Thesis (doctoral)--Freie Universität Berlin, 1980.

Thesis--University of Turku, 1986. / Also published in: Proceedings of the Finnish Dental Society, 1986, Vol. 82, Suppl. VII. Includes bibliographical references.

Thesis--University of Turku, 1986. / Also published in: Proceedings of the Finnish Dental Society, 1986, Vol. 82, Suppl. VII. Includes bibliographical references.

A radioimmunoassay was developed, specific for the gastrointestinal polypeptide, motilin. Antisera were raised in guinea pigs and rabbits. The immunogen was porcine motilin, conjugated to bovine serum albumin by the carbodiimide condensation reaction. The routine antiserum behaved identically towards endogenously- released motilin and the pure standard preparation. A radioactive tracer of high 125 specific activity was obtained after incorporation of - iodine into the motilin molecule by the chloramine-T method. The optimum conditions for all other assay variables were established to produce the most sensitive displacement Cstandard) curve. Motilin antiserum, coupled directly to an agarose matrix, retained full antibody activity and sensitivity. It is a feasible technique for use in both the radioimmunoassay and in the extraction of motilin from both serum and tissue extracts. The fasting serum levels of IR- motilin was 190 - 131 pg/ml in men and 294 -'.44 pg/ml in dogs (mean - SD) . The increase in motor activity in the extrinsically denervated fundic pouch of the dog after duodenal alkalinization was associated with a concomitant elevation in serum IR- motilin levels. This increase in serum IR- motilin was in the same range as that achieved by the exogenous administration of the porcine polypeptide which produced the same motor response. Duodenal acidification produced an apparent increase in serum IR-motilin with no associated increase in gastric motor activity. Only one peak of motilin immunoreactivity was detected when serum containing alkali-stimulated motilin or a partially purified duodenal extract were subjected to gel filtration on Sephadex G-50. The distribution of motilin throughout the hog gastrointestinal tract, determined by radioimmunoassay on partially purified extracts, agreed with the immunocytochemical findings that motilin was predominantly located in the duodeninn and jejunum, with traces.in the upper ileum. Virtually the intact molecule was required for the expression of full biological potency. The individual amino acids were important inasmuch as they contributed to the charge distribution and conformation of the molecule. The physiological release and function of motilin have yet to be determined. Elevated levels of circulating IR- motilin have not been associated with any gastro-intestinal function, although they appear to be depressed by feeding. Motilin has been implicated in the control of the interdigestive phase of gastric motor activity. It may be acting in a local or paracrine manner. Motilin has not been implicated in any .•cU'in±cal.rst"ait"eC&s sjffetfce i The hormonal status of gastric inhibitory polypeptide (GIP) has been studied with the existing radioimmunoassay, modified to improve the label specific activity (by ion exchange chromatography). Direct coupling of GIP antisera to agarose beads was unsatisfactory, antibody activity and sensitivity being greatly reduced by the close proximity of the solid matrix. The postulated role of GIP as the enterogastrone1 of Kosaka and Lim, suggested by studies with exogenously-administered polypeptide, was confirmed by experiments in the dog. Pentagastrin-stimulated gastric acid secretion was inhibited by intra-duodenal infusion with glucose or fat; this inhibition being associated with a significant elevation in the circulating serum IR- GIP levels, within the range produced by ingestion of a mixed meal. GIP does not appear to be involved in the inhibition of gastric acid secretion produced by duodenal acidification. Endogenous;GIP.stimulated by either fat or glucose exhibited at least 3 Immunoreactive components after column chromatography. The IR- GIP eluting in the void volume appeared to represent a non-specific complex between GIP and a serum protein and is possibly biologically inactive. A second IR-GIP component with a molecular weight of 7500-8000 (ProGIP), eluted ahead of the established form of GIP (molecular weight = 5105). ProGIP has been found to be relatively unstable. ProGIP and GIP^QQQ have also been detected in extracts of hog duodenal mucosa. The established insulinotropic effect of GIP correlates best with that percentage of the total IR- GIP composed of ProGIP and GIP500(). The relative proportions of IR- GIP500Q and IR- ProGIP in serum samples taken at different times after ingestion of either fat or glucose, suggest that ProGIP is either a precursor of GIP or that the ProGIP-producing cells occupy a more distal region of the duodenal and jejunal mucosa than the GIP- producing cells. Exogenous administration of synthetic somatostatin in dogs and man will inhibit both.GIP release by either fat or glucose and the insulino-tropic action of GIP at the level of the 8/-cell. Naturally-occurring intestinal or pancreatic somatostatin may contribute to the control of GIP release and serve to modulate the GIP- mediated response of the gastric parietal or pancreatic β-cell. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 6 uppsatser.

OBJECTIVE: The principal objective of this study was to measure various aspects of immunity in children with invasive infections due to Haemophilus influenzae type b and Staphylococcus aureus. These serious infections are a significant cause of childhood morbidity and mortality in all populations and affect healthy as well as compromised children. Evidence suggests that imbalances or deficiencies in certain aspects of immunity such as IgG subclasses, the capacity to make specific subclass antibodies, antibody affinities, complement isotypes, immunoglobulin allotypes or mannose binding protein may place certain children at risk for developing invasive disease. Investigation of these factors in a group of children with infection necessitated that normal ranges be established for children of comparable ages from the same population. A secondary objective of this study has therefore been to establish normal percentiles for the IgG subclasses in age, race and sex matched healthy controls. METHODS: Patients admitted to the Red Cross War Memorial Children's Hospital with septic meningitis due to Haemophilus influenzae type b and osteomyelitis/septic arthritis due to Haemophilus influenzae type b or Staphylococcus aureus formed the study population. Section A of this thesis describes the methods for establishing, validating and standardizing ELISAs for measuring the IgG subclasses (lgGl, IgG2, IgG3 and IgG4) and subclass antibodies specific to Haemophilus influenzae polyribosylribitol phosphate, Staphylococcus aureus teichoic acid and tetanus toxoid. The relative affinity of antibodies in these ELISAs was determined by the incorporation of diethylamine (DEA). In order to determine the immunoglobulin allotypes ELISAs were developed to measure the G1m(f), G2m(n) and Km(3) allotypes. The frequency of these allotypic markers in the different ethnic groups was established. The relationship between immunoglobulin allotypes and IgG subclass values were investigated in both patient and control groups. RESULTS: ELISA assays to measure IgG subclasses; IgG, IgG 1 and IgG4 tetanus toxoid antibodies; IgG, IgG 1 and IgG2 H. influenzae type b polyribosylribitol phosphate capsular polysaccharide antibodies; IgG, IgG1 and IgG2 S. aureus teichoic acid antibodies and G1m(f), G2m(n) and Km(3) allotypes were successfully established. Where possible the assays were standardized with reference sera and specimens were exchanged with international laboratories. Age, race and sex related percentile charts and tables of normal ranges for IgG and IgG subclasses of Black and Coloured children were established. The IgG and IgG 1 values were higher than those previously reported for children in developed countries. Black children with H. influenzae meningitis had significantly lower IgG 1, IgG2 and IgG3 levels compared to the controls and although similar trends were seen for IgG and IgG4 levels they were not statistically significant. Coloured children with H. influenzae meningitis and Coloured and Black children with H. influenzae osteomyelitis/septic arthritis also showed a similar tendency of lower IgG and IgG subclass levels than the controls but these trends were also not significantly different. All patients responded to tetanus toxoid antigen suggesting normal immunocompetence to protein antigens. H. influenzae type b capsular polysaccharide antibodies were low in children with H. influenzae type b meningitis and osteomyelitis/septic arthritis and did not increase during the illness. IgG and IgG 1 teichoic acid antibodies were raised in patients with S. aureus osteomyelitis/septic arthritis although no further rise in these antibodies was seen when measured several weeks after the illness. The antibody affinity ELISAs showed that IgG 1 tetanus toxoid antibody had a greater affinity than IgG4 tetanus toxoid antibody, the IgG 1 and IgG2 H. influenzae capsular polysaccharide antibodies were of similar affinity and the IgG 1 teichoic acid antibody was of higher affinity than the IgG2 antibody. The G1m(f) and G2m(n) positive allotypes were uncommon in Black but common in the Coloured populations whereas Km(3) was common in both groups. There was a significantly decreased frequency of the G2m(n) positive allotype in Coloured patients with H. influenzae type b meningitis and H. influenzae type b osteomyelitis/septic arthritis which was not found in patients with S. aureus osteomyelitis/septic arthritis. In both Coloured and Black children with H. influenzae meningitis there was a significantly decreased frequency of the Km(3) allotype. No differences in C4 isotypes and mannose binding protein levels were evident in the patient and control groups. CONCLUSION: This study has developed simple, specific and reproducible ELISAs to measure IgG subclasses and subclass antibodies specific to tetanus toxoid, H. influenzae polyribosylribitol phosphate and S. aureus teichoic acid. Age, sex and race related normal ranges for IgG subclasses in the local Black and Coloured populations have been established. Black children with H. influenzae type b meningitis had significantly lower IgG 1, IgG2 and IgG3 levels compared to the controls. There was a clear association between a decrease of the G2m(n) allotype and the Km(3) allotype and susceptibility to invasive infections caused by H. influenzae.