental groups which have been vaccinated against Newcastle disease in different time periods: O1-1, O2-2, O3-1 after seven days, O1-2 , O2-2, O3-2 after fourteen days, O1-3, O2-3, O3-3 after twenty-one days of vaccination against Gumboro disease. From the first to the fourty second day of the experiment, blood was extracted form the chicks every seven days, their body weight was measured and burza and spleen were sampled. Blood serum were assayed for the amount of the antibody titer by enzyme-linked immunosorbent assay test (ELISA) for Gumboro disease, and with Hemagglutination-inhibition test (HI) for Newcastle disease. The biggest titer for Gamboro disease were showed by experimental group O1, then O2 and group O3 the lowest. In the experimental subgroups O3-1, O3-2 and O3-3 immunosuppressive effect was exhibited on the immune response to Newcastle disease. Relative weight and bursal index were determined for burza of Fabricius to determine whether there was atrophy after administration of the vaccine. The lowest bursal index was established in the experimental group O3, followed by O2 and O1. Such sequence was establilshed also for histopathological changes in the burza. The obtained results indicate that the use of the intermediate-plus vaccine induces the best immune response, but also the lowest relative value of the mass and burzal index, as well as a lower body weight compared to the inermediate vaccine. Applied vaccines did not affect the relative weight of the spleen and have not led to the appearance of histopathological changes in it.</p>