Today the immunosuppressive treatment is kept to a level were the incidence of acute rejection is below 20% within the first year after transplantation. As a consequence, a group of transplanted patients is over-immunosuppressed and at risk for infections. There is therefore an urgent need for tools which are able to determine the cellular immune response after organ transplantation. This knowledge would facilitate the task of prospectively opimize the immunosuppressive treatment and identify patients at risk of developing rejection episodes or infections. To address this issue, a rat-kidney transplantation model for acute rejection was developed to study immune responses to allografts. Infiltrating lymphocytes were analysed using an in vitro culture system which allowed cells to propagate from the biopsies to culture medium. The numbers of outgrowing cells were correlated with morphological and immunohistochemical signs of rejection. When immunosuppressive treatment was administered for 2 and four days after acute rejection, histology did not reveal any improvement, however cellular propagation was reduced by 50 and 75%, respectively. Using the tissue culture technique in human transplanted kidney grafts, which was originally developed for the animal model, the number of propagated cells measured was profoundly higher in grafts with acute cellular rejection than from grafts in other groups. In some cases the number of propagated cells was better correlated with the clinical outcome than the diagnosis made by morphological evaluation. To determine immune responses to cytomegalovirus (CMV), we utilised Human Leukocyte Antigen (HLA) tetramer staining and stimulation of T cells with viral antigens. Both of these techniques independently detected CMV specific T cells in immunosuppressed and healthy individuals with latent or active infection. Although the frequency of CMV specific T cells did not differ between groups, there was a functional impairment in immunosuppressed patients as evidenced by reduced interferon-gamma production. In conclusion, these techniques can be used to determine the cellular immune response to allografts and cytomegalovirus and prove valuable for the optimization of immunosuppressive protocols and antiviral treatment.

The purpose of this study was to gain more knowledge about the deleterious effects of decreased muscle protein concentration on skeletal muscle function, by measuring the concentrations of myosin and actin in single pig muscle fibres. The pigs were earlier used in an experimental animal model to study the early stages of acute quadriplegic myopathy (AQM), a disease that is found in mechanically ventilated intensive care unit patients. Percutaneous biopsies were taken from these pigs and where now used in this study. Even though the method used was accurately tested and theoretically working, certain problems arose. These problems were unexpected and caused problems to the study. The method used to measure the concentration of myosin and actin, an ELISA, gave no logical results. The reason could not be found and because of the time limit of this project no results from the AQM-pigs were gained. The efforts to make the method work is described and discussed.

Cattle are vitally important to American agriculture industry, generating over 24.6 billion pounds of beef (by carcass weight), and 79.5 billion dollars in 2005, and over 27 billion dollars in milk sales in 2004. As of July 2006, the U.S. beef and dairy industry is comprised of 104.5 million head of cattle, 32.4 million of which were processed in 2005. The health of the animals has always been an important concern for breeders, as healthy animals grow faster and are more likely to reach market weight. Animals that exhibit natural resistance to disease do not require chemicals to stimulate normal weight gain, and are less prone to disease related wasting. The major histocompatibility complex (MHC) is a collection of genes, many of which function in antigen processing and presentation. The bovine MHC (BoLA) differs from typical mammalian MHCs in that the class II region was disrupted by a chromosomal inversion into two subregions, designated BoLA IIa and BoLA IIb. BoLA IIb was transposed to a position near the centromere on bovine chromosome 23,while BoLA IIa retains its position in BoLA. Comparative sequence analysis of BoLA IIb with the human MHC revealed the location of the region containing the proximal inversion breakpoint. Gene content, order and orientation of BoLA IIb are consistent with the single inversion hypothesis when compared to the corresponding region of the human class II MHC (HLA class II). BoLA IIb spans approximately 450 kb. The genomic sequence of BoLA IIb was used to detect sequence variation through comparison to other bovine sequences, including data from the bovine genome project, and two regions in the BAC scaffold used to develop the BoLA IIb sequence. Analysis of the bovine genome project sequence revealed a total of 10,408 mismatching bases, 30 out of 231 polymorphic microsatellites, and 15 sequences corresponding to the validated SNP panel generated by the bovine genome sequencing project. The two overlapping regions in the BoLA IIb BAC scaffold were found to have 888 polymorphisms, including a total of 6 out of 42 polymorphic microsatellites indicating that each BAC derived from a different chromosome.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009.

Dendritic cells (DCs) are a migratory group of bone-marrow-derived leukocytes specialized for uptake, transport, processing and presentation of antigens to T cells. Exposure of DCs to bacterial pathogens can induce DC maturation characterized by cytokine production, up-regulation of co-stimulatory molecules and an increased ability to activate T cells. DCs have the ability to restrict growth of L. pneumophila (Lp), an intracellular Gram-negative bacillus that causes a severe form of pneumonia known as Legionnaires' disease, in murine ER-derived organelles (121) but replicate in human DCs (145). Even in human cells, however, lysis of the DCs does not occur for at least 24 hours which may allow DCs time to participate in the transition from innate to adaptive immunity (145). The primary polyphenol in green tea extract is the catechin (-)-epigallocatechin-3-gallate (EGCG) which accounts for most of the numerous reported biological effects of green tea catechins, including anti-bacterial, anti-tumor, and neuroprotective effects. Primary murine bone marrow derived DCs from BALB/c mice were treated in vitro with Lp, or stimulated for comparison with Escherichia coli lipopolysaccharide (LPS). CD11c, considered an important marker of mouse DCs, and surface expression of co-stimulatory molecules CD40, CD80, CD86, as well as class I/ II MHC molecules was determined by flow cytometry. Treatment of the cells with EGCG inhibited the microbial antigen induced up-regulation of CD11c, CD40, CD80, CD86 and MHC I/ II molecules. EGCG also inhibited, in a dose dependent manner, induced production of the Th1 helper cell activating cytokine, IL-12, and the chemokines RANTES, MIP1a, and MCP-1. However, EGCG upregulated TNFa production. In addition, EGCG inhibited both Lp and LPS induced expression of both TLR2 and TLR4 as well as LPS-induced NF-kB activation; all of which are important mediators of DC maturation. The modulation of phenotype and function of DCs by EGCG has implications for host interaction with microbial pathogens like Lp, which involve TLR interaction.

This thesis investigates the impact of avian malaria (Haemosporidia) parasites on the outcome of interspecific competition between two closely related bird species, pied (Ficedula hypoleuca) and collared (F. albicollis) flycatchers. I further investigated how variation in timing of breeding, life history strategies and immune competence genes (MHC genes) modulate the fitness effects of malaria parasites in one of the two species i.e. collared flycatchers. Collared flycatchers colonized the Baltic island Öland in the late 1950-ties and has since then been expanding their breeding range while competitively excluding pied flycatchers from the favourable habitats (deciduous forests). I investigated the underlying mechanisms behind this exclusion by combining detailed long-term breeding data with modern molecular genetic techniques identifying both the presence/absence and lineage specificity of haemosporidian blood parasites. I found that the rapid decline of pied flycatchers can be explained by the combined effects of competition over nestling sites, hybridization and haemosporidian infections. Haemosporidian infections have a negative impact on survival of pied flycatcher females but no detectable effect on collared flycatchers’ longevity or reproductive success. This may be due to the fact that collared flycatchers carry (and are potentially exposed to) a higher diversity of parasites than pied flycatchers, which in turn may select for a higher diversity of MHC genes and hence a better overall protection from the negative impact of parasites. Indeed, functional MHC diversity correlates negatively with malaria prevalence among collared flycatchers from Gotland. Moreover, I found that both, malaria infection intensity and immunoglobulin level influences how infected collared flycatchers respond to increased nestling food-demands. The latter results mean that there is variation in allocation strategies (i.e. in resource allocation between reproductive effort and immune competence) within the collared flycatcher population. Hence, this population has the ability to respond to novel selection pressures in terms of optimal allocation of resources into immune functions. In summary, my results show that local parasites may facilitate the expansion of a new colonizer. This is important in the context of global climate change that will probably increase the colonization rate of southern species and lead to novel host-parasite interactions.

A major objective in Theileria parva subunit vaccine development is to induce a vaccine antigen specific response mediated by cytotoxic CD8+ T cells (CTL). Therefore it is essential to be able to measure the frequency of the responding CD8+ T cells after vaccination and correlate it with a clinical outcome on challenge. Recently concluded immunogenicity and efficacy studies of T. parva specific CTL antigens showed successful induction of CTL responses in some animals, which correlated with reduced disease severity after challenge. To provide correlates of immunity antigen-specific CD8+ T cell mediated IFN-γ responses and CTL lytic responses were measured over the course of the experiments. Several challenges presented in these trials aimed at optimising vaccine efficacy. While the IFN-γ ELISPOT is a sensitive and reliable assay widely used in vaccine research, the use of chromium/indium release assay remains to be the only assay in use that measures T. parva-specific CTL activity. Hence the overall goal of the study was to develop novel reagents and novel assays to identify parasite-specific CD8+ T lymphocytes with lytic potential. To address this objective, bovine perforin, granzymes A and B, as specific effector proteins expressed in activated CTL were cloned and expressed using a baculovirus expression system. Sequence analysis of the cloned cDNAs showed the isolated cDNA belonged to the perforin and granzyme sub-families respectively. Perforin cDNA demonstrated 85% homology to human perforin with presence of conserved regions resembling calcium binding motif, membrane attack complex component as well complement protein. The sequences encoded by the cloned granzyme A and B cDNAs have the features of a trypsin like serine protease and demonstrates over 70% homology to the human cDNA over the active enzyme region as well catalytic residues characteristic of serine proteases. The expressed polypeptides of all three proteins were used to produce specific antibodies for use as reagents in immunoassays including ELISpot and intracellular staining for flow cytometric analysis. While the antibodies showed reactivity to the recombinant proteins, these reagents displayed different functionality in the recognition of the native protein. Peptide-major histocompatibility complexes (MHC) class I tetrameric complexes (tetramers) are proving invaluable as fluorescent reagents for enumeration, characterisation and isolation of peptide-specific CD8+ T cells and have afforded advantages to phenotype antigen-specific T cells with minimal in vitro manipulation. Fluorescent bovine tetramers were shown to specifically stain antigen-specific CTL by directly binding the T cell receptor (TCR). Analyses of CD8 T-cell responses in live-vaccine immunised cattle also showed that this method is robust and demonstrates changes in the kinetics and specificity of the CD8+ T cell response in primary and secondary infections with T. parva. On average, results of functional assays and tetramer staining followed parallel trends, measured roughly the same populations and allowed for surface and intracellular staining for CD8 T cell marker and perforin, respectively, demonstrating a method that reliably quantifies the frequency, phenotype and function of specific CD8+ T cells. The technical simplicity, rapidity and ability of the flow cytometric technique described in this thesis to measure low frequency antigen-specific responses suggests that tetramer staining, combined with functional assays could be broadly applicable to the valuation of vaccination efficacy to determine which protocols are most successful in inducing CTL responses.

The major histocompatibility complex region (MHC) is a multi gene family present in all jawed vertebrates, with a fundamental role in vertebrate immunity. More than two decades of studies have resulted in the characterization of over a dozen MHC regions, and models of evolution explaining that the MHC has gradually increased in size and gene content since its origins without addressing their genomic context or the environmental selective forces. Furthermore, a compelling reconstruction of the evolutionary history of the MHC has been hampered due to phylogenetic gaps and the absence of comparative phylogenetic methods applied to comparative genomics. Here I reconstruct 320 MY of MHC evolution using 42 amniote genomes using improved gene annotations, genomic alignments and phylogenetic algorithms to reconstruct the evolution of the MHC at three levels of phylogenetic resolution. The first one describes 25 MY of evolution of the primate MHC using eight Human and four non-Human primate MHC haplotypes. Results suggests that highly dense gene segments have a strikingly conserved gene organization, and six conserved and highly rearranging segments overlap genes that are most commonly associated to disease. Phylogenomic analysis implies that the MHC has remained stable in gene content and size, with significantly increased duplication rates in the primate ancestors. The second one describes 280 MY of MHC evolution through the first characterization of reptilian MHC region, which combines mammalian, reptilian, Bird and amphibian characteristics, which favors the hypothesis of the existence of a primordial MHC in which natural killer receptors, CD1 and lectin genes co-exist. The Anolis MHC expands our understanding of the origins of the exceptionally small Bird MHC regions and provides further information about the organization and size of the ancestral amniote MHC. The third one compares 42 amniote MHC regions and map gene duplications and losses to further evaluate the mode and tempo of the evolution of the region. Comparative phylogenetic methods imply that the genomic and environmental factors affect the diversification of MHC during 320 My of evolution.

Natural killer (NK) cells can kill virus-infected cells without prior antigenic exposure, and are therefore important for controlling viral replication prior to the onset of adaptive immune responses. Primate NK cells express activating and inhibitory killer-cell immunoglobulin-like receptors (KIRs) that bind to specific major histocompatibility complex (MHC) class I molecules. The importance of KIR interactions with MHC class I in human immunodeficiency virus (HIV) pathogenesis is demonstrated by the association of select KIR and MHC class I genotypes with delayed progression to acquired immunodeficiency syndrome (AIDS).

No description available.