Biochemical markers for cardiovascular risk following treatment in endogenous Cushing's syndrome.

OBJECTIVE: Cardiovascular disease has been reported to be more common in patients with endogenous Cushing's syndrome (CS) compared to the normal population. In addition to altered lipid profile, inflammation seems to play an important pathogenic role in atherogenesis, but the role of inflammation in CS-associated cardiovascular disease is still not clear. To further elucidate these issues we measured several markers of inflammation in CS patients at baseline and following operative treatment and potential cure. SUBJECTS: Twenty-eight CS patients (22 women, 6 men) were included in the study and 21 of these patients (15 women, 6 men) were also followed longitudinally for a mean 33 months (range 5-69 months) after operative treatment. For comparison, blood samples were also collected from 24 healthy controls (21 women, 3 men). RESULTS: We show a distinct cytokine profile in CS patients before and after operative treatment. Thus, while interleukin (IL)-8 and osteoprotegerin (OPG) were significantly increased in CS patients before operation and fell during follow-up, levels of C-reactive protein (CRP) and soluble intracellular adhesion molecule 1 (sICAM) were significantly decreased at baseline, reaching normal levels after operation. While soluble CD40 ligand was within normal limit at baseline, this marker of platelet-mediated inflammation was markedly elevated during follow-up. CONCLUSIONS: Our findings suggest a complex interaction between CS and inflammation. In particular, the raised levels of IL-8 and OPG in CS patients, despite glucocorticoid excess, may represent an inflammatory and pro-atherogenic phenotype. However, the clinical relevance of these findings will have to be clarified.

Interleukin-1 receptor antagonist is associated with fat distribution in endogenous Cushing's syndrome: a longitudinal study.

The weight gain and visceral obesity associated with Cushing's syndrome (CS) has been linked to elevated plasma leptin levels, although the mechanism behind a central leptin resistance in these patients is unknown. Several studies describe interactions among the hypothalamic-pituitary-adrenal axis, leptin, and the IL-1 system. To investigate these interactions, we have evaluated changes in regional fat distribution, by DEXA, and the role of circulating cortisol, leptin, IL-1beta, and IL-1 receptor antagonist (IL-1Ra), in relation to these changes, in 27 (19 DEXA; 27 serum measurements) patients with CS, before and after surgical treatment (mean follow-up, 31 months; range, 5-80), and compared them with measurements of age-, sex-, and body mass index-matched healthy controls (also obtained longitudinally). We found that surgical treatment caused a decrease in all fat parameters, without changing lean body mass, and these changes were significantly larger than the so-called natural changes occurring in control subjects. These changes in CS patients were paralleled by decreases in cortisol, leptin, and IL-1Ra, whereas IL-1beta increased. Stepwise linear regression showed that serum IL-1Ra was strongly associated with regional fat distribution, and especially truncal fat mass, both at baseline and during treatment. In conclusion, the present study shows that treatment significantly changes body composition in CS patients by decreasing fat mass, especially in the truncal region, without major effects on lean body mass. We also show that circulating IL-1Ra is strongly associated with these changes, signifying a relationship among the hypothalamic-pituitary-adrenal axis, IL-1 system, and regional fat distribution in these patients.

Eradication of virulent footrot from sheep and goats in an endemic area of Nepal and an evaluation of specific vaccination.

Programmes based on the identification and treatment of cases and the culling of animals refractory to treatment had failed to eradicate virulent footrot from two districts in the western region of Nepal. From 1993 to 1996 vaccination against two endemic virulent strains of Dichelobacter nodosus was tested for its potential to contribute to the eradication of footrot from the region. Only sheep and goats which had been free of signs of footrot at three inspections at monthly intervals before their annual migration to alpine pastures were eligible for inclusion. From November 1992, the treatment of cases identified during inspections included the injection of specific vaccine. Successfully treated cases migrated with their flocks but were excluded from the vaccine trial. Non-responding cases were culled. Forty combined flocks of sheep and goats (approximately 9500 animals) were used initially to compare three vaccination regimens. Eleven flocks (sheep and goats) were treated with two doses of specific vaccine (group A), nine (sheep and goats) were treated with commercial vaccine followed by specific vaccine (group B) and 10 (sheep and goats) were treated with two doses of commercial vaccine (group C) in March to April 1993 before the annual migration; 10 flocks (sheep and goats) remained unvaccinated (group D). Only sheep and goats free of signs of footrot were allowed to migrate. Nevertheless, virulent footrot recurred in many flocks three months later. However, its prevalence was significantly lower in group A than in the other three groups combined. Groups A, B and C then received the specific vaccine before their migrations in 1994 to 1996; group D remained unvaccinated. The annual programme of inspection and identification and treatment of cases continued for seven years, but the vaccinations ceased after four years. There was no recurrence of virulent footrot after November 1993. After the first season the virulent strains of D nodosus used in the specific vaccine could no longer be isolated, although antigenically distinct, benign strains of the organism persisted in cases of benign footrot.

[Surgical treatment of gastrointestinal malignant melanoma]. / Kirurgisk behandling av malignt melanom i bukorganer.

Malignant melanoma is the most common metastatic lesion of the intestine, found at autopsy in approximately 60% of patients who die from melanoma. Common symptoms include bleeding, perforation and ileus/subileus. Patients with such symptoms should be operated on immediately, if they are not too reduced because of advanced disease. Long-term survival (five years) has been achieved in 34 of 179 (19%) of stage IVA melanoma patients after a radical operation procedure. Surgical removal of gastrointestinal metastases provides effective palliation in 80-95% of patients, who undergo laparotomy. We describe two patients with gastrointestinal metastases from malignant melanoma. A 33 year-old man had a large melanoma metastasis removed (non-radically) from the small intestine. He recovered rapidly, and experienced good palliative effect for three months, but died from a recurrence of the disease six months after operation. The other patient had a melanoma metastasis in the right lobe of the liver, which was treated by right hemihepatectomy. There has been no recurrence ten months after operation. We recommend surgical removal of abdominal melanoma metastasis if the surgery can be performed without unacceptable risk.

Disease resistance in Merino sheep. III. Genetic variation in resistance to footrot following challenge and subsequent vaccination with an homologous rDNA pilus vaccine under both induced and natural conditions.

SUMMARY: Eight traits representing clinical indicators of resistance to footrot were examined in 1562 Merino sheep, representing the progeny from 162 sires in four major bloodlines. Over a 4-year period, sheep were exposed to virulent isolates of Dicbelobacter nodosus under both an experimental challenge in which footrot was induced, and a separate natural challenge involving a different isolate of D. nodosus. Five footrot traits and three healing traits were each recorded on seven occasions following induced challenge, and on five occasions following natural challenge. All sheep were vaccinated with a primary and booster injection of an homologous rDNA pilus vaccine, 9 and 6 weeks after initiation of the induced and natural challenge respectively. The major fixed effects which influenced variation in resistance were (in order of importance) time of inspection after challenge, year and group in which sheep were challenged, and sex of the animal. Date of birth, birth-rearing type and age or dam were unimportant in the expression of footrot. Half-sib heritability estimates of resistance to footrot were low to moderate for single observations recorded pre-vaccination (0.07-0.22), and slightly lower for inspections made after vaccination (0.07-0.15). Repeatability estimates for footrot traits during a challenge ranged from 0.31 to 0.70 for inspections pre-vaccination, and 0.19 to 0.35 for inspections post-vaccination. Genetic correlations among footrot traits recorded at repeat inspections were high for observations pre-vaccination (range 0.87-1.00) and slightly lower for observations made after vaccination (0.52-1.00). Heritability estimates derived from repeat measurements approached 0.30 for most traits, except for traits describing healing, which had a heritability of almost zero. Heritability estimates of liability to footrot ranged between 0.09 and 0.41 depending on the time after challenge when the inspections were made. The genetic correlation between induced and natural footrot ranged from 0.14 to 0.95, depending on the period over which inspections were made, with an average of 0.67. In addition to within-flock genetic variation in resistance to footrot, significant differences were observed between different bloodlines within the experimental flock. It was concluded that there is substantial genetic variation in resistance to challenge with virulent isolates of D. nodosus. However, practical restrictions of exploiting available genetic variation may limit the widespread adoption of direct selection. ZUSAMMENFASSUNG: Krankheitsresistenz in Merinos III. Genetische Variabilität in Moderhinke Resistenz nach Infektion und folgender Impfung mit homologer rDNA pilus Vakzine unter induzierten und natürlichen Bedingungen Acht Merkmale, die als klinische Hinweise auf Moderhinkeresistenz betrachtet werden, wurden in 1562 Merino Schafen aus 162 Vatertieren von vier wichtigen Linien untersucht. Über eine 4-Jahresperiode wurden die Schafe virulenten Isolaten von Dichelobacter nodosus unter Versuchsbedingungen ausgesetzt und eine getrennte natürliche Infektion mit verschiedenen Isolaten von D. nodosus durchgeführt. Fünf Moderhinkemerkmale und drei Gesundungsmerkmale wurden nach Infektion bei sieben Gelegenheiten festgehalten und an fünf nach natürlicher Infektion. Alle Schafe wurden mit einer primären und einer booster Injektion homologer rDNA pilus Vakzine geimpft, 9 und 6 Wochen nach der induzierten und natiirlichen Infektion. Die wichtigsten fixen Effekte, welche die Variabilität der Resistenz beeinflussen, waren, nach Wichtigkeit gereint, Zeit der Prüfung nach Impfung, Jahr und Gruppe in welcher Schafe geimpft wurden und Geschlecht. Geburtsdatum, Aufzuchttyp und Mutterschaf-alter waren im Hinblick auf Moderhinke unwichtig. Halbgeschwister-Heritabilitätsschätzungen ihrer Resistenz waren niedrig bis mittel für Einzelbeobachtungen vor der Impfung (0,07-0,22) und geringfügig geringer für Beurteilung nach Impfung (0,07-0,15). Wiederholbarkeitsschätzungen für Moderhinkemerkmale bewegten sich von 0,31 bis 0,70 für Inspektionen vor und 0,19-0,35 für Inspektionen nach Impfung. Genetische Korrelationen zwischen Moderhinkemerkmalen bei verschiedenen Untersuchungen waren fur Beobachtungen vor der Impfung hoch (0,87-1) und geringfügig niedriger nachher (0,52-1). Heritabilitätsschätzungen von wiederholten Messungen erreichten 0,30 für die meisten Merkmale außer für jene, welche Heilung beschreiben, die nahezu keine Heritabilität zeigen. Heritabilitätsschätzungen für Moderhinkeempfindlichkeit variierten zwischen 0,09 und 0,41 in Abhängigkeit von der Untersuchungszeit nach den Impfungen. Die genetische Korrelation zwischen induzierter und natürlicher Moderhinke schwankte von 0,14 bis 0,95 in Abhängikeit von der Dauer der Beobachtungsperiode, durschnittlich 0,67. Zusätzlich zur genetischen Variabilität innerhalb der Herde wurden signifikante Unterschiede zwischen verschiedenen Linien innerhalb der Versuchsherde gefunden. Darauf wird es geschlossen, daß substantielle genetische Variabilität für Resistenz gegenüber virulenten Isolaten von D. nodosus existiert. Allerdings können praktische Hindernisse die Ausnutzung der vorhandenen genetischen Variabilität durch direkte Selektion einschränken.

Antigenic stability of fimbriae of Bacteroides nodosus.

Successful vaccination of sheep against footrot and attempts to eradicate the disease depend on there being a limit to the antigenic diversity of the causative bacterium, Bacteroides nodosus. Fimbrial antigenic variation was therefore investigated in vivo, both under conditions of chronic infection and under the pressure of a vaccine-induced immune response, to ascertain whether this represented an obstacle to such goals. Material was available from 5 experiments and although B. nodosus appeared to have undergone changes in its fimbrial antigens in one of these, the possibility that superinfection was responsible for the variation detected could not be ruled out because all sheep in this case were maintained at pasture. Overall, the results provided no evidence of fimbrial antigenic shift in B. nodosus in vivo and in conclusion, the survival of the organism in the sheep's foot, both in long-term natural infection and following vaccination, must therefore be related to factors other than the ability to undergo antigenic variation in order to evade the host's immune response.

Western blot (immunoblot) analysis of the fimbrial antigens of Bacteroides nodosus.

The roles of the fimbrial subunit and the putative basal protein antigens in the serological classification of Bacteroides nodosus have been examined by Western blot (immunoblot)-antibody binding studies of fimbriae isolated from a wide range of strains representative of different serogroups and serotypes. Fimbrial subunits were recognized by antiserum against the homologous serogroup but not generally by heterologous antisera, whereas recognition of the basal antigen was independent of serological classification. Secondary cross-reaction patterns among fimbrial subunits indicated that some serogroups may be more closely related than others. Examples include serogroups C and G and serogroups D and H. Similar analyses of isolates classified within serotypes A1 and A2, with serotype-specific antisera, showed that this subdivision is also determined by the fimbrial subunit and that significant variation does occur even at this level. These studies suggest that the various serogroups and serotypes of B. nodosus comprise a series of overlapping sets of antigenically related strains.

Protection of sheep against footrot with a recombinant DNA-based fimbrial vaccine.

Recombinant Pseudomonas aeruginosa cells containing the Bacteroides nodosus fimbrial subunit gene under the transcriptional control of a strong promoter produce large amounts of B. nodosus-type fimbriae. We have carried out vaccination trials which show that these fimbriae are just as effective as either natural fimbriae or whole cell preparations of B. nodosus in inducing protective immunity against homologous footrot challenge. The recombinant-produced fimbriae are also effective therapeutically in accelerating the rate of healing of pre-existing footrot lesions. These results confirm that the structural subunit of the fimbrial strand is a primary protective antigen against footrot, and demonstrate the practicality and potential of recombinant DNA approaches to the development of new vaccines against B. nodosus and other Type 4 fimbriate pathogens.

Variation in the structural subunit and basal protein antigens of Bacteroides nodosus fimbriae.

The fimbriae of Bacteroides nodosus play a major role in protective immunity against ovine footrot and are an important determinant in the serological classification system that divides field isolates into at least eight serogroups and 16 serotypes. Purified fimbriae contain two polypeptide antigens, the structural subunit of the fimbrial strand (molecular weight about 17,000) and a basal protein (molecular weight about 80,000), both of which exhibit structural variation. Fimbriae were prepared from all prototype strains, as well as from a number of other isolates representative of each of the B. nodosus serotypes, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Substantial variation was observed in the electrophoretic mobility of the fimbrial subunits from the prototypes of each of the eight serogroups. With the exception of serogroup H, which is an unusual case, the apparent molecular weights of the fimbrial subunits ranged from about 16,500 in serogroup D to 19,000 in serogroup F (serotype 1); in serogroup A, B, C and E, the apparent molecular weights were clustered in the range of 17,000 to 17,500, whereas serogroup G was about 18,500. Serogroup H fimbriae appeared to consist of two smaller polypeptides, which in the prototype (H1) had apparent molecular weights of about 6,000 and 10,000 and which seem to have arisen as a consequence of an internal proteolytic nick in the original subunit. Electrophoretic variation in the fimbrial subunit was also observed between different serotypes, although with the exceptions of serogroups F and H, this was not as pronounced as between the serogroups. Examination of a number of isolates classified within the same serotypes showed that some variation, although minor, also occurred at this level. The basal antigen exhibited significant variation at all levels of the serotypic hierarchy in a manner apparently unrelated to the classification system. Among the range of isolates examined, the apparent molecular weight of this antigen varied from about 77,000 to 88,000.