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Articles |
in Serum of Patients with Inflammatory Bowel Disease as Measured by a Highly Sensitive Immuno-PCR
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Division of Laboratory Diagnosis,
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Department of Clinical Laboratory Medicine, and
3
Fourth Department of Internal Medicine, Sapporo Medical University, School of Medicine, Sapporo 060-8543, Japan.
aAddress correspondence to this author at: Department of Clinical Laboratory Medicine, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-Ku, Sapporo 060-8543, Japan. Fax 81-11-622-7502; e-mail watanabn{at}sapmed.ac.jp.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF-) in the pathogenesis of inflammatory bowel disease (IBD) is uncertain. We measured TNF- in serum from IBD patients by immuno-PCR to analyze the relationship between TNF- and pathophysiologic state in IBD.
Methods: Serum samples were collected from 54 healthy blood donors, 29 patients with ulcerative colitis (UC; 46 samples), and 7 patients with Crohn disease (CD; 8 samples). DNA label was generated by PCR amplification using biotinylated primer and was bound with streptavidin to biotinylated third antibody. TNF- sandwiched by antibodies was detected by PCR amplification of the DNA label.
Results: TNF- could be measured in all samples. The median serum concentration in IBD patients overall was 
390-fold higher than in healthy donors (median increase, 380-fold for UC, 640-fold for CD). The median serum TNF- concentration was 1.7-fold higher in the active stage of UC than in the inactive stage (P <0.05), and this difference could be detected in individual patients.
Conclusions: Sensitive measurement of serum TNF- could provide an important pathophysiologic marker for the presence and activity of IBD.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF-) mRNA and protein expression beyond the amounts observed in healthy control subjects (2)(4)(5). Plevy et al. (6) recently found that TNF- augmented production of interferon-
and interleukin-2 in mononuclear cells isolated from intestinal mucosal tissues of healthy subjects. These investigators also found that the treatment of CD patients with anti-TNF- antibodies (cA2) could inhibit production of TNF- and interferon- in mononuclear cells obtained from patients as well as improve the CD activity index and endoscopic findings (6). The same group presented initial evidence that one effect of TNF- in IBD is induction of T-helper 1 cytokines, which are important in the onset and progression of IBD. These observations suggested that accurate measurement of TNF- in serum could provide pathogenetically and clinically important information in IBD (7).
Unfortunately, serum TNF- concentrations, particularly those in healthy individuals, are too low to measure by conventional methods such as ELISA, so the pathophysiologic significance of TNF- in IBD patients remains incompletely understood. We recently established a method for measurement of TNF- in serum using an immuno-PCR, which has a detection limit 5 x 104-fold lower than that of a conventional ELISA. This method can reliably determine TNF- concentrations in serum samples from healthy subjects (8). In this study, we measured serum TNF- concentrations in serum samples from IBD patients by immuno-PCR and correlated these data with other findings; we also compared the concentrations of TNF- in these samples with those in control sera.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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human recombinant tnf- and antibodies
Human recombinant TNF-, mouse anti-human recombinant TNF- monoclonal antibody (mAb), and rabbit anti-human recombinant TNF- polyclonal antibody (pAb) for the ELISA and immuno-PCR assay were generously provided by ASAHI Chemical Industry Co. (Tokyo, Japan).
elisa
ELISA was performed according to our previously described method (8). The absorbance at 492 nm was measured in an EAR400 ELISA reader (SLT Labinstruments GmbH). The detection limit of this ELISA was 50 ng/L, as determined by triplicate measurements and represented by the absorbance at 492 nm.
immuno-pcr
Immuno-PCR was performed according to our previously described method (8). A schematic representation of this method is shown in Fig. 1
. Briefly, appropriately diluted serum was reacted with a mouse mAb (100 mg/L; Fig. 1
, 1st mAb) and rabbit pAb (3 mg/L; Fig. 1
, 2nd pAb). Biotinylated goat anti-rabbit mAb (0.5 mg/L; Biosouse) was then added, and incubation was continued at 25 °C for 90 min. After the plate was washed five times, 0.1 mg/L streptavidin (Chemicon International) was added and incubated at 25 °C for 30 min. The plate was washed five times, and then 0.5 ng/L biotinylated DNA (DNA label) in 1 g/L bovine serum albumin in phosphate-buffered saline containing 1 g/L single-stranded DNA was bound with streptavidin and incubated at 25 °C for 30 min. The DNA label was generated in advance by the method described in our previous report (8).
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The plate was washed and then subjected to PCR using a Gene Amp PCR System 9600-R (Perkin-Elmer Cetus). PCR was carried out under the following reaction conditions: 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3.0 mM MgCl2, 0.2 mM each deoxyribonucleotide, 0.2 mM each forward (5'-AGCGCGCGTAATACGACTC-3') and reverse primer (5'-ACCATGATTACGCCAAGCG-3'), and 1 U of AmpliTaq DNA polymerase in a total volume of 50 µL. The temperature profile was as follows: initial denaturation at 95 °C for 5 min; followed 40 cycles of denaturation at 94 °C for 15 s, annealing at 58 °C for 15 s, and extension at 72 °C for 30 s, with a final extension at 72 °C for 5 min. The 196-bp PCR product was electrophoresed on a 3% agarose gel at 100 V for 80 min. The ethidium bromide-stained gel was scanned immediately with an Eagle Eye II (Stratagene). The detection limit of this immuno-PCR was 5 x 104-fold lower than that of the ELISA for TNF-, as described in our previous report (8). A comparison of the detection ranges of the ELISA and the immuno-PCR is shown in Fig. 2
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measurement of c-reactive protein
The concentration of C-reactive protein (CRP) in sera from patients with IBD was measured by a latex agglutination method using LPIAACE CRP-L reagent (Dia-Iatron Co. Ltd.) in a Hitachi 7350 (Hitachi Industry). The detection limit for CRP was 1 mg/L, and the assay was linear to 300 mg/L.
statistical analysis
The Mann-Whitney U-test was used for comparison of serum TNF- concentrations in healthy donors and IBD patients.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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concentrations in healthy donors and ibd patients concentrations were measured in 54 samples from healthy blood donors and a total of 54 samples from 36 patients with IBD (20 inactive and 26 active UC; 4 inactive and 4 active CD; Fig. 3
). Of the 54 samples from the from the IBD patients, 50 (93%) had TNF- concentrations below the detection limit of the conventional ELISA; the TNF- in these samples was measured by immuno-PCR. Overall, the median concentration of serum TNF- in IBD patients (7.8 ng/L) was 390-fold higher than that in healthy donors (0.02 ng/L). The concentrations in both the UC and the CD patients were significantly higher than those in healthy donors (P <0.001). The median concentrations in UC and CD patients were 380- and 640-fold higher, respectively, than in healthy donors (Table 1
). In addition, UC and CD patients with both active or inactive disease showed significantly higher TNF- concentrations than healthy donors (P <0.001). Furthermore, a significant difference in TNF- concentration was seen between active and inactive cases, especially in UC patients (P <0.05).
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tnf- concentrations in serial samples from patients with active and inactive uc
In six UC patients, serum samples could be obtained during both the inactive and the active stages. In five of six patients, serum TNF- in the active stage was notably higher than in the inactive stage (Fig. 4
). However, TNF- concentrations in four of these five active-stage samples remained below the detection limit of the ELISA.
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correlation between tnf- and crp in ibd patients
As a general comparison of TNF- with other molecules related to inflammation, serum TNF- concentrations were considered together with CRP concentration in our IBD patients (Table 2
). All of the IBD patients showed an increased TNF- concentration, whereas CRP concentrations remained below the detection limit in some cases. This was especially notable in UC patients, whose CRP concentrations were below the detection limit in 37 of 46 cases (80%).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in IBD is poorly understood because of difficulties in measuring changes in the serum concentrations typically present in IBD. The present study is the first to report that the serum concentration of TNF- in IBD patients was strikingly higher than in controls (390-fold higher). A highly sensitive immuno-PCR assay was used because the relative insensitivity of the conventional serum assay methods used previously greatly impeded measurement of this cytokine in IBD. One group has reported that serum TNF- concentrations in IBD patients often were below the detection limit of their assay (9). Other investigators were able to measure serum TNF- concentrations in some IBD patients, but a portion of the samples showed concentrations below the detection limit (10)(11). In our IBD samples, 50 of 54 sera (93%) had a TNF- concentration below the detection limit of conventional ELISA, but all of our samples could be measured by immuno-PCR.
Whether serum TNF- concentrations differ between UC and CD has not been known. In this study, the median concentrations of serum TNF- in UC and CD were 380- and 640-fold higher, respectively, than those in healthy donors. Murch et al. (4) previously detected TNF- protein in mucosal tissues obtained from UC and CD patients by immunohistochemistry and ELISA, demonstrating that TNF--positive cells were more numerous in submucosa and lamina propria from CD patients than in specimens from UC patients. Dionne et al. (2) examined TNF- mRNA expression in mucosal biopsy specimens from patients with IBD and found that TNF- mRNA expression was higher in CD patients than in UC patients. In addition, a recent study has suggested that T-helper 1 cytokines induced by TNF- are important in the pathogenesis of IBD, especially CD (6). The distinctly larger increases in TNF- found in our CD sera support these previous findings.
Earlier studies investigated the relationship between serum TNF- concentrations and IBD disease activities using ELISA and RIA (9)(10). However, the degree to which the serum TNF- concentration decreases in the inactive stage of disease remained unclear. In the present study, serum TNF- concentrations in the active stage were apparently higher than those in the inactive stage, and there was a significant difference between the active and inactive stages in UC patients. Fortunately, we had sera obtained from six patients with UC in both the active and inactive stages to measure TNF-. A difference in serum TNF- concentration was evident between the inactive and active stages in almost all of these serially sampled patients; this difference could not be detected by conventional ELISA. Furthermore, the TNF- concentrations in the inactive stages of all cases remained higher than that in the healthy subjects. These results suggest that TNF- could aid diagnosis of disease and assessment of activity in individual patients with IBD.
CRP is well known as a general inflammatory marker and clinical indicator in IBD. We compared the frequency with which serum TNF- and CRP concentrations were increased in UC and CD. Of the UC and CD samples without detectable CRP, 80% and 25%, respectively, showed increased TNF- concentrations. Schreiber et al. (12) recently reported that acute relapse within 1 year could be predicted by increased secretion of TNF- as measured by ELISA, but not by increased CRP. Our sensitive assay may further enhance this predictive ability and shed light on pathogenesis.
In conclusion, our study indicated that the TNF- concentration could be measured by immuno-PCR in all serum samples from IBD patients. This information may be highly informative concerning the course of illness in individual patients as well as for fundamental understanding of IBD.
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Footnotes |
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, tumor necrosis factor-; mAb, monoclonal antibody; pAb, polyclonal antibody; and CRP, C-reactive protein. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and ELISA (•).
