<p>A 95-kDa protein was purified from normal human granulocytes. The protein reacted with a monoclonal antibody against CEACAM8. MALDI-Tof and MS/MS analyses revealed the protein to be a CGM6 gene product. Thus, the protein was proved to be identical to CEACAM8. </p><p>An ELISA for CEACAM8 was developed with detection range of 1-64μg/L. Data are presented on the levels of CEACAM8 in the blood of healthy individuals and patients undergoing surgery, as well as in patients with acute infection. The highly elevated levels of CEACAM8 in the blood of these patients were significantly correlated with the surface expression of CEACAM8 on neutrophils and the number of circulating neutrophils, which suggests that CEACAM8 could serve as a biological marker for granulocyte activitiy in vivo. </p><p>The cellular content of CEACAM8 in neutrophils was estimated to be 82.4 ± 8.9 ng/10⁶ cells. Subcellular localisation and mobilisation studies showed that the majority of CEACAM8 is present in the secondary granules of human neutrophils, with a small amount on the plasma membranes. Upon stimulation, CEACAM8 translocated to the plasma membranes from the secondary granules and was also released extracellularly (5.5 ± 0.7% of the total content of CEACAM8).</p><p>In eosinophils, the cellular content of CEACAM8 was estimated to be 73.8 ± 6.0 ng/10⁶ cells. In these cells, CEACAM8 is mainly stored in secretory vesicles. Upon activation, eosinophils released 5.1 ± 1.1% of the total content of CEACAM8. </p><p>Administration of granulocyte colony-stimulating factor (G-CSF) to healthy individuals resulted in an increased content of CEACAM8 in neutrophils on day 1, and decreased on day 4. However, the content of CEACAM8 in light membrane fractions was increased on day 4. The translocation of CEACAM8 observed <i>in vivo</i> after G-CSF administration is probably not directly related to this cytokine but to other cytokines such as TNF-a. </p>

Immunoglobulin Y (IgY) is the major antibody found in eggs from chicken (Gallus domesticus). IgY can be used as an alternative to mammalian antibodies normally used in research, and its use in immunotherapy has recently been proposed. Compared to mammalian antibodies, IgY possesses several biochemical advantages and its simple purification from egg yolk prevents a stressful moment in animal handling, as no bleeding is necessary. Small amount of antigen (1 mg) can be used to elicit an immune response in chickens and there are low intra-individual differences regarding antibody concentration found in yolk. By studying two chicken breeds and their cross, a genetic correlation was shown regarding the IgY concentration, which implies a possibility by breeding to increase IgY concentrations. By using IgY instead of goat antibody as capture antibody in ELISA, it is possible reduce interferences by complement activation. After oral administration of IgY to healthy volunteers, IgY activity was present in saliva 8 hours later, indicating a protective effect. This effect has been studied in an open clinical trial with cystic fibrosis patients. Specific IgY against Pseudomonas aeruginosa given orally prolongs the time of intermittent colonization by six months, decrease the number of positive colonizations and might be a useful complement to antibiotic treatment. Immunoglobulin therapy may diminish the development of antibiotic resistant microorganisms. The use of immunoglobulin therapy broadens the arsenal available to combat pathogens in medicine and IgY is a promising candidate, both as an alternative to antibiotics and as a useful tool in research and diagnostics.

A 95-kDa protein was purified from normal human granulocytes. The protein reacted with a monoclonal antibody against CEACAM8. MALDI-Tof and MS/MS analyses revealed the protein to be a CGM6 gene product. Thus, the protein was proved to be identical to CEACAM8. An ELISA for CEACAM8 was developed with detection range of 1-64μg/L. Data are presented on the levels of CEACAM8 in the blood of healthy individuals and patients undergoing surgery, as well as in patients with acute infection. The highly elevated levels of CEACAM8 in the blood of these patients were significantly correlated with the surface expression of CEACAM8 on neutrophils and the number of circulating neutrophils, which suggests that CEACAM8 could serve as a biological marker for granulocyte activitiy in vivo. The cellular content of CEACAM8 in neutrophils was estimated to be 82.4 ± 8.9 ng/106 cells. Subcellular localisation and mobilisation studies showed that the majority of CEACAM8 is present in the secondary granules of human neutrophils, with a small amount on the plasma membranes. Upon stimulation, CEACAM8 translocated to the plasma membranes from the secondary granules and was also released extracellularly (5.5 ± 0.7% of the total content of CEACAM8). In eosinophils, the cellular content of CEACAM8 was estimated to be 73.8 ± 6.0 ng/106 cells. In these cells, CEACAM8 is mainly stored in secretory vesicles. Upon activation, eosinophils released 5.1 ± 1.1% of the total content of CEACAM8. Administration of granulocyte colony-stimulating factor (G-CSF) to healthy individuals resulted in an increased content of CEACAM8 in neutrophils on day 1, and decreased on day 4. However, the content of CEACAM8 in light membrane fractions was increased on day 4. The translocation of CEACAM8 observed in vivo after G-CSF administration is probably not directly related to this cytokine but to other cytokines such as TNF-a.

Försämrat immunförsvar och ökad risk att drabbas av bakterie- och virusinfektioner förekommer hos patienter med blodsjukdomarna multipelt myelom, Waldenströms makroglobulinemi samt hos vissa patienter med blodsjukdomen monoklonal gammopati av oklar signifikans. Infektionerna kräver ofta antibiotikabehandling och behandling med antivirala medel. I dagsläget är det svårt att förutsäga vilka av patienterna som kommer att drabbas av svåra och ibland livshotande infektioner. Därför ges många av patienterna förebyggande antibiotikabehandling. I studiens början sattes en enzyme-linked immunosorbent assay (ELISA) för detektion av antikroppar mot Moraxella catarrhalis upp. I studien undersöktes om antikroppstitrar i serum mot bakterien Moraxella catarrhalis var lägre hos patientgrupperna än hos friska kontrollpersoner i samma ålder och om variationer förekom mellan patientgrupperna samt hur kontrollgrupper i olika åldrar skiljde sig från varandra. Kontrollgrupperna som undersöktes var mellan 20-40 år, 40-60 år samt 60 år och äldre. Resultatet var att patienterna med multipelt myelom hade lägst antikroppstitrar, patienter med monoklonal gammopati av oklar signifikans hade något högre och patienter med Waldenströms makroglobulinemi hade ännu högre antikroppstitrar. Kontrollgruppen äldre än 60 år hade högre antikroppstitrar än både kontrollgruppen 20-40 år och 40-60 år. Lägst antikroppstitrar hade kontrollgrupp 40-60 år men ingen signifikant skillnad påvisades mellan kontrollgrupp 20-40 år och 40-60 år.

Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are two structurally related groups of chemicals, generally referred to as `dioxins´. These are of great concern due to their high toxicity and global spread. Other groups of compounds with similar chemical structure and toxicity mechanisms are the brominated analogues polybrominated dibenzo-p-dioxins (PBDDs) and polybrominated dibenzofurans (PBDFs), and the dioxin-like polychlorinated biphenyls (PCBs). Numerous studies have been undertaken to investigate sources and transport routes of dioxins. However, much remains to be done, including analytical, inventories of dioxin-like compounds, such as PBDD/Fs, and the development of more convenient analytical methods. The currently standard procedure for analyzing dioxins (and dioxin-like compounds) is to use Soxhlet extraction followed by multi-step clean-up and gas chromatography - high resolution mass spectrometry (GC- HRMS) for detection. Unfortunately, this method is very solvent, labor and time-consuming, making it very expensive. The main aim of the studies this thesis was to develop pressurized liquid extraction (PLE) with integrated clean up techniques for fast, convenient preparation of dioxin samples. PLE with integrated clean-up has previously been used for extracting dioxins from biological samples, but in these studies the possibility of extending its use to abiotic samples was explored. The results show that PLE with an integrated carbon trap is suitable for analyzing dioxins in various types of soil samples, sediment and flue gas samples. The results also showed that it has potential for analyzing dioxins in fly ash. The thesis focuses on developments of the methodology for dioxin analysis, but also includes results obtained from PBDDs and dioxin-like PCB analyses. In addition, the possibility of using various other kinds of detection techniques rather than GC-HRMS, such as enzyme-linked immunosorbent assays (ELISAs) or two-dimensional gas chromatography with micro electron capture detection (GCxGC-µECD) was explored. The results indicate that ELISA and GCxGC-µECD could serve as complementary detection systems in some cases. However, it is not yet possible to fully replace GC-HRMS. A further refinement of the PLE with in-cell clean-up technique is the modular approach developed in these studies. With this technique it is possible to include various steps for both clean-up and fractionation. For example, sulphuric acid impregnated silica could be combined with active carbon for the simultaneous removal of lipids (along with other interferences) and fractionation of PCBs and PCDD/Fs. It was shown that the method could provide data that agreed reasonably well with both reference values and values obtained using traditional methods. In general PLE proved to have high extraction efficiency and to yield very similar congener profiles to the reference method. In addition, it was shown that it allowed one-step extraction and clean-up of a salmon sample. Such single-step procedures are the ultimate goals for any extraction technique, and it would be highly desirable to develop one-step methods that could be extended to other types of samples. For the rest of the matrices tested (soil, sediment, mussel and crab tissue and flue gas) the method was successful, however a final polishing step is currently required, involving either dilution or clean-up using miniaturized multilayer silica columns, to obtain extracts that are pure enough for GC-HRMS analysis. Using the developed modular-PLE system substantial costs could be saved. It was estimated that the method could reduce the cost of preparing samples by up to 90%, which would greatly facilitate large-scale inventories.

The Arctic mutation in the Amyloid-β (Aβ) domain of the Amyloid-β precursor protein (APP) causes Alzheimer’s disease (AD) and confers unique biochemical characteristics to Aβ peptides. The aims of this thesis were to evaluate a transgenic model with the Arctic mutation, and to use it to gain new insights into the mechanisms of early (pre-plaque) and late-stage Aβ pathogenesis in AD. The Arctic mutation made Aβ more prone to aggregate, to accumulate in intracellular compartments and to form extracellular plaques when the models tg-ArcSwe and tg-Swe were compared. By inhibiting APP processing genetically or pharmacologically, the intraneuronal granular immunoreactivity with antibodies binding the Aβ domain was shown to largely represent Aβ, and not APP or APP-fragments. At two months of age, the intracellularly accumulated Aβ decreased rapidly, likely because it was still accessible to intracellular clearance. Extracellular Aβ deposits emerged at 5-6 months of age and the amyloid fibril structure was more compact than in tg-Swe. Moreover, Aβ deposits in tg-ArcSwe were more resistant to chemical extraction than those of established models carrying the Swedish APP mutation only, e.g. tg-Swe mice. The stability of deposits better reflects the biochemistry of senile plaques in AD. Thus, the tg-ArcSwe model may better predict the outcome of clinical trials, particularly therapies designed to enhance clearance of Aβ aggregates and deposits. Postmortem brain of Arctic mutation carriers contained extensive parenchymal plaque pathology. Differential immunostaining patterns with C- and N-terminal Aβ antibodies revealed a subset of plaques that were unique to the brains of Arctic mutation carriers. Aβ deposits in the cerebral vessel walls were congophilic and mainly composed of full-length Aβ. In contrast, N-terminally truncated Aβ was more prominent in the parenchymal plaques, all of which essentially lacked amyloid cores. A heterogeneous assembly of mutant and wild-type Aβ was shown to favor the formation of diffuse deposits in bitransgenic mice, and such mechanisms may at least partly explain observations of plaques lacking amyloid cores in postmortem Arctic mutant brain. In the bitransgenic mice, a low level of Arctic Aβ was sufficient to facilitate aggregation of wild-type Aβ. This observation, but also our findings of differences in amyloid fibril structure in tg-ArcSwe and tg-Swe, further highlights similarities between AD and prion disorders in which PrPsc refolds PrPc and facilitates fibril formation. / (Faculty of medicine)

La périostine est une protéine matricellulaire préférentiellement exprimée aux sites de contraintes mécaniques, notamment le périoste, et dans le stroma associé à de nombreux types de cancers. En premier lieu, nous nous sommes attachés à évaluer la pertinence de cette protéine en tant que biomarqueur du métabolisme osseux et de la réaction stromale dans les métastases osseuses. Nous avons développé le premier dosage ELISA de la périostine circulante chez la souris présentant des caractéristiques analytiques (spécificité, précision) conformes aux exigences réglementaires. Ce dosage nous a permis de préciser l'implication de la périostine dans le métabolisme osseux et les métastases osseuses de cancer du sein. Nos données in vitro et in vivo suggèrent que la périostine n'est pas un indice direct du remodelage osseux, contrairement aux marqueurs biologiques conventionnels, mais une composante de l'ossification primaire. Nous avons aussi montré dans les métastases osseuses d'origine mammaire que la périostine est surexprimée par les cellules stromales de la métastase, comme cela a pu être observé au niveau des tumeurs primaires. Enfin, nous avons confirmé par une approche bioinformatique la relation étroite entre périostine et réaction stromale dans la plupart des tumeurs chez l'Homme. La périostine et d'autres protéines conjointement exprimées pourraient donc constituer un panel de marqueurs biologiques de la progression tumorale, certains pouvant se révéler comme nouvelles cibles thérapeutiques en oncologie.

The teneurin C-terminal associated peptides (TCAP) are a family of four predicted peptides that are expressed in all metazoans where the teneurins have been studied to date. Of the four peptides, TCAP-1 has been studied most extensively. In vitro, TCAP-1 increases neuronal proliferation and neurite outgrowth. In vivo, the peptide reduces CRF-induced behavioural responses in rats. Despite the large body of evidence indicating a strong biological role for TCAP-1, little is known about the chemistry and solubility of the peptide, or the signaling pathway(s) mediating these effects. The aim of this research was to appropriately solubilize the peptide and to develop detection assays for its study in greater detail. I have now established an appropriate formulation of TCAP-1 and developed an immunoassay to assess its concentrations in tissues and in circulation. Also, by examining a number of transcriptional response elements, I have found two assays for probing the signal transduction mechanisms of this peptide.

The teneurin C-terminal associated peptides (TCAP) are a family of four predicted peptides that are expressed in all metazoans where the teneurins have been studied to date. Of the four peptides, TCAP-1 has been studied most extensively. In vitro, TCAP-1 increases neuronal proliferation and neurite outgrowth. In vivo, the peptide reduces CRF-induced behavioural responses in rats. Despite the large body of evidence indicating a strong biological role for TCAP-1, little is known about the chemistry and solubility of the peptide, or the signaling pathway(s) mediating these effects. The aim of this research was to appropriately solubilize the peptide and to develop detection assays for its study in greater detail. I have now established an appropriate formulation of TCAP-1 and developed an immunoassay to assess its concentrations in tissues and in circulation. Also, by examining a number of transcriptional response elements, I have found two assays for probing the signal transduction mechanisms of this peptide.

<p>More than 20 million infants in the world (15.5 % of all births) are born with low birth weight. Ninety-five % of them are in developing countries. Oral colonization of Gramnegative anaerobes has&nbsp / been implicated as a risk factor for preterm delivery of low birth weight (PLBW) infants. The objective of this study was to investigate the association between periodontal pathogens and pre-term&nbsp / delivery of low birth weight (PLBW) infants. The study sample included 200 randomly selected women admitted to the department of obstetrics-gynecology of the teaching hospital of Butare in&nbsp / Rwanda. Mothers were asked to complete a questionnaire in order to identify factors which might pose a health risk to them and their infants. Gingival crevicular fluid (GCF) was collected from each quadrant of the mother&rsquo / s month (using paper points) within 24 hours of delivery. Ten ml of foetal cord serum samples were collected at delivery and 10 ml of maternal serum samples were&nbsp / collected within 48 of delivery. GCF was examined by PCR for the presence of 5 periodontopathogens and ELISA was used for the evaluation of cytokines (IL-6 and IL-10) and immunoglobulins (IgM, IgG) in foetal cord and maternal blood against the periodontopathogens. P. intermedia showed significant associations either on its own or in combinations with most indicators of&nbsp / periodontal disease used in this study, while Aa and members of the red complex were significantly associated with gum bleeding and reduced frequency of tooth brushing. A strong association&nbsp / between PLBW and maternal and foetal cord serum sample levels of IL-10 was observed. Also, a good association was observed between PLBW and FCB sample levels of IL-6. Significant&nbsp / associations were observed between PLBW and maternal IgG against the different peridontopathogens. The findings of this study may suggest that the levels of maternal IgG and foetal IgM&nbsp / against the different periodontopathogens are associated with dissemination of maternal periodontopathogens to the foetus thereby illiciting an inflammatory response which contributes to&nbsp / PLBW. </p>