Patients with clinical exacerbation of UC (n = 18) or CD (n = 18), necessitating hospitalization and requiring high-dose prednisolone to induce remission, were included consecutively at the Medical Department V, Aarhus University Hospital in the period December 2000 to July 2001. Patients were excluded by the presence of obvious bacterial infection or acute abdomen.
All patients had a well-established diagnosis of UC or CD according to clinical, biochemical, endoscopy, histopathological criteria, and small bowel follow through.
The group of patients with UC consisted of 6 women and 12 men, median 39 years of age (range 21–78). There were 12 women and 6 men among the patients with CD, median 29 years of age (range 18–78).
The disease activity was evaluated clinically by the Harvey-Bradshaw Index in both UC and CD patients [19]. The UC patients had a median score of 7 (range 5–19), and CD patients revealed a median score of 7 (range 3–24). Seventeen UC patients had extensive colitis (pancolitis) and 1 left sided colitis. Patients with CD had either colon affection, small bowel affection, or a combination.
At inclusion, UC patients were treated with combinations of steroids (rectal enema)(n = 6), 5-aminosalicylic acid (n = 12), antibiotics (n = 1), whereas 6 patients received no immunomodulatory medication at all. The CD patients were treated with combinations of steroids (rectal enema)(n = 1), 5-aminosalicylic acid (n = 7), azathioprine (n = 1), antibiotics (n = 1), whereas 4 patients received no immunomodulatory medication at all.
All treatments except for rectal steroid enema (except for 1 patient) and antibiotics were continued. The treatment with prednisolon was started instantly, and administrated intravenously for the first 3–7 days (1 mg/kg body-weight), followed by oral administration by day 7 where the dose were tapered to 40–60 mg/day, and further tapering typically 5 mg/week until 0.
Blood samples were collected from all patients before high-dose prednisolone treatment was initiated, after 7 days of high-dose prednisolone treatment, after 6 weeks when the prednisolone dosage was approximately 0.5 mg/kg, and after 12 weeks when prednisolone was stopped for most patients (except in 6 UC- and 4 CD patients). The patients still treated with prednisolon after 12 weeks were all well characterized by clinical and/or biochemical relapse during the last tapering period.
Plasma samples from healthy blood donors (n = 38) from the Department of Clinical Immunology, Aalborg Hospital were included as controls. In addition, plasma samples from some of these donors were heat inactivated at 56°C for 30 min and used as negative internal controls.
The blood samples were collected in citrate tubes by venepuncture without stasis, placed on ice for a maximum of 10 min, and centrifuged at 4°C and 2,000 g for 20 min. Duplicate blood samples were collected in glass tubes and allowed to clot at room temperature before centrifuged. Plasma/serum was withdrawn and stored in aliquots of 0.5 ml at minus 80°C.
Plasma/serum samples were analyzed for alternative- and classical C pathway mediated factor C3 activation capacity (C3-AC), mannan-binding lectin (MBL), MBL-C4-AC, leucocyte count, C-Reactive Protein (CRP) and orosomucoid.
The AC-derived measurements were performed at the Surgical Gastroenterological Research Unit whereas the MBL-assay was performed as a routine method at the Department of Clinical Immunology, Aalborg Hospital, as described in details elsewhere [20–22].
C3-AC-assay
The enzyme linked immunosorbent assay (ELISA) measures plasma derived C3b/iC3b deposition on IC during in vitro C activation. The activation capacity of the C system is defined as the amount of C3b/iC3b generated and bound to the solid-phase IC as a function of incubation time. The assay differentiates between activation mediated by AP and CP. Briefly, the plasma was diluted 1/5 in MgEGTA-buffer (10 mM Mg2+ and 10 mM EGTA) when measuring the AP and 1/200 in CaMg-buffer (0.30 mM Ca2+ and 1.0 mM Mg2+) when measuring the CP. The AP is activated at low plasma dilutions only and the CP activation requires Ca2+. Upon C activation, the generated C3b molecules bind covalently to the IC and are eventually degraded to iC3b. Bound C3b/iC3b fragments were detected by the addition of biotinylated rabbit anti-C3c-antibodies (Dako, Glostrup, Denmark), avidin alkaline phosphatase, and para-nitrophenylphosphate as enzyme substrate. In standard dose response curves the absorbance values for plasma dilutions 1/5 (AP) and 1/200 (CP) were designated 100%. Plasma from 1 healthy donor was heat inactivated at 56°C for 30 min, and used as negative control. Test samples were analyzed in duplicate and the mean value was converted to 'per cent of the standard' [20].
A second ELISA measured the plasma concentration of MBL. Briefly, microplates were coated with anti-MBL-antibodies, plasma was diluted 1/100 in dilution-buffer (6.7 g NaCl, 4.6 g NaH2PO4·2H2O, 1.1 g KH2PO4, 5 mM EDTA, 200 μl mouse IgG). Bound MBL were detected by the addition of biotinylated anti-MBL-antibodies, avidin-peroxidase conjugate, and 1,2-phenylene diamine dihydrochloride as enzyme substrate. A plasma standard diluted 1/25-1/3,200 was included in all plates. Plasma from 3 healthy donors with high, moderate and low concentration of MBL were included as controls. Test samples were analyzed in duplicate and the mean value was used in the calculations [21].
The third ELISA measured the plasma derived C4b/iC4b of the MBL-pathway deposited on mannan during in vitro C activation. Briefly, microtiter plates were coated with mannan, and plasma was diluted 1/10 in diluent-buffer (10 mM tris hydroxy aminomethan, 10 mM CaCl2, 1 M NaCl, 15 mM NaN3, pH 7.8). The assay does not measure activation of the AP at the plasma dilution (1/10) used and the CP was not initiated due to the high NaCl-concentration in the diluent buffer (1 M). Bound C4b/iC4b fragments were detected by the addition of biotinylated rabbit anti-C4c-antibodies (Dako, Glostrup, Denmark), avidin alkaline phosphatase, and para-nitrophenylphosphate as enzyme substrate. A plasma standard diluted 1/5-1/100 was included. Test samples were analyzed in duplicate and the mean value was converted to 'per cent of the standard'. Plasma from 2 healthy donors with high and low MBL-C4-AC were included as controls. Plasma from 1 healthy donor was heat inactivated at 56°C for 30 min, and used as negative control [22].
The measurements of leucocyte count, CRP, and orosomucoid were performed by routine methods at the Department of Clinical Chemistry, Aarhus University Hospital.
Ethics
This investigation has been approved by the regional Ethical Committees of Northern Jutland and Aarhus Counties, and wasin accordance with the standards of the Declaration of Helsinki II.
Statistics
Non-parametric descriptive (median – range) and comparative statistics were used with a probality value of < 0.05 considered statistically significant. Data were analyzed by non-parametric methods. The calculations and analyses were performed with Prism 3.0 (GraphPad Software Inc., Microsoft Corp., USA). First, overall comparison over time (before treatment, 1 week, 6 weeks and 12 weeks after treatment) of a variable in the same group of patients (e.g. CD patients) was done by Friedman's two-way analysis. Secondly, paired comparison in a group of patients of a variable between two different time points (matched pair) were by Wilcoxon signed-rank test. Furthermore, the Mann-Whitney U test was used comparing one variable between two different groups at a specific time point (e.g. plasma value of MBL before prednisolone treatment in CD patients versus UC patients). Correlation between two variables were calculated using Spearman's rank correlation coefficient rho. Finally, to allow for multiple comparisons the Bonferroni correction was used, the corrected p-values are stated.