<p>The current research is directed towards dissection of host genetic factors involved in host immune response and the malaria disease outcome. A possible association between FcγRIIa polymorphism and anti-malarial antibody (A.M.A) responses were investigated in Sudanese patients in relation to clinical outcome of falciparum malaria. The frequency of the R/R131 genotype was significantly higher in patients with severe malaria as compared with mild malaria. A.M.A IgG3 was shown to be associated with reduced risk of clinical malaria in individuals carrying the H/H131 genotype. Low levels of IgG2 reactive with the Pf332-C231 antigen were associated with lower risk of severe malaria in individuals carrying the H131 allele. </p><p>Fulani and Masaleit, two sympatric ethnic groups in Sudan, are characterized by marked differences in susceptibility to falciparum malaria. We investigated whether the two populations differ in the frequency of GM/KM allotypes. The distribution of GM/KM phenotypes differed significantly among the two groups, with Gm 6 being significantly lower among the Fulani, and the combined frequency of Km 1,3 and Gm 1,17 5,6,13,14 phenotypes was found to be higher among Masaleit.</p><p> In interethnic study we investigated whether the two groups differ in the frequency of FcγRIIa and HbAS genotypes. The frequency of the H/H131, R/R13 and HbAS genotypes differed significantly among the two groups. Moreover, the Fulani showed higher levels of A.M.A IgG2 and lower IgG1 and IgG3 when compared to their sympatric non-Fulani neighbours.</p><p>A tri-allelic SNP (C/T/A) in the CRP gene was investigated for possible ethnic associations. The A allele, which is associated with higher basal CRP levels, was found to be less frequent in the Fulani compared with non-Fulani ethnic groups both in Sudan and Mali. </p><p>In conclusion, our results suggest possible associations between FcγRIIa, CRP genotypes, GM/KM allotypes, and anti-malarial antibody responses and the clinical outcome of falciparum malaria.</p>

<p>With the studies performed in this theses the local drug delivery technique FibMat developed by the biotech company AddBIO, was shown to be applicable to other plasma proteins and drugs than the fibrinogen-bisphosphonate combination that is today being commercialized. Hence the potential for a broader field of application was demonstrated. The application targeted today is as a surface modification giving improved strength to bone around screws used in bone implants. The effect of changing protein and manufacturing conditions was studied with null ellipsometry. It was demonstrated that with changes in incubation temperature, pH and salinity the fibrinogen could be successfully exchanged for the plasma proteins human serum albumin and immunoglobulin G. With liquid scintillation counting it was shown that the developed protein multilayers were able to absorb and release the bone strengthening drug alendronic acid in levels comparable to that of the fibrinogen based ditto. Disk susceptibility tests with the bacteria S. Aureus showed a potential for antibacterial functionalization with gentamicin. The release was, in the case of the fibrinogen multilayer, detectable up to 48 hours. Similar test revealed an inability of silver nanoparticle incorporated protein multilayers to achieve inhibitory levels.</p>

<p>In this thesis, two different vaccines were evaluated as adjuvant therapy for patients with colorectal cancer. The ability of the two candidate vaccines to generate antigen-specific cellular and humoral responses, respectively, was studied. The effectiveness of granulocyte colony stimulating factor (GM-CSF) as a cytokine adjuvant to augment the immune response was also examined.</p><p>The first vaccination strategy involved immunization with the recombinant tumor-associated protein, carcinoembryonic antigen (CEA). Recombinant CEA was administered at 4 different dose levels 7 times during one year. Peripheral blood samples were regularly analyzed during 36 months. This vaccination regimen induced a strong immunoglobulin 1 (IgG1) and IgG4 response, a moderate IgG2 response and a weak IgG3 response against CEA. GM-CSF markedly augmented the effect on IgG1 and IgG4 as well as the T cell response. In contrast, dose of rCEA had no or modest effect on induced immune responses. The response gradually increased during the 12 months immunization period. Responses of all three IgG subclasses and of T cells were protracted up to 36 months. The anti-CEA IgG titers related significantly to survival. Functional HLA-DR epitopes of CEA could be defined. These major histocompatibility class II epitopes may serve as putative components of a peptide-based vaccination strategy. </p><p>The other vaccine strategy consisted of the tumor-associated antigen epithelial cell adhesion molecule (Ep-Cam) expressed as a transgene in a viral vector, ALVAC. Patients were immunized subcutaneously/intradermally 3 times over 6 weeks and monitored for immune responses for 46 weeks. No anti-Ep-Cam specific humoral response was induced, but Ep-Cam specific type 1 T cells (interpheron-gamma production) were induced, mainly in the GM-CSF group. The cytotoxic cellular response appeared late, or a few months after the last immunization.</p><p>Both vaccines were well tolerated. Since GM-CSF was an important component for both regimens, immungenicity of this cytokine was assessed. Multiple immunizations with low dose GM-CSF were associated with a low incidence of GM-CSF antibodies that did not neutralize the biological effect of GM-CSF. </p><p>In conclusion, both vaccines are promising candidate vaccines. GM-CSF is necessary to induce a strong humoral and cellular immune response. Large clinical trials are urgently warranted to evaluate the clinical efficacy.</p>

<p>Mammalian pregnancy is an immunological paradox. The foetus, which expresses both paternal and maternal cell-surface molecules, has to be protected from rejection by the maternal immune system. At the same time, the mother has to have an efficient immune defence and must provide her offspring with antibodies.</p><p>The first part of this thesis investigates some of the mechanisms involved in the foetal avoidance of maternal rejection reactions. Placental absence of MHC class II expression, as well as a bias for Th2-cytokines at the maternal-foetal interface are suggested to be important for foetal survival. The results showed that placental MHC class II expression cannot be induced <i>in vivo</i>. Transfections of trophoblast cells with MHC class II genes, however, resulted in detectable MHC class II cell-surface expression, indicating that a post-transcriptional block does not exist in these cells.</p><p>By using IL-4- and IL-10-double deficient mice, it was shown that neither maternal nor foetal expression of these cytokines were crucial for completion of allogeneic pregnancy.</p><p>In the second part of the thesis, the effect of transmission of immunoglobulin G (IgG) from the mother to the offspring was studied. It was observed that viable maternal Ig-secreting cells occasionally infiltrated the B cell-deficient offspring and remained functional for long periods. In this study "green fluorescent mice" were used as a tool. Furthermore, neonatal ingestion of wild type milk increased the survival of adoptively transferred B-lineage cells in B cell-deficient mice, suggesting that suckling of IgG-containing milk could be used to facilitate B cell-reconstitution in B cell-deficient mice. Finally, results from studies on normal mice showed that absence of maternal IgG-transmission during their neonatal development resulted in elevated serum-IgG production, as well as enhanced immune reactions upon immunisations in adult life. This showed that maternal IgG can have long-term immunoregulatory effects in the offspring.</p>

Mammalian pregnancy is an immunological paradox. The foetus, which expresses both paternal and maternal cell-surface molecules, has to be protected from rejection by the maternal immune system. At the same time, the mother has to have an efficient immune defence and must provide her offspring with antibodies. The first part of this thesis investigates some of the mechanisms involved in the foetal avoidance of maternal rejection reactions. Placental absence of MHC class II expression, as well as a bias for Th2-cytokines at the maternal-foetal interface are suggested to be important for foetal survival. The results showed that placental MHC class II expression cannot be induced in vivo. Transfections of trophoblast cells with MHC class II genes, however, resulted in detectable MHC class II cell-surface expression, indicating that a post-transcriptional block does not exist in these cells. By using IL-4- and IL-10-double deficient mice, it was shown that neither maternal nor foetal expression of these cytokines were crucial for completion of allogeneic pregnancy. In the second part of the thesis, the effect of transmission of immunoglobulin G (IgG) from the mother to the offspring was studied. It was observed that viable maternal Ig-secreting cells occasionally infiltrated the B cell-deficient offspring and remained functional for long periods. In this study "green fluorescent mice" were used as a tool. Furthermore, neonatal ingestion of wild type milk increased the survival of adoptively transferred B-lineage cells in B cell-deficient mice, suggesting that suckling of IgG-containing milk could be used to facilitate B cell-reconstitution in B cell-deficient mice. Finally, results from studies on normal mice showed that absence of maternal IgG-transmission during their neonatal development resulted in elevated serum-IgG production, as well as enhanced immune reactions upon immunisations in adult life. This showed that maternal IgG can have long-term immunoregulatory effects in the offspring.

In this thesis, two different vaccines were evaluated as adjuvant therapy for patients with colorectal cancer. The ability of the two candidate vaccines to generate antigen-specific cellular and humoral responses, respectively, was studied. The effectiveness of granulocyte colony stimulating factor (GM-CSF) as a cytokine adjuvant to augment the immune response was also examined. The first vaccination strategy involved immunization with the recombinant tumor-associated protein, carcinoembryonic antigen (CEA). Recombinant CEA was administered at 4 different dose levels 7 times during one year. Peripheral blood samples were regularly analyzed during 36 months. This vaccination regimen induced a strong immunoglobulin 1 (IgG1) and IgG4 response, a moderate IgG2 response and a weak IgG3 response against CEA. GM-CSF markedly augmented the effect on IgG1 and IgG4 as well as the T cell response. In contrast, dose of rCEA had no or modest effect on induced immune responses. The response gradually increased during the 12 months immunization period. Responses of all three IgG subclasses and of T cells were protracted up to 36 months. The anti-CEA IgG titers related significantly to survival. Functional HLA-DR epitopes of CEA could be defined. These major histocompatibility class II epitopes may serve as putative components of a peptide-based vaccination strategy. The other vaccine strategy consisted of the tumor-associated antigen epithelial cell adhesion molecule (Ep-Cam) expressed as a transgene in a viral vector, ALVAC. Patients were immunized subcutaneously/intradermally 3 times over 6 weeks and monitored for immune responses for 46 weeks. No anti-Ep-Cam specific humoral response was induced, but Ep-Cam specific type 1 T cells (interpheron-gamma production) were induced, mainly in the GM-CSF group. The cytotoxic cellular response appeared late, or a few months after the last immunization. Both vaccines were well tolerated. Since GM-CSF was an important component for both regimens, immungenicity of this cytokine was assessed. Multiple immunizations with low dose GM-CSF were associated with a low incidence of GM-CSF antibodies that did not neutralize the biological effect of GM-CSF. In conclusion, both vaccines are promising candidate vaccines. GM-CSF is necessary to induce a strong humoral and cellular immune response. Large clinical trials are urgently warranted to evaluate the clinical efficacy.

The current research is directed towards dissection of host genetic factors involved in host immune response and the malaria disease outcome. A possible association between FcγRIIa polymorphism and anti-malarial antibody (A.M.A) responses were investigated in Sudanese patients in relation to clinical outcome of falciparum malaria. The frequency of the R/R131 genotype was significantly higher in patients with severe malaria as compared with mild malaria. A.M.A IgG3 was shown to be associated with reduced risk of clinical malaria in individuals carrying the H/H131 genotype. Low levels of IgG2 reactive with the Pf332-C231 antigen were associated with lower risk of severe malaria in individuals carrying the H131 allele. Fulani and Masaleit, two sympatric ethnic groups in Sudan, are characterized by marked differences in susceptibility to falciparum malaria. We investigated whether the two populations differ in the frequency of GM/KM allotypes. The distribution of GM/KM phenotypes differed significantly among the two groups, with Gm 6 being significantly lower among the Fulani, and the combined frequency of Km 1,3 and Gm 1,17 5,6,13,14 phenotypes was found to be higher among Masaleit. In interethnic study we investigated whether the two groups differ in the frequency of FcγRIIa and HbAS genotypes. The frequency of the H/H131, R/R13 and HbAS genotypes differed significantly among the two groups. Moreover, the Fulani showed higher levels of A.M.A IgG2 and lower IgG1 and IgG3 when compared to their sympatric non-Fulani neighbours. A tri-allelic SNP (C/T/A) in the CRP gene was investigated for possible ethnic associations. The A allele, which is associated with higher basal CRP levels, was found to be less frequent in the Fulani compared with non-Fulani ethnic groups both in Sudan and Mali. In conclusion, our results suggest possible associations between FcγRIIa, CRP genotypes, GM/KM allotypes, and anti-malarial antibody responses and the clinical outcome of falciparum malaria.

Njurarna är ett organsystem med viktiga funktioner som exempelvis utsöndring av flertalet vattenlösliga substanser. För att sjukdomssymtom ska uppträda krävs mer än tre fjärdedelars bortfall av njurfunktionen, eftersom njurarna har en enorm reservkapacitet. Genom att analysera bland annat proteinerna albumin, immunoglobulin G, kappa och lambda i urin utreds om njurfunktionen fungerar som den ska. Analys av dessa proteiner kan ske med analysinstrumenten IMMAGE 800 från Beckman Coulter och BN ProSpec från DADE BEHRING. Båda dessa analysinstrument använder sig av metoden nefelometri, som är en metod där ljusspridning i en vätska eller gas kan mätas. Syftet med föreliggande studie var att analysera urinprover på både IMMAGE 800 och BN ProSpec och sedan jämföra resultaten. Under denna studie kalibrerades standardkurvor, genomfördes kvalitetskontroller och 37 prov analyserades. Samma prov analyserades flera gånger, både under samma dag och vid ett antal kommande dagar för att erhålla precisionen. Korrelationskoefficienten blev 0,999 för U-albumin; 0,998 för U-IgG; 0,947 för U-kappa och 0,883 för U-lambda. ProSpec kan således användas vid analys av U-albumin, U-IgG, U-kappa och U-lambda då den uppfyller EQUALIS kvalitetsmål.

Le [18F]Fluoro-éthoxybenzovesamicol ([18F]FEOBV) a été identifié comme étant un des agents radioactifs les plus prometteurs pour l'imagerie du transporteur vésiculaire de l'acétylcholine en utilisant la tomographie par émission de positrons (TEP). Nous rapportons ici que cette approche est en mesure de détecter de subtiles pertes de terminaux cholinergiques, comme celles associés avec le vieillissement ou suivant la lésion partielle du noyau basal de Meynert (NBM). Vingt-et-un rats adultes ont été distribués également en trois groupes : 1) Rats âgés (18 mois); 2) Jeunes rats (3 mois); et 3) Rats avec une lésion unilatérale du NBM induite par une infusion locale de saporine-IgG 192. Pour les rats normaux et ceux avec lésion, la plus haute valeur de liaison du [18F]FEOBV a été observée dans le striatum, suivi de valeurs similaires dans le cortex frontal et le thalamus, puis de valeurs plus faibles pour les hippocampes et le cortex temporo-pariétal. Les rats avec lésion du NBM ont démontré une liaison de [18F]FEOBV diminuée principalement dans la partie ventrale du cortex frontal. Cette perte est plus importante du côté de la lésion, mais également présente dans la région homologue de l'hémisphère controlatéral. Le vieillissement a entraîné une diminution de liaison de [18F]FEOBV localisée dans les hippocampes. Le [18F]FEOBV semble être une marqueur très prometteur pour la quantification in vivo du transporteur vésiculaire de l'acétylcholine dans le cerveau; l'imagerie TEP avec cet agent permet la détection de réductions physiologique et pathologique de la densité des terminaisons cholinergiques. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Tomographie par émission de positrons, transporteur vésiculaire de l'acétylcholine, neurodégénérescence, vieillissement, saporine-IgG 192, [18F]Fluoro-éthoxybenzovesamicol

Mantle cell lymphoma (MCL) is one of several sub-types of B-cell lymphomas. The malignancy is very aggressive and average survival time is short. The hallmark of MCL is over expression of cyclin D1, however about 15% of all MCL cases do not display this over expression and are easily misdiagnosed. Recently the transcription factor Sox11 has been shown to be specifically over expressed in the nucleus of MCL-tumour cells, and polyclonal rabbit anti-Sox11 antibodies have been used to successfully identify MCL in both cyclin D1 positive and negative cases. Howev-er, human recombinant MCL-specific antibodies as have several advantages over these polyclonal rabbit antibodies; they can easily be produced in large quantities in vitro, their specificity is constant from batch to batch and they can possibly be used for therapeutic purposes. Because of this, it is desirable to produce human recombinant antibodies against proteins over expressed in MCL. In this study human recombinant IgGs have been produced towards two pro-teins over expressed in MCL, Sox11 and KIAA0882. This was done by cloning of single chain variable fragments (scFvs), previously selected from a large scFv library through phage display selection against Sox11- and KIAA0882-protein epitope signature tag (PrEST), into vectors containing human IgG constant regions followed by expression of human IgG antibodies in human embryonic kidney (HEK) 293 cells. One IgG clone for each antigen was shown to be functional and specific. Both clones were shown to have overlapping binding epitopes with their polyclonal rabbit antibody counterpart (rabbit anti-Sox11/KIAA0882) through competitive ELISA. The anti-Sox11 IgG was able to detect two bands in cell lysate in Western blot, of which one probably is Sox11 while the other band possibly could be Sox4. However, this needs to be confirmed in future experiments. The affinity of the anti-Sox11 IgG was measured in Biacore and compared to the affinity of its original scFv. This gave a rough estimation of the affinities, but the values are unreliable and the measurements need to be redone. Although more work has to be put into evaluating the potential of the produced IgGs, they compose a promising starting point to an improved understanding and improved diagnosis of MCL.