<p>Malaria remains a challenging health problem in malaria endemic regions. Infection with malaria invariably leads to anaemia. The groups at risk of developing malarial anaemia include children below the age of five years and pregnant women, especially primigravidae. Several factors have been suggested to be responsible for its aetiology, including increased destruction of infected and normal red blood cells together with bone marrow suppression. However, until recently, the molecular mechanisms involved have remained elusive. The aim of the work presented herein was to investigate the mechanisms responsible for the destruction of normal red blood cells in anaemia, and more specifically to define the role of the ring surface protein (RSP/RAP) -2 and other members of the low molecular weight rhoptry associated protein (RAP) complex, RAP-1 and -3.</p><p>In the first study we showed that antibodies to the RAP complex could mediate the destruction of RSP-2 tagged erythroid cells by phagocytosis or by complement activation and then lysis. In addition, antibodies to RAP-1 and RAP-2 could induce the death of RSP-2/RAP-2 tagged erythroblasts. We further investigated the frequency and functionality of naturally occurring RSP-2/RAP-2 antibodies in the sera of anaemic and non-anaemic Cameroonian children. We found that all sera investigated contained RSP-2/RAP-2 reactive antibodies by both immunoflorescence and flow cytometry. The anaemic group of children had significantly higher levels of antibodies of the IgG isotype than the non-anaemic individuals, while the levels of IgM were similar in both groups. With respect to IgG subclasses, low levels of IgG1 and -3 antibodies were detected. Higher levels of IgG3 were seen in the non-anaemic individuals as compared to anaemic subjects. With regards to antibody functionality, the non-anaemic individuals recognised a greater proportion of RSP-2/RAP-2 tagged erythrocytes and activated complement to a greater extent than the anaemic individuals.</p><p>From our findings, we can conclude that antibodies to the RAP complex are potentially involved in erythroid cell destruction during malaria which may result in anaemia, and that high levels of such antibodies may be detrimental to the host.</p>

No description available.

No description available.

Today, atopic allergy is the most common chronic disease among children in the developed world. The increase in allergy prevalence during the past decades in these countries might be associated with lower microbial exposure. The gut flora, consisting of approximately 800 different species of bacteria, has been postulated to be important for the development of a fully functional immune system. Essentially, these bacteria are in constant contact with the gut flora associated lymphoid tissue, the largest lymphoid tissue of the human body. Following birth, the sterile gut of the newborn is immediately colonised by various bacterial species. Actually, alterations in the infant gut flora have been associated with allergy development. Human milk is the major food in infancy and could thus influence the composition of the infant gut flora. Immunomodulatory components in human milk might differ between mothers and could therefore explain the contradictory results seen regarding breastfeeding and allergy development. Oligosaccharides, the third most abundant solid component in human milk, survive the passage through the stomach and are utilised by the gut microbiota. We analysed nine abundant neutral oligosaccharides in colostrum samples from allergic and non-allergic women and related to subsequent allergy development in their children. We found a considerable variation in the concentration of neutral oligosaccharides in colostrum, which was not to be explained by the allergic status of the women. Neither was the consumption of neutral colostrum oligosaccharides related to the allergy development in children. Relevant bacterial species in early faecal samples were analysed, with Real-time PCR, and related to allergy development in children followed up to five years of age. Infants who harboured Lactobacilli (L.) group I (L. rhamnosus, L. paracasei, L. casei) at 1 week of age and Bifidobacterium adolescentis at 1 month of age developed allergic disease less frequently during their first five years than infants who did not harbour these bacteria at the same time (p=0.004 and p=0.008 respectively). In conclusion, the work presented in this thesis implies the importance of a diverse gut flora early in life for the development of a fully functional immune system. However, consumption of colostrum with high amounts of neutral oligosaccharides does not protect against early allergy development.

Tuberculosis (TB) remains one of the world’s most serious infectious diseases. It is estimated that a third of the world’s population is latently infected and 8 million new cases are recorded each year. Although BCG vaccination triggers protective immune responses in the neonates, it confers protection against only certain forms of childhood TB. Protection mediated by BCG, against pulmonary TB, is controversial as reported with variable efficacy ranging from 0-80%. In addition to the problems associated with the BCG vaccine, diagnosis of TB cannot be performed readily with the available tools. At present, an effective control of TB is highly dependent on the development of a new TB-vaccine as well as proper identification and treatment of individuals with active disease. Therefore, we particularly focused on identification of biomarker (s) of infection and the development of better vaccines, with special emphasis on the immune responses in the respiratory tract. In the first study, we aimed to identify immune biomarker (s) of infection for better diagnosis of TB. Mice were infected with BCG administered i.n. or i.v., and the bacterial burden in the lungs, spleen and liver was examined. We measured IL-12, IFN-γ, TNF, soluble TNF receptors (sTNFR) and mycobacteria-specific antibodies in the broncho-alveolar lavage (BAL) and in serum in order to find immune correlates of infection. Results showed that sTNFR and mycobacteria-specific antibodies in BAL, but not in serum, might be useful in distinguishing active from latent infection or exposure to mycobacterial antigens. In the second study, we investigated whether we could improve the currently used BCG vaccine. For this purpose, we tested a combination of neonatal vaccination protocol using BCG and posterior boosting with the protein heparin-binding hemagglutinin adhesion (HBHA). It has been described that immunization with native (n) HBHA but not recombinant (r) HBHA conferred protection against M. tuberculosis challenge in mice. This protection was comparable to that afforded by the BCG vaccine. In order to improve the protective efficacy of the nHBHA vaccine we followed heterologous prime-boost strategy, comprising BCG vaccination at the neonatal age, followed by nHBHA boosting at the infant and adult ages. We also examined whether the rHBHA protein could boost BCG-mediated protective immunity. Cellular immune responses and protection as measured by control of bacterial growth in the lungs of the treated animals were followed. Our results showed an improved effect of BCG-priming on HBHA-immunization. The BCG/HBHA immunization protocol was more effective in induction of HBHA-specific immune responses, as well as in protection than when the animals received only BCG or HBHA alone. Importantly, our study revealed that nHBHA does not require co-administration with adjuvant provided that mice were primed with live BCG before boosting. Finally, we hypothesized that in utero sensitization of the fetal immune system with nHBHA may improve nHBHA-specific immune responses after birth. The pregnant mother was immunized with nHBHA 1 week before delivery. After birth, the offspring received two doses (week 1 and week 4) of nHBHA formulated with cholera toxin. We examined HBHA-specific recall responses and protection after challenge with a high dose of BCG. We found that immune responses were improved by priming the pregnant mother, and that this also provided better protection than when the offspring received only BCG or HBHA neonatal vaccinations.

Till date, there are no effective control strategies against the deadly disease of malaria, and millions of children across Africa, Oceania, Asia, and Latin America are at the mercy of this long term enemy of man every second that passes by. Other control measures combined with vaccination might help improve control strategy against malaria, but the development of vaccines face various challenges as well, due to the complexity of the parasites’ life cycle and other host factors. The asexual blood stage antigen Pf332 of Plasmodium falciparum, is expressed during the trophozoite stage, and transported from the parasitophorous membrane to the outer erythrocyte membrane during schizogony. Previous studies have suggested this antigen as a potential vaccine candidate, because Pf332-reactive human monoclonal antibody (mAb 33G2) inhibits parasite growth and cytoadherence in vitro. Elucidating and understanding the immunological capabilities of antigen Pf332, as a vaccine candidate was the aim of the studies presented in this thesis. In our first study we identified and characterized the immunogenicity of a non-repeat fragment of antigen Pf332, termed Pf332-C231, a 231 amino acids long fragment corresponding to 13 percent of the total protein. Various analyses carried out with this fragment reveal that recombinant C231 was immunogenic in rabbits. In addition, anti- C231 antibodies have in vitro inhibitory capabilities. In immunoflourescence and immunoblot assays, rabbit anti-C231 antibodies were able to recognize the native protein. In the other study, we examined the distribution of antibodies regarding recombinant C231 and crude P. falciparum extract in a malaria endemic area of Senegal. IgG antibody reactivity with crude P. falciparum antigen was detected in the sera of all the  donors while many of the children lacked or had low levels of such antibodies against C231. The distribution of the anti-C231 antibodies in the different IgG subclasses differed from that shown by crude P. falciparum antigen. The crude P. falciparum antigen gives a higher IgG3 response than IgG2 for all age-groups, while C231 gave similar levels of IgG2 and IgG3. Correlation studies showed that the levels of anti-C231antibodies were associated with protection from clinical malaria, but this only reached significance with IgE. These findings further emphasize the inclusion of antigen Pf332 as a subunit vaccine candidate against P. falciparum malaria.

Malaria remains a challenging health problem in malaria endemic regions. Infection with malaria invariably leads to anaemia. The groups at risk of developing malarial anaemia include children below the age of five years and pregnant women, especially primigravidae. Several factors have been suggested to be responsible for its aetiology, including increased destruction of infected and normal red blood cells together with bone marrow suppression. However, until recently, the molecular mechanisms involved have remained elusive. The aim of the work presented herein was to investigate the mechanisms responsible for the destruction of normal red blood cells in anaemia, and more specifically to define the role of the ring surface protein (RSP/RAP) -2 and other members of the low molecular weight rhoptry associated protein (RAP) complex, RAP-1 and -3. In the first study we showed that antibodies to the RAP complex could mediate the destruction of RSP-2 tagged erythroid cells by phagocytosis or by complement activation and then lysis. In addition, antibodies to RAP-1 and RAP-2 could induce the death of RSP-2/RAP-2 tagged erythroblasts. We further investigated the frequency and functionality of naturally occurring RSP-2/RAP-2 antibodies in the sera of anaemic and non-anaemic Cameroonian children. We found that all sera investigated contained RSP-2/RAP-2 reactive antibodies by both immunoflorescence and flow cytometry. The anaemic group of children had significantly higher levels of antibodies of the IgG isotype than the non-anaemic individuals, while the levels of IgM were similar in both groups. With respect to IgG subclasses, low levels of IgG1 and -3 antibodies were detected. Higher levels of IgG3 were seen in the non-anaemic individuals as compared to anaemic subjects. With regards to antibody functionality, the non-anaemic individuals recognised a greater proportion of RSP-2/RAP-2 tagged erythrocytes and activated complement to a greater extent than the anaemic individuals. From our findings, we can conclude that antibodies to the RAP complex are potentially involved in erythroid cell destruction during malaria which may result in anaemia, and that high levels of such antibodies may be detrimental to the host.

Onchocerciasis, or river blindness, is a parasitic disease that affects more than 20 million people globally. The induction of pathology is directly related to the presence and destruction of the microfilarial stages (mf) of this filarial nematode. The disease presents clinically with a wide spectrum of dermal and ocular manifestations, the basis of the variation is believed to involve the immune system. The clinical presentations of infected hosts relate to the intensity of the reactions against the parasite. Anti-microfilarial drugs are also thought to somehow involve the immune system in their pharmacological action. In this study we have investigated some of the factors that might contribute to the pathogenesis, with the aim of gaining a better understanding of the role of immune response in these host inflammatory reactions to Onchocerca volvulus parasite. In the first study we have highlighted the clinically most severe form of dermal onchocerciasis, known as reactive onchocercal dermatitis (ROD), one that is often ignored and has not been properly identified. This form has special characteristics and important biological information that could greatly assist the general understanding of the disease as a whole. Amongst the three major foci of the disease in the study country, Sudan, the prevalence of ROD was found to be associated with different environmental and epidemiological characteristics; strikingly higher in the hypo-endemic areas. Including ROD cases in the prevalence will upgrade the level of endemicity of a locality, and often bring patients much in need of treatment into mass treatment programs that currently only treat localities with medium to high levels of endemicity. In the following research studies, we tried to address the immunological characteristics of the clinically different onchocerciasis patients. Then we also investigated the role of genetic polymorphism in the gene encoding receptor that links innate and adaptive immunity, namely, FcγRIIa. Patients with either of two major forms of the clinical spectrum-mild and severe dermatopathology were studied by assaying the antigen-driven proliferation of peripheral blood mononuclear cells and the ability of patients’ serum antibodies to promote cytoadherence activity to mf in vitro. Immune responses of those with severe skin disease were found to be stronger compared with the mild dermatopathology group. Mectizan® treatment was followed by an increase in immune responsiveness in those with initially poor responses. Thus the degree of dermatopathology is related to the host’s immune response against mf and immunocompetence may be necessary for Mectizan® to clear the infection efficiently. The infection has also been associated with increased levels of circulating immune complexes (CIC) containing parasite antigens and a cytokine response that involves both pro-and anti-inflammatory cytokines. Our fourth paper investigated the effect of IC from the O. volvulus infected patients on the production of pro-and anti-inflammatory cytokines. CIC were increased in all patients studied. The precipitate from plasma treated with polyethylene glycol (PEG) were added to peripheral blood mononuclear cell (PBMC) cultures, and the levels of IL-10, tumor necrosis factor TNF-α, IL-1β and their endogenous antagonists soluble TNF-Rp75 and IL-1-receptor antagonist (IL-1ra) were measured. A significant induction of all cytokines measured occurred in the onchocerciasis patients compared to healthy controls. However, the IL-1ra level was suppressed. The suppression of the production of IL-1ra suggests that the IC containing antigens may have a selectively suppressive effect on the production of this anti-inflammatory cytokine; thus implicating its possible role in counteracting inflammatory responses associated with the disease, and suggesting a potential therapeutic significance. FcgRIIa receptors are involved in many important biological responses, and considered as important mediators of inflammation. A polymorphism in the gene encoding this receptor, that is either arginine (R) or histidine (H) at position 131, affects the binding to the different IgG subclasses. We therefore hypothesized that this polymorphism might be one of the underlying mechanisms to the varied clinical presentations seen in this disease. FcgRIIa genotyping was carried out by gene specific polymerase chain reaction (PCR) and allele-specific restriction enzyme digestion of DNA from clinically characterized patients. The genotype R/R frequencies were found to be significantly higher among patients with the severe form of the disease (including ROD), and it was particularly associated with one tribe (Masaleet) compared to Fulani. Moreover, the H allele was found to be associated with lower risk of developing the severe form. As no significant difference was seen between onchocerciasis cases and controls, the study also implies that this polymorphism influences protection from developing the severe form rather than being related to protection from the infection.

During infancy, before adaptive immunity has matured, innate immunity is thought to be relatively more important. Human natural killer (NK) cells are innate immune cells involved in the control of virus-infected cells and can influence adaptive immunity mainly through cytokine production. This thesis aimed at investigating function and phenotype of NK cells in children from birth and during early childhood and to see if these features are altered in children that develop early allergy, in children latently infected by herpes viruses or born by preeclamptic mothers. Our results suggest that NK-cell populations are dynamic during the first years of life and start to resemble the phenotype of adults after five years of age. Early alterations in the NK-cell populations could lead to insufficient Th1 priming, with an increased risk to develop allergic disease. Early infection by common herpes viruses can influence NK-cell function and might be one important factor involved in early maturation processes of adaptive immunity. The altered NK-cell function and cytokine levels, noticed in CB from pathological pregnancies, suggest that NK cells could be influenced already in utero. These early alterations of innate immunity may affect the development of the child’s immune system, sometimes with beneficial outcome but could in some cases promote pathology.

Background: Type 1 diabetes (T1D) is a chronic, autoimmune disease caused by a T cell mediated destruction of ß-cells in pancreas. The development of T1D is determined by a combination of genetic susceptibility genes and environmental factors involved in the pathogenesis of T1D. This thesis aimed to investigate diverse environmental and immunological risk factors associated with the development of T1D. This was accomplished by comparing autoantibody development, T cell responses and the function of CD4+CD25+ regulatory T cells between healthy children, children at risk of T1D and T1D patients. Results: Induction of autoantibodies in as young children as one year old, was associated with previously identified environmental risk factors of T1D, such as maternal gastroenteritis during pregnancy and early introduction of cow’s milk. We did not see any general increase in the activity of peripheral blood TH subtypes in children with HLA class II risk haplotypes associated with T1D, nor were HLA class II risk haplotypes associated with any aberrant cytokine production in response to antigenic stimulation of peripheral blood mononuclear cells. However children with a HLA class II protective haplotype showed an increased production of IFN-γ in response to enteroviral stimulation. CTLA-4 polymorphisms connected with a risk of autoimmune disease were associated with enhanced production of IFN-γ. Healthy children with ß-cell autoantibodies had a lower expression level of GATA-3 compared to health children with HLA risk genotype or children without risk. Instead, children with manifest T1D showed lower expression levels of T-bet, IL-12Rß1 and IL-4Rα. Both T1D and healthy children showed the same expression of the regulatory markers Foxp3, CTLA-4 and ICOS in peripheral blood mononuclear cells, and the amount of CD4+CD25+ T cells did neither reveal any differences. The regulatory T cells seemed also to be functional in children with T1D, since increased proliferation after depletion of CD4+CD25high cells from PBMC was demonstrated in T1D as well as in healthy children.However, T1D children did have more intracellular CTLA-4 per CD4+CD25high T cell, increased levels of serum C-reactive protein and higher spontaneous expression of IFN-α in CD25depleted PBMC, all which are signs of activation of the immune system. This suggests a normal or enhanced functional activity of regulatory T cells in T1D at diagnosis. Conclusions: Our findings emphasize that environmental risk factors do have a role in the development of ß-cell autoimmunity. Our results do not support a systemic activation of the immune system in pre-diabetes or T1D, but instead a possible up-regulation of regulatory mechanisms seems to occur after diagnosis of T1D, which probably tries to dampen the autoimmune reaction taking place.