Genomic alterations accompanying tumour evolution in colorectal cancer: tracking the differences between primary tumours and synchronous liver metastases by whole-exome sequencing.

BACKGROUND: Colorectal cancer (CRC) patients with metastatic disease can become cured if neoadjuvant treatment can enable a resection. The search for predictive biomarkers is often performed on primary tumours tissue. In order to assess the effectiveness of tailored treatment in regard to the primary tumour the differences in the genomic profile needs to be clarified. METHODS: Fresh-frozen tissue from primary tumours, synchronous liver metastases and adjacent normal liver was collected from 21 patients and analysed by whole-exome sequencing on the Illumina HiSeq 2500 platform. Gene variants designated as 'damaging' or 'potentially damaging' by Ingenuity software were used for the subsequent comparative analysis. BAM files were used as the input for the analysis of CNAs using NEXUS software. RESULTS: Shared mutations between the primary tumours and the synchronous liver metastases varied from 50 to 96%. Mutations in APC, KRAS, NRAS, TP53 or BRAF were concordant between the primary tumours and the metastases. Among the private mutations were well-known driver genes such as PIK3CA and SMAD4. The number of mutations was significantly higher in patients with right- compared to left-sided tumours (102 vs. 66, p = 0.004). Furthermore, right- compared to left-sided tumours had a significantly higher frequency of private mutations (p = 0.023). Similarly, CNAs differed between the primary tumours and the metastases. The difference was mostly comprised of numerical and segmental aberrations. However, novel CNAs were rarely observed in specific CRC-relevant genes. CONCLUSION: The examined primary colorectal tumours and synchronous liver metastases had multiple private mutations, indicating a high degree of inter-tumour heterogeneity in the individual patient. Moreover, the acquirement of novel CNAs from primary tumours to metastases substantiates the need for genomic profiling of metastases in order to tailor metastatic CRC therapies. As for the mutational status of the KRAS, NRAS and BRAF genes, no discordance was observed between the primary tumours and the metastases.

Danish Prostate Cancer Registry - methodology and early results from a novel national database.

BACKGROUND: Systematized Nomenclature of Medicine (SNOMED) codes are computer-processable medical terms used to describe histopathological evaluations. SNOMED codes are not readily usable for analysis. We invented an algorithm that converts prostate SNOMED codes into an analyzable format. We present the methodology and early results from a new national Danish prostate database containing clinical data from all males who had evaluation of prostate tissue from 1995 to 2011. MATERIALS AND METHODS: SNOMED codes were retrieved from the Danish Pathology Register. A total of 26,295 combinations of SNOMED codes were identified. A computer algorithm was developed to transcode SNOMED codes into an analyzable format including procedure (eg, biopsy, transurethral resection, etc), diagnosis, and date of diagnosis. For validation, ~55,000 pathological reports were manually reviewed. Prostate-specific antigen, vital status, causes of death, and tumor-node-metastasis classification were integrated from national registries. RESULTS: Of the 161,525 specimens from 113,801 males identified, 83,379 (51.6%) were sets of prostate biopsies, 56,118 (34.7%) were transurethral/transvesical resections of the prostate (TUR-Ps), and the remaining 22,028 (13.6%) specimens were derived from radical prostatectomies, bladder interventions, etc. A total of 48,078 (42.2%) males had histopathologically verified prostate cancer, and of these, 78.8% and 16.8% were diagnosed on prostate biopsies and TUR-Ps, respectively. FUTURE PERSPECTIVES: A validated algorithm was successfully developed to convert complex prostate SNOMED codes into clinical useful data. A unique database, including males with both normal and cancerous histopathological data, was created to form the most comprehensive national prostate database to date. Potentially, our algorithm can be used for conversion of other SNOMED data and is available upon request.

Inflammation and uPAR-Expression in Colorectal Liver Metastases in Relation to Growth Pattern and Neo-adjuvant Therapy.

Proteolytic activity and inflammation in the tumour microenvironment affects cancer progression. In colorectal cancer (CRC) liver metastases it has been observed that three different immune profiles are present, as well as proteolytic activity, determined by the expression of urokinase-type plasminogen activator (uPAR).The main objectives of this study were to investigate uPAR expression and the density of macrophages (CD68) and T cells (CD3) as markers of inflammation in resected CRC liver metastases, where patients were neo-adjuvantly treated with chemotherapy with or without the angiogenesis inhibitor bevacizumab. Chemonaive patients served as a control group. The markers were correlated to growth patterns (GP) of liver metastases, i.e. desmoplastic, pushing and replacement GP. It was hypothesised that differences in proteolysis and inflammation could reflect tumour specific growth and therapy related changes in the tumour microenvironment. In chemonaive patients, a significantly higher level of uPAR was observed in desmoplastic liver metastases in comparison to pushing GP (p = 0.01) or replacement GP (p = 0.03). A significantly higher density of CD68 was observed in liver metastases with replacement GP in comparison to those with pushing GP (p = 0.01). In liver metastases from chemo treated patients, CD68 density was significantly higher in desmoplastic GP in comparison to pushing GP (p = 0.03). In chemo and bevacizumab treated patients only a significant lower CD3 expression was observed in liver metastases with a mixed GP than in those with desmoplastic (p = 0.01) or pushing GP (p = 0.05). Expression of uPAR and the density of macrophages at the tumour margin of liver metastasis differ between GP in the untreated patients. A higher density of T cells was observed in the bevacizumab treated patients, when desmoplastic and pushing metastases were compared to liver metastases with a mix of the GP respectively, however no specific correlations between the immune markers of macrophages and T cells or GP of liver metastases could be demonstrated.

Growth pattern of colorectal liver metastasis as a marker of recurrence risk.

Despite improved therapy of advanced colorectal cancer, the median overall survival (OS) is still low. A surgical removal has significantly improved survival, if lesions are entirely removed. The purpose of this retrospective explorative study was to evaluate the prognostic value of histological growth patterns (GP) in chemonaive and patients receiving neo-adjuvant therapy. Two-hundred-fifty-four patients who underwent liver resection of colorectal liver metastases between 2007 and 2011 were included in the study. Clinicopathological data and information on neo-adjuvant treatment were retrieved from patient and pathology records. Histological GP were evaluated and related to recurrence free and OS. Kaplan-Meier curves, log-rank test and Cox regression analysis were used. The 5-year OS was 41.8% (95% CI 33.8-49.8%). Growth pattern evaluation of the largest liver metastasis was possible in 224 cases, with the following distribution: desmoplastic 63 patients (28.1%); pushing 77 patients (34.4%); replacement 28 patients (12.5%); mixed 56 patients (25.0%). The Kaplan-Meier analyses demonstrated that patients resected for liver metastases with desmoplastic growth pattern had a longer recurrence free survival (RFS) than patients resected for non-desmoplastic liver metastases (p=0.05). When patients were stratified according to neo-adjuvant treatment in the multivariate Cox regression model, hazard ratios for RFS compared to desmoplastic were: pushing (HR=1.37, 95% CI 0.93-2.02, p=0.116), replacement (HR=2.16, 95% CI 1.29-3.62, p=0.003) and mixed (HR=1.70, 95% CI 1.12-2.59, p=0.013). This was true for chemonaive patients as well as for patients who received neo-adjuvant treatment.

Histologic and immunohistochemical classification of 41 bovine adrenal gland neoplasms.

Tumors of the adrenal glands are among the most frequent tumors in cattle; however, few studies have been conducted to describe their characteristics. The aim of this study was to classify 41 bovine adrenal neoplasms from 40 animals based on macroscopic and histologic examination, including electron microscopy and immunohistochemistry for melan A, synaptophysin, chromogranin A, vimentin, pan-cytokeratin, 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase), and Ki-67. The tumors were classified as 23 adrenocortical adenomas, 12 adrenocortical carcinomas, 2 schwannomas, 2 pheochromocytomas (1 malignant), and 1 ganglioneuroma. Five histologic features were characteristic of metastasizing adrenocortical tumors: invasion of the capsule, vascular invasion, diffuse growth pattern, spindle-cell morphology, and nuclear pleomorphism. Adrenocortical tumors with at least 3 of these features were classified as malignant. Immunohistochemically, adrenocortical tumors expressed melan A (16/19), vimentin (14/26), cytokeratin (11/26), and chromogranin A (9/27), whereas pheochromocytomas expressed chromogranin A (2/2), synaptophysin (2/2), and vimentin (1/2). Both schwannomas expressed CNPase. An immunohistochemistry panel consisting of antibodies against melan A, synaptophysin, and CNPase was considered most useful to classify bovine adrenal tumors. However, the distinction between benign and malignant adrenocortical tumors was based on histologic features as in human medicine.

CD4⁺ and CD8⁺ regulatory T cells (Tregs) are elevated and display an active phenotype in patients with chronic HCV mono-infection and HIV/HCV co-infection.

The aim of this study was to examine regulatory T cells (Tregs) in peripheral blood and liver tissue in patients with chronic hepatitis C virus (HCV) mono-infection and in patients with HIV/HCV co-infection. In a cross-sectional study were included 51 patients with chronic HCV infection, 24 patients with HIV/HCV co-infection and 24 healthy individuals. CD4⁺ and CD8⁺ Tregs were determined using flow cytometry. Fibrosis was examined by transient elastography. Inflammation, fibrosis and Tregs were determined in liver biopsies from 12 patients. Increased frequency of CD4⁺ and CD8⁺ Tregs was found in HIV/HCV co-infected patients [median: 6.4% (IQR: 5.7-6.9) and 1.0% (0.7-1.2), respectively] compared to HCV mono-infected patients [5.6% (4.2-6.3), P = 0.01 and 0.5% (0.3-0.7), P < 0.001, respectively]. Furthermore, HCV mono-infected patients had increased frequencies of Tregs compared with healthy controls (P < 0.05). However, no associations between the frequency of Tregs and fibrosis were found. Furthermore, characterization of CD4⁺ Tregs using CD45RA demonstrated a higher frequency of activated Tregs in both HCV mono-infected and HIV/HCV co-infected patients compared with healthy controls. Finally, number of intrahepatic Tregs was associated with both peripheral CD8⁺ Tregs and intrahepatic inflammation. In conclusion, HCV mono-infected patients and particularly HIV/HCV co-infected patients have increased the frequency of CD4⁺ and CD8⁺ Tregs compared with healthy controls. Furthermore, CD4⁺ Tregs in infected patients displayed an active phenotype. Tregs were not associated with fibrosis, but a positive correlation between intrahepatic Tregs and inflammation was found. Taken together, these results suggest a role for Tregs in the pathogenesis of chronic HCV infection.

Effects of a recombinant FVIIa analogue, NN1731, on blood loss and survival after liver trauma in the pig.

BACKGROUND: We considered whether haemorrhage after a liver trauma would be reduced by early administration of a pro-haemostatic agent and evaluated the effect of i.v. vs i.m. administration of the coagulation factor VIIa analogue NN1731 on haemorrhage after a liver trauma in the pig. METHODS: The pharmacokinetics of i.v. and i.m. NN1731 was evaluated in eight minipigs, and the effects of dose and administration route of NN1731 (i.v. 180 microg kg(-1), n=6; i.m. 540 microg kg(-1), n=4, or 2000 microg kg(-1), n=6) vs vehicle (n=16) were studied on a liver laceration injury in pigs. To simulate a pre-hospital setting, the administration of NN1731 was delayed by 1 min for i.m. administration and 7 min for i.v. administration, at which time fluid resuscitation also began. RESULTS: In the minipigs, NN1731 exposure was similar after i.v. 180 microg kg(-1) and i.m. 540 microg kg(-1), with a bioavailability of approximately 35%. The injury and blood loss at 7 min was comparable between the four groups of pigs; however, after 60 min, the blood loss was lower in the i.v. treated animals: 1.3 (0.3) (i.v.) vs 2.2 (0.8) litres (i.m.(540), i.m.(2000), and vehicle) (P<0.001). Also, the survival time was increased: 117 (14) (i.v.) vs 84 (28) min (i.m.(540), i.m.(2000), and vehicle) (P<0.001). CONCLUSIONS: After a liver trauma in the pig, i.v. administration of NN1731 reduced the bleeding and increased the survival time. In contrast, i.m. administration had no effect, presumably because reduced muscle perfusion during haemorrhage reduced the uptake of NN1731.

EBV-associated gastric carcinoma in high- and low-incidence areas for nasopharyngeal carcinoma.

BACKGROUND: Approximately 10% of gastric carcinomas are associated with Epstein-Barr virus (EBV). The Inuit in Greenland have a high incidence of EBV-associated nasopharyngeal carcinoma. METHODS: We conducted a population-based case-control study comparing gastric carcinomas in Greenland and in Denmark. RESULTS: The prevalence rate of EBV-associated gastric carcinomas was 8.5% in both populations. CONCLUSION: The findings of this study argue against a general susceptibility to EBV-associated carcinomas among the Inuit.

MTHFR polymorphisms and 5-FU-based adjuvant chemotherapy in colorectal cancer.

BACKGROUND: Methylenetetrahydrofolate reductase is a pivotal enzyme in folate metabolism and 5-fluorouracil (5-FU) cytotoxicity. Two common single-nucleotide polymorphisms (SNPs), MTHFR 677C>T (rs1801133) and 1298A>C (rs1801131), reduce enzyme activity. Initially, these SNPs were claimed to predict clinical efficacy, but further studies have yielded contradictory results. We tested whether these two polymorphisms are determinants of clinical outcome in a large patient group with a long follow-up time. PATIENTS AND METHODS: We included 331 patients who had been treated with adjuvant 5-FU/leucovorin chemotherapy after intended curative resection between 1997 and 2003. Clinical data, including relapse rates, overall survival, and tumor stage, were collected. DNA was extracted from formalin-fixed tumor tissue and analyzed for the MTHFR 677C>T and 1298A>C SNPs with real-time PCR. RESULTS: The MTHFR 677C>T and 1298A>C polymorphisms were not associated with survival or relapse-free survival (P > 0.2). The 677 CC genotype was associated to toxicity (odds ratio = 1.83, P = 0.01). CONCLUSIONS: The MTHFR 677C>T and 1298A>C polymorphisms probably do not predict efficacy of adjuvant 5-FU treatment in colorectal cancer after complete resection; however, the 677C>T polymorphism may be associated with lower toxicity in 5-FU treatment. Implementation of SNP analysis for these polymorphisms for individualized treatment is premature.

Juvenile nasopharyngeal angiofibromas in Denmark 1981-2003: diagnosis, incidence, and treatment.

CONCLUSIONS: Juvenile nasopharyngeal angiofibroma (JNA) is a rare tumor in young males, with a non-negligible potential for recurrence. Preoperative embolization is a safe procedure that diminishes the peroperative blood loss and the need for blood transfusion. The endoscopic approach was used with good results in JNA stage I and II (Chandler). OBJECTIVES: To estimate the incidence rate of JNA in the Danish population and to describe symptoms and treatment. PATIENTS AND METHODS: This was a national retrospective cohort study. All cases of JNA diagnosed in Denmark from 1981 to 2003 were identified. Data were extracted from medical records. RESULTS: Forty-five male (no female) JNA cases were identified. In 43 cases, clinical data were recovered. Median age was 15 years. The incidence rate in Denmark was 0.4 cases per million inhabitants per year and 3.7 cases per million males (aged 10-24) per year. All patients underwent surgery, and the endoscopic approach was increasingly being used. The embolization procedure proved to be safe and decreased the intraoperative blood loss statistically to 650 ml in the embolized group from an average of 1200 ml in the non-embolized group (p<0.05). Similarly, the need for peroperative blood transfusion was reduced (p<0.005). The primary recurrence rate was 23% and no patients died.

Detection rate of periintestinal lymph nodes.

AIM: Compared to standard two-dimensional (2D) endosonography, three-dimensional (3D) endosonography has been presented as a possible improvement regarding imaging of the gastrointestinal (G-I) tract and detection of metastatic lymph nodes. The aim of this study was to evaluate the efficacy of detecting periintestinal lymph nodes in surgical specimens using 3D endosonography. PATIENTS AND METHODS: Surgical specimens from 31 patients with malignant G-I tumours were investigated by 3-D endosonography and histology with focus on the presence of periintestinal lymph nodes and presence of metastasis. The specimens were scanned submerged into water. Position and size of the lymph nodes were mapped on a photo of the specimen both by the pathologist and the examiners. RESULTS: Three-dimensional endosonography detected 48 out of 60 malignant lymph nodes (80.0 %), and 110 out of 219 benign lymph nodes (50.2 %). The positive predictive value for an endosonographic finding interpreted as a lymph node was 0.97. CONCLUSION: The detection rates for periintestinal lymph nodes were relatively high and seemed superior to the one usually assigned to 2D endosonography. Although distinguishing between metastatic and non-metastatic lymph nodes remains a problem, all patients with histologically confirmed metastasis to lymph nodes were detected by 3D endosonography, and the technique thus seems suitable for grouping of patients prior to surgery.

Intercellular adhesion molecule-1 (ICAM-1) in ulcerative colitis: presence, visualization, and significance.

The intensive research of recent years has suggested that the cause of ulcerative colitis (UC) involves a genetic predisposition to an uncontrolled or unbalanced immune response to luminal or epithelial antigens or against other external factors. Intercellular adhesion molecule-1 (ICAM-1) is pivotal for the influx of neutrophil granulocytes into colonic mucosa, and gene analyses have found polymorphisms in the gene encoding ICAM-1, indicating that changes in ICAM-1 function may be involved in the pathogenesis of UC. Clinical trials of the ICAM-1 antisense oligonucleotide Alicaforsen, which inhibits the synthesis of ICAM-1, have shown positive results in the treatment of patients with left-sided (distal) UC. In addition to emphasizing the central role of ICAM-1 in active stages of UC, the results provide hope for the development and introduction of a more specific and efficient treatment for UC than those currently available. This review discusses the results from studies on the expression of ICAM-1 in colonic tissue from patients with UC.

Spontaneous aggregation of leukocytes in active ulcerative colitis might be ICAM-1 dependent.

OBJECTIVE: In active stages of ulcerative colitis (UC), a tendency for neutrophils to aggregate in the colonic lamina propria is mediated by yet unidentified surface receptors. The aim was to assess the spontaneous leukocyte aggregation and the aggregation induced by bacteria-derived products in UC and to evaluate the involvement of ICAM-1 and beta(2)-integrins in this aggregation. MATERIALS AND METHODS: Blood was drawn from 10 patients with quiescent UC, 10 patients with active UC, and 10 healthy volunteers. The blood was stimulated with LPS or fMLP with subsequent blocking of CD11b or ICAM-1 with specific antibodies. The aggregation was assessed on glass slides with an automated image analyzer (Inflamet). RESULTS: The spontaneous leukocyte aggregation was increased in quiescent and active UC as compared to healthy controls (p < 0.05). Although not statistically significant, LPS and fMLP seemed to increase the leukocyte adhesiveness, and also a tendency towards inhibition of the leukocyte aggregation was observed by blocking ICAM-1. CONCLUSIONS: Increased adhesiveness of circulating leukocytes seems to be involved in the pathogenesis of UC, and ICAM-1 is suggested to be a part of this phenomenon. The results indicate an altered basic neutrophil response in UC.

Correlation between circulating soluble ICAM-1 and prednisolone-induced amelioration of ulcerative colitis.

BACKGROUND: A soluble form of intercellular adhesion molecule-1 (sICAM-1) shed from endothelial cells is present in the circulation. Whether the circulating molecules represent passive turnover of surface ICAM-1 or may have some active functions in the inflammatory process is unknown. Glucocorticoids (e.g. prednisolone) are cornerstones in the treatment of acute exacerbations of ulcerative colitis (UC), and influence of the leucocyte/endothelial interaction appears to be part of their mode of action. The aim of the present study was therefore to evaluate the ICAM-1-shedding through measurements of sICAM-1 concentrations during prednisolone treatment of UC patients. METHODS: Prednisolone (40 mg) was prescribed to 15 patients with severe disease activity. At inclusion, and after 2 weeks of treatment, plasma sICAM-1 levels were measured using the ELISA technique. RESULTS: The concentrations of sICAM-1 were significantly decreased during treatment from median 256.2 (ng/ml) (interquartile range 239.7-321.0 ng/ml) to 220.4 ng/ml (196.0-276.3 ng/ml) (P < 0.01). This reduction correlated with a decrease in disease activity (r(s) = 0.8; P < 0.003). CONCLUSIONS: sICAM-1 seems to be a poor diagnostic tool, but since plasma sICAM-1 concentrations decreased during the treatment period, it might prove to be applicable as an activity marker in the individual patient.

Upregulation of interleukin-12 and -17 in active inflammatory bowel disease.

BACKGROUND: Cytokines are essential mediators of the intestinal inflammation during active episodes of inflammatory bowel disease (IBD). Interleukin (IL)-12 and IL-17 are potent immunoregulatory cytokines whose roles in the pathogenesis of IBD are unknown. The aim of this study was to evaluate the colonic expression of IL-12 and IL-17 genes in IBD. METHODS: Fifty-one patients (22 with ulcerative colitis (UC), 17 with Crohn disease (CD), and 12 controls) who underwent colonoscopy were included. IBD disease activity was determined using a clinical grading scale. The degree of inflammation, as well as the content of CD4+ T cells (synthesizing IL-17) and CD68+ macrophages (synthesizing IL-12) in colonic biopsies, was determined. The amounts of IL-12 and IL-17 mRNA were assessed by RT-PCR, using GAPDH as an internal standard. RESULTS: In colonic specimens, IL-17 mRNA expression was increased in moderately and severely active UC (P = 0.03) and in all degrees of activity in CD (P < 0.04). Levels of IL-12 mRNA were upregulated in both active UC and active CD compared to controls (P < 0.02). In cases of remission, IL-12 mRNA expression was similar to that found in control samples. Compared to controls, histological examination showed significant differences in signs of chronic and acute inflammation in UC (P < 0.01) and CD (P < 0.02), revealing a high correlation between clinical disease activity and histological scoring (r2 = 0.92, P < 0.005). Whereas CD4+ T cells were observed in lymphocyte aggregates located profound in the lamina propria, CD68+ macrophages were primarily found just underneath the surface epithelium. The density of CD4+ and CD68+ cells correlated significantly with the amounts of IL-17 and IL-12 mRNA, respectively (P < 0.05). CONCLUSION: The expression of both IL-12 and IL-17 mRNA is induced in active UC and CD and may thus be involved in sustaining the intestinal inflammation in IBD. Inhibition of IL-12 or IL-17 might be future therapeutic targets in IBD.

Hidrosadenoma of the anal canal: a case report with review of the literature.

Hidrosadenoma of the anal canal is an extremely rare tumour. Only nine cases with similar histologic structure have been described in the literature, most representing tumours resected from the anal or rectal mucosa. We present a case of anal hidrosadenoma with immunohistochemical staining features identifying it as a true sweat gland tumour.

Review article: the treatment of inflammatory bowel disease with 6-mercaptopurine or azathioprine.

The thioguanine derivative, azathioprine, is a prodrug of 6-mercaptopurine that is further metabolized by various enzymes present in the liver and gut. Azathioprine and 6-mercaptopurine have been used in the treatment of inflammatory bowel disease, i.e. ulcerative colitis and Crohn's disease, for more than 30 years. However, widespread use of azathioprine or 6-mercaptopurine in inflammatory bowel disease is of more recent origin, the primary reason being a long-standing debate on the efficacy of these agents in inflammatory bowel disease. Both drugs are slow acting, which is why clinical efficacy cannot be expected until several weeks or even months of treatment have elapsed. Consequently, azathioprine and 6-mercaptopurine have no place as monotherapy in the treatment of acute relapsing inflammatory bowel disease. Today, azathioprine and 6-mercaptopurine are the most commonly used immunomodulatory drugs in the treatment of inflammatory bowel disease. Their clinical effects are probably identical, although their exact mode of action is still unknown. The mode of action of azathioprine is thought to be multifactorial, including conversion to 6-mercaptopurine (which acts as a purine antimetabolite), possible blockade of thiol groups by alkylation, inhibition of several pathways in nucleic acid biosynthesis (preventing proliferation of cells involved in the determination and amplification of the immune response) and damage to DNA through the incorporation of thiopurine analogues. However, 6-thioguanine nucleotides may accumulate in toxic doses in myeloid precursor cells, resulting in life-threatening myelosuppression. Azathioprine and 6-mercaptopurine are further known to alter lymphocyte function, reduce the number of lamina propria plasma cells and affect natural killer cell function. The purpose of this comprehensive review is to suggest guidelines for the application of azathioprine and 6-mercaptopurine in the treatment of inflammatory bowel disease.

Impaired sensitivity to beta 2 integrin-blocking in ICAM-1-mediated neutrophil migration in ulcerative colitis.

BACKGROUND: Factors influencing the directed migration of neutrophils into colonic tissue in ulcerative colitis (UC) are poorly described. ICAM-1 has recently been shown to possess chemotactic properties, and the aim of this study was to evaluate the involvement of beta 2 integrins in this ICAM-1-mediated migration. METHODS: The chemotactic effect of ICAM-1 on neutrophils isolated from 13 UC patients and 17 healthy volunteers was studied in microchemotaxis chambers. Physiological concentrations of ICAM-1 (0.05-500 pM) were separated from neutrophils by nitrocellulose filters, and cell migration was evaluated using the leading front technique. beta 2 integrins on neutrophils were blocked with antibodies to CD11a, CD11b, CD11c and CD18, and migration towards ICAM-1 was examined. RESULTS: Migration towards ICAM-1 was equal for UC and control neutrophils, showing a bell-shaped ICAM-1 dosemigratory response curve with peak migration at 5 pM ICAM-1 (30.0 microns; interquartile range 22.9-35.7; P < 0.001). Blockade of the CD11 subunits on control cells inhibited the chemoattractant effect of ICAM-1 by 43.6%-58.0%, whereas the migration was decreased by only 20% in UC under similar blocking conditions (P < 0.01). Anti-CD18 mAbs had no effect. Inhibition of protein kinases with staurosporin only slightly decreased the ICAM-1-mediated migration, whereas incubation with staurosporin and CD11 antibodies showed additive effects on UC neutrophils and synergistic effects on control cells. No quantitative differences in beta 2 integrin expression were detected between control and UC neutrophils. CONCLUSIONS: The chemotactic property of ICAM-1 was shown to be CD11-dependent and UC neutrophils were found to be less dependent on CD11/ICAM-1-mediated migration than were control neutrophils.

Colonic expression and synthesis of interleukin 13 and interleukin 15 in inflammatory bowel disease.

UNLABELLED: A dysregulated local immune reaction with unbalanced cytokine expression seems essential in inflammatory bowel disease (IBD), i.e. ulcerative colitis (UC) and Crohn's disease (CD). Since the roles of interleukin (IL-)13 and IL-15 remain unclear, this study aimed at studying intestinal expression of IL-13 and IL-15 in IBD. METHODS: In colonic biopsies from 24 UC, 18 CD, and 12 controls IL-13 and IL-15 were measured using ELISA, and their gene expressions were assessed by RT-PCR. Leukocytes were visualised histochemically. RESULTS: Concentrations of IL-13 were decreased in UC (median 56 pg/mg tissue; interquartile range 30-99 pg/mg) compared to CD (82 pg/mg tissue; 41-122;P=0.004) and controls (83 pg/mg tissue; 18-134;P>0.05), and lower in active UC (53 pg/mg tissue; 33-96) than in inactive UC (80 pg/mg tissue; 65-99;P=0.02). IL-15 concentrations were higher in CD patients (34 pg/mg tissue; 24-53) as compared to controls (20 pg/mg tissue; 15-21;P=0.001) whilst being 22 pg/mg tissue (15-32) in UC. IL-13 mRNA and IL-15 mRNA were detected in 20% and 15%, respectively. Infiltration of leukocytes correlated inversely with IL-13 levels (P=0.02). CONCLUSION: Active UC is associated with decreased colonic IL-13 suggesting that IL-13 levels are diminished as a part of UC exacerbations, or that exacerbations follow active downregulation of IL-13.