The mechanistic factors responsible for the increase in allergic diseases are still not fully understood, but a reduced microbial stimulation seems to be one of the key issues. Research is now aiming at investigating the relationship between the innate immune system, involving the toll-like receptors, and allergy development. Further, the maternal influence on the child, possibly through in utero effects, but also through the breast milk, has shown to be of great importance. This thesis aimed at understanding how the maternal immune system is influenced by early exposures and allergic disease, but also to investigate the consequences of the maternal phenotype on the innate immune system of the developing child. The Th1/Th2 cytokine pattern in allergic diseases has been extensively studied. Here we were interested in comparing the innate cytokines in allergic and non-allergic women, and to see if the allergic status was influencing the effect of pregnancy differently. We demonstrate that IL-1β, IL-6, IL-10 and IL-12 production in cells from adult women are not influenced by allergic status, neither during pregnancy nor 2 years after. However, pregnancy had an apparent effect on cytokine levels, regardless of allergic status. Also, total IgE levels in allergic women were significantly lower 2 years after pregnancy in comparison with the levels during pregnancy, pointing to the fact that pregnancy indeed has an immunomodulatory role. We further wanted to investigate the immune system of mothers who had migrated to Sweden in comparison with indigenous mothers. The reason for our interest here was that children born from immigrated mothers have shown to have an increased risk of developing diseases such as allergy and Crohn’s disease. The results showed that immigrants from a developing country had significantly higher levels of breast milk IL-6, IL-8 and TGF-β1. Further, regardless of maternal country of birth, a larger number of previous pregnancies was associated with down-regulation of several substances, statistically significant for soluble CD14 and IL-8. The results suggest that maternal country of birth may indeed influence adult immune characteristics, potentially relevant to disease risk in offspring. The influence of allergic status of the mother on the expression of CD14, TLR2 and TLR4 was further investigated in monocytes from mothers and their newborn babies upon microbial stimulation. We could not find any differences in monocytic TLR levels between the groups. No significant differences regarding cytokine levels between allergic and non-allergic mothers in response to stimuli were found either. However, the cytokine and chemokine release triggered by TLR2 stimulation in CB revealed that CBMC from children with maternal allergic disease released significantly less IL-6, and a trend towards less IL-8. As we could not find differences in TLR levels attributed to maternal allergy, but an impaired IL-6 response, we turned our focus on an intracellular event taking place after TLR ligation. The results confirmed our results of decreased IL-6 levels in CB from children to allergic mothers. At 2 years of age, the children of allergic mothers still displayed a diminished IL-6 response. Additionally, they also had a decreased activity of p38 MAPK. p38 has an important role in driving Th1 responses, suggesting that the p38 pathway could be one of the responsible mechanisms behind the impaired responses correlated to allergic heredity found in CB as well as at 2 years of age. Infancy is a crucial time period for the developing immune system. Further, the relative composition of the two major monocytic subsets CD14++CD16- and CD14+CD16+ is altered in some allergic diseases. TLR levels are different in the two subsets, proposing a possible link to the reduced responding capacity of monocytes from children with allergic heredity. We followed up our earlier studies of children at birth and at 2 years of age by looking at 5 year old children. There were no differences regarding monocytic subsets, nor in TLR levels in unstimulated cells. However, when stimulating the cells with PGN, both monocytic subsets in allergic subjects were less capable of upregulating TLR2 compared to the age-matched controls. Taken together, the work in this thesis suggests that the maternal immune system is affected by the process of pregnancy and childhood exposures. It further suggests that maternal allergy affects the young child, in terms of impaired responses to microbial stimuli, which later in infancy correlates with allergic disease in the child. These impaired innate responses could lead to a diminished Th1 response, or alternatively to a deficiency in regulatory mechanisms, and thereby cause allergic disease.

Type 1 diabetes (T1D) is a chronic, autoimmune disease caused by a T cell mediated attack on the insulin producing pancreatic ß-cells. Even though reasonable quality of life can be acquired with modern insulin therapy, prevention of acute and late serious complications is facilitated by preservation of residual insulin secretion. Preventing β-cell destruction is therefore an important goal of T1D therapy. Characterisation of immunological changes in the course of T1D is essential for understanding the underlying pathogenic mechanisms and for evaluating the efficacy of therapeutic intervention.   This thesis aimed to study the immune profile in individuals at increased risk of T1D and in patients diagnosed with the disease. In addition, the immunological effects of treatment with the B vitamin, Nicotinamide, and by antigen-specific immunotherapy using GAD65, have been studied in high-risk individuals and in T1D patients, respectively. We have found that individuals at high risk of T1D had an increased T helper (Th) 1 like immune profile, defined by high secretion of interferon (IFN) -γ. At the time of clinical onset of T1D, the Th1 dominance was diminished. We further demonstrate that children with newly diagnosed T1D had a suppressed Th1 like profile, detected by chemokine and chemokine receptor profile. This was accompanied by an induced population of CCR7+ and CD45RA+ naïve, CD8+cytotoxic T (Tc) cells and a reduced CD45RO+ memory Tc cell pool.   It has previously been shown that oral Nicotinamide had no clinical effect in prevention of T1D. However, we found that the treatment was associated with a decreased secretion of IFN-γ. We have previously shown that subcutaneous injections with GAD-alum in T1D children induced a better preservation of endogenous insulin secretion compared with placebo. Here, we demonstrate that the treatment induced an early antigen-specific Th2 and regulatory immune profile. After a few months, and still after more than two years, the recall response to GAD65 was characterised by a broader range of cytokines. GAD-alum treatment also induced a GAD65-specific CD4+CD25highFOXP3+ cell population and reduced the levels of CD4+CD25+ cells.   In conclusion, a Th1 like immune profile in pre-diabetic individuals indicates an imbalance of the immune system. At time of clinical onset, and in the period afterwards, reduction of the Th1 associated immune response could be an effect of a suppressed destructive process, selective recruitment of effector T cells to the pancreas or a defective immune regulation. The protective effect of GAD-alum in T1D children seems to be mediated by an early skewing of GAD65-induced responses towards a Th2 phenotype. Further, induction of GAD65-specific T cells with regulatory characteristics might be able to suppress autoreactive responses and inflammation in the pancreas.

More than a century after the identification of the tubercle bacillus and the first attempts at vaccination, tuberculosis (TB) still remains one of the world’s most serious infectious diseases. TB, caused by the bacterium Mycobacterium tuberculosis, is typically a disease of the lung, which serves both as port of entry and as the major site of disease manifestation. The currently used vaccine, BCG, is administered parenterally and induces a systemic immune response. However, it fails to protect against pulmonary TB, thereby raising the question whether vaccination targeting the mucosal immunity in the lungs could be favourable. The respiratory mucosal surfaces represent the first line of defence against a multitude of pathogens. Secretory IgA, in mucosal secretions has an important function by blocking entrance of pathogenic organisms and preventing infections. Additionally, a role for IgA in modulation of immune responses is currently being revealed. In this work, we investigated the relevance of mucosal IgA in protection against mycobacterial infections using mice deficient for IgA and the polymeric Ig receptor, the receptor responsible for mucosal secretions of IgA. Gene-targeted mice were more susceptible to mycobacterial infections in the respiratory tract and displayed reduced production of proinflammatory, and protective, factors such as IFN-γ and TNF-α in the lungs. The mechanisms explaining the defective proinflammatory responses in the lungs of deficient mice might involve impaired signalling through Fcα receptors, or homologous receptors, which could lead to inadequate activation of pulmonary macrophages. This could subsequently result in suboptimal induction and production of cytokines and chemokines important for attraction and migration of immune cells to the site of infection. Induction of optimal adaptive immune responses to combat mycobacterial infections requires prompt innate immune activation. Toll-like receptors (TLRs) are vital components of the innate branch of the immune system, ensuring early recognition of invading pathogens. Using TLR-deficient mice we demonstrated an important role for TLR2, and partly TLR4, in protection against mycobacterial infection in the respiratory tract. TLR2-deficient mice failed to induce proper proinflammatory responses at the site of infection, and macrophages derived from the knockout mice displayed impaired anti-mycobacterial activity. Experimental evidence has concluded that the immune response upon an infection can influence the outcome of succeeding infections with other pathogens. Concurrent infections might additionally interfere with responses to vaccinations and have deleterious effects. We developed an in vitro model to study the effect of a malaria infection on a successive M. tuberculosis infection. Our results demonstrate that a malaria blood-stage infection enhances the innate immune response to a subsequent M. tuberculosis infection with a Th1 prone profile. Reduced infectivity of malaria-exposed dendritic cells implies that a malaria infection could impose relative resistance to ensuing M. tuberculosis infection. However, a prolonged Th1 response may interfere with malaria parasite control. The outcome of this work emphasizes the importance of generating effective immune responses in the local mucosal environment upon respiratory mycobacterial infections. It furthermore puts new light on the immunological interaction between parasites and mycobacteria, which could have implications for future vaccine research.

Host defense against Mycobacterium tuberculosis (Mtb) is mediated by a combination of innate and adaptive immunity. In this thesis we investigated the role of components of innate system such as TLR2 signalling and alveolar epithelial cells type II (AEC II) in the immune responses against mycobacterial infections. Since TLR2 has been shown to be important in the defense against mycobacterial infections; in paper I we investigated the role of TLR2 to generate acquired immune responses. We compared both humoral and cellular immune responses in TLR2-/- and WT (wild type) mice immunized with the mycobacterial antigens 19kDa (TLR2 ligand) or Ag85A (non-TLR2 ligand). We did not find any differences in the humoral responses in both mouse strains. However, we found some deficiencies in the T cell memory compartment of TLR2-/- mice immunized with 19kDa. In addition, the antigen presenting cells (APC) compartment in TLR2-/- mice, for instance bone marrow derived macrophages (BMM) and pulmonary macrophages (PM) in this study, has also shown deficiencies. This effect was more evident when PM were used as APC. We next evaluated the responses in both BMM and PM upon stimulation with anti-CD40 and TLR ligands where PM were the low responders to TLR2 ligand and to anti-CD40 both in the production of different cytokines and in the up-regulation of the co-stimulatory molecules. Together, our results have demonstrated the importance of TLR2 in the generation of specific immune responses. In paper II, we investigated the role of AEC II in the defense against mycobacterial infections. AEC II have been suggested to play an important role in the local immune responses to inhaled pathogens. First, we compared murine AEC II with PM in their ability to take up and control mycobacterial growth and their capacity as APCs. AEC II were able to internalize and control bacterial growth as well as presenting antigen to memory T cells. In addition, both cells types were compared in their capacity to produce cytokines, chemokines and other factors where AEC II exhibited a different pattern of secretion than PM. Also, a more complete profile of AEC II responses reveled that AEC II were able to secrete different factors important to generated various effects in others cells. The major finding in this study was that upon TNF, AEC II produced MCP-1 a chemokine involved in the recruitment monocytes/macrophages to the sites of infection. Since TNF is predominantely produced by macrophages, we speculate that both cell types may communicate and influence each other. In conclusion, our results provide more evidence of the important role of AEC II in the immune responses in the respiratory tract.

No description available.

No description available.

Human papilloma virus (HPV) is a small DNA virus that is known as the causative agent in cervical cancer. The virus is found worldwide and causes more than 300 000 deaths yearly. Although there are many types of HPV, HPV16 is found in 99 % of cervical cancer cases. There are several vaccines available but as of now there is no antiviral treatment for the disease. Thus, it is further studies are required to find potential targets or biomarker which can be used to develop new treatments. The viral genome is divided into early and late genes. Where the early genes are active during the early stage of infection and the late genes are upregulated at the late stage of infection. The early (E) genes E6 and E7 are oncogenic. E6 prevents the cells from going into apoptosis and E7 drives proliferation. The E1 and E2 genes are involved in the replication of the viral genome. The HPV16 genome is polycistronic, meaning that the mRNAs that the virus produces encodes for several proteins. Gene regulation is thus very important for the virus. The HPV only have two promoters which means that it has to utilize other methods to control its gene expression. It uses alternative splicing to control the expression of its genes. Using different splice sites the virus can produces many different pre-mRNAs. The splice sites are in turn controlled by regulatory elements called enhancers and silencers. These elements acts as binding places for cis-acting elements (RNA) and trans-acting factors (protein).   This report focuses on the regulation of the E1 gene. It investigates whether the trans-acting factor TRAP150 affects the regulation of the E1 gene. Using reporter plasmids to with a reporter Luciferase gene the expression of the gene can be studied.   The results indicate that TRAP150 does induce splicing of E1, although it is not clear how it does this. It also seems that the splicing variant E1C upregulated by the presence of TRAP150. Using deletion mutants to study the TRAP150 interaction, it was also found that the N-terminal is essential for splicing to happen. This also concurs with previously reported results.

Hantaviruses are of major global health concern, causing over 150.000 infections annually.They cause two distinct acute inflammatory diseases, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Impaired endothelial barrier function is an important hallmark of both diseases, leading to vascular leakage and formation of oedema in major organs. What causes vascular leakage and determines the disease outcome remains unknown.Recent studies have identified IL-6 as a marker for disease severity and suggested an impact of IL-6 in hantavirus pathogenesis. IL-6 signaling occurs via the membrane-bound IL-6 receptor (IL-6R), known as classical signaling, or via the soluble IL-6R, known as transsignaling. As IL-6 trans-signaling has been shown to mediate mainly proinflammatory effects,we sought to investigate the role of IL-6 trans-signaling on vascular endothelial cells in hantavirus infection. In the present study, we examined the plasma concentration of sIL-6R in acute and convalescent Puumala virus (PUUV) infection. Further we examined IL-6 and CCL2 secretion, ICAM-1 and VE-cadherin expression and the barrier function upon IL-6 trans-signaling in PUUV infected endothelial cells. For the first time we showed increased levels of sIL-6R in acute HFRS and proved endothelial cell activation and barrier dysfunction upon PUUV infection and IL-6 trans-signaling. IL-6 and CCL2 secretion as well as ICAM-1 expression were increased, and VE-cadherin structures and barrier functions were disrupted.Our findings suggest an impact of IL-6 trans-signaling on vascular leakage and reveal new possible treatment targets for hantavirus infections.

Introduction: IL-35 is an anti-inflammatory cytokine that is secreted by regulatory B (Breg) and T (Treg) cells, as well as tolerogenic dendritic cells (tolDCs). Previously our group has shown that systemic administration of IL-35 can reverse established type 1 diabetes (T1D) in mouse models, namely in non-obese diabetic (NOD) and multiple low-dose streptozotocin (MLDSTZ) induced diabetes mice. Furthermore, the circulating levels of IL-35 are lowered in patients with T1D compared to healthy individuals. Whether the decreased levels of IL-35 in both patients and diabetic mice are due to an altered expression of IL-35 receptors (IL35R) in T1D is still unclear. Herein, we aimed to study the expression of IL-35R on Breg, Treg cells and tolDC in pre-clinical T1D and characterize these receptors on tolDC. Methods: NOD and MLDSTZ mice were used for determining the expression of IL- 35R. Thymic glands, spleens and pancreatic draining lymph nodes were harvested from mice for immune cell isolation. This was followed by fluorescent flow cytometry analysis of IL- 35R+ cells. Results: The analysis revealed that the proportion of IL-35R+ Breg and Treg cells do not differ between 12-week-old female and male NOD mice. There was an increasing trend for the proportion of IL-35R+ Treg cells in the spleens of STZ-treated mice while the proportion of splenic IL-35R+ Breg cells were decreased in MLDSTZ mice compared with controls. We also observed a reduced expression of the IL-12Rβ2 subunit in the splenic Breg cells of STZtreated mice. Conclusion: Our data illustrate that in the disease condition the expression of IL-35R may be altered. The expression of the receptor needs to be further investigated in clinical samples in order to determine whether similar trends can be observed in humans.

The use of mice as a model organism in biomedical research is widespread due to their similarities with humans in anatomy and physiology. However, the genetic isolation of inbred strains from natural populations raises concerns about the reliability of these models. Studies have shown differences in immune responses between wild and laboratory mice, and the use of exploratory tools for large datasets can help to analyze and interpret these differences. The aim statistical analysis of dataset can provide insights into the differences between variables and individuals and guide further investigations. The results showed that LDA provided clear separation between the different groups, and successfully differentiated between stimulation types and mouse strains, with distinct clustering of data points. The KNN algorithm performed best for smaller values of K. However, the selected gender characteristic did not possess strong discriminatory power in separation and further investigation into alternative features or methodologies may be necessary. In conclusion, the aim of providing comparative immunological analysis of wild and laboratory mice types is achieved. This study underscores the importance of careful statistical analysis, acknowledges the limitations of imputation methods, and highlights the potential of LDA and KNN algorithms in analyzing immune response data. As well, highlighting the need for improved models that are able to capture the complexities of immune responses and their relevance to human immunology.