Albert Roessner

Inhibition of p38 MAP kinase- and RICK/NF-κB-signaling suppresses inflammatory bowel disease

Ulcerative colitis and Crohns disease are the two entities of chronic inflammatory bowel diseases (IBD). One of the main pathogenic mechanisms is probably a dysregulated immune response triggered by products of the enteric bacterial flora. The goal of this study was to evaluate the effects of the p38 mitogen‐activated protein kinase (MAPK) inhibitor SB203580 on inflammatory responses using the DSS‐induced experimental colitis model in mice reflecting human IBD. We found that SB203580 improved the clinical score, ameliorates the histological alterations, and reduces the mRNA levels of proinflammatory cytokines. In addition to p38 kinase activity, the “classical” and the “alternative” NF‐κB pathways were also strongly activated during colitis induction. All three pathways were drastically down‐regulated by SB203580 treatment. An analysis of the molecular basis of NF‐κB activation revealed that Rip‐like interacting caspase‐like apoptosis‐regulatory protein kinase (RICK), a key component of a pathway leading to NF‐κB induction, is also strongly inhibited by SB203580. Since RICK is an effector kinase of NOD2, an intracellular receptor of bacterial peptidoglycan, these results support the notion that NOD signaling could play a pivotal role in the IBD pathogenesis. Thus, RICK could represent a novel target for future therapies in human IBD.

High Prognostic Value of p16INK4 Alterations in Gastrointestinal Stromal Tumors

PURPOSE Gastrointestinal stromal tumors (GISTs) represent a distinctive (but histologically heterogeneous) group of neoplasms, the malignant potential of which is often uncertain. To determine the prognostic relevance of p16INK4 alterations in GISTs, we investigated a larger group of GISTs and correlated the genetic findings with clinicopathological factors and patient survival. MATERIAL AND METHODS We evaluated the methylation status of the promotor by methylation-specific polymerase chain reaction (PCR), the presence of mutations by PCR-SSCP-sequencing, the loss of heterozygosity at the p16INK4 locus (using the c5.1 marker), and the immunohistochemical expression of p16INK4 protein in 43 GISTs in 39 patients. RESULTS p16INK4 alterations were found in 25 of 43 GISTs (58.1%), with benign, borderline, or malignant GISTs showing no differences in the type and frequency of alteration. p16INK4 alterations were correlated with a loss of p16INK4 protein expression (P <.01). Patients who had tumors with p16INK4 alterations had a poorer prognosis than patients with tumors without such alterations (P =.02). There was a high predictive value for p16INK4 alterations only in the group of benign and borderline GISTs (P <.01) with regard to clinical outcome. Univariate Coxs proportional hazard regression analysis revealed a strong correlation between p16INK4 alterations, tumor size, mitotic index, and overall survival (P <.02), whereas multivariate Coxs analysis confirmed only p16INK4 alterations as an independent prognostic factor. CONCLUSION We believe that the evaluation of p16INK4 alteration status is a helpful prognosticator, particularly in the benign and borderline groups of GISTs.

Determinants and consequences of atrial fibrosis in patients undergoing open heart surgery

OBJECTIVE Atrial fibrillation (AF) is a frequent complication following open-heart surgery (OHS). Increased atrial fibrosis may indicate the presence of an intrinsic arrhythmogenic substrate. The aim of this prospective study was to determine whether atrial fibrosis is associated with increased prevalence of AF after OHS. METHODS Right atrial appendages were obtained from 259 patients undergoing OHS; none of the patients had a history of AF. Atrial fibrosis was quantitatively analyzed with point counting. All patients were followed prospectively until hospital discharge. None of the patients received anti-arrhythmic prophylaxis. Post-operative AF was defined as an episode of AF lasting > or = 5 min. RESULTS Quantitation of atrial fibrosis yielded a mean volume percentage of 15.8 +/- 4.3% (V%; range 4.6-32.4%). Patient age was found to correlate with the amount of atrial fibrosis (r = 0.165; P < 0.01) and surface P-wave duration (r = 0.249; P < 0.01). The degree of fibrosis combined with P-wave duration predicted post-operative AF (P < 0.01). Age (> 60 years) and P-wave duration (> or = 100 ms) were independent predictors of post-operative AF (age: relative risk 2.20; P-wave: relative risk 2.69; P < 0.05). The patients were divided into three groups: group 1, V% = 4.6-13.8%; group 2, V% = 13.9-23.1%; group 3, V% = 23.2-32.4%. A total of 52 patients (20.1%) developed AF, which occurred least commonly in group 1 (16.3%) and group 2 (21.2%) as compared with group 3 (33.3%). CONCLUSIONS Atrial fibrosis provides a pathophysiological substrate for post-operative AF. The results support the importance of P-wave duration as a predictor of post-operative AF, and explain the increased prevalence of AF in elderly patients after OHS.

Differential pattern of DNA-Aneuploidy in human malignancies

The differential pattern of DNA-aneuploidy, detected by flow cytometry (FCM) regarding its frequency, grade and multiclonality, was investigated and correlated to tumor type, malignancy grade, tumor stage and prognosis in a multi-institutional study at the University of Münster. High resolution measurements using admixed normal blood reference cells were undertaken in 2413 cases of 13 different malignant diseases and in 776 benign lesions or samples. The incidence of DNA-aneuploidy was highest in melanomas, carcinomas, testicular tumors, sarcomas (75%-95%) and myelomas (65%). Acute leukemias showed an intermediate DNA-aneuploidy rate of 40% with special subgroups represented by common ALL (44%), p less than 0.05) and myelomonocytic/monocytic AML (47%, p less than 0.01). The lowest DNA-aneuploidy-rate was found in basal cell skin carcinomas (19%) and congenital melanocytic nevi (9%). No case of DNA-aneuploidy was observed in the 776 benign lesions or samples.--DNA-indices giving the grade of DNA-aneuploidy with 1.0 for normal diploid G1/0 cells were found distributed predominantly between 1.0 and 2.0 in the solid tumors, except testicular tumors, clustering around a triploid maximum at 1.5. DNA-indices of myelomas and acute leukemias generally ranged below 1.25 with lower DNA-aneuploidy grades in AML than in ALL (p less than 0.01).--In melanomas the aneuploidy rate was higher (86%) in metastases than in the primary tumors (54%, p = 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Development of Osteoarthritis in the Knee Joints of Wistar Rats After Strenuous Running Exercise in a Running Wheel by Intracranial Self-Stimulation

The influence of excessive running load on the development of knee osteoarthritis (OA) was investigated in male Wistar rats. Running exercises were performed in a running wheel using intracranial self-stimulation to motivate Wistar rats to run daily distances of 500 m at 5 days/week. Hereby, ten rats ran a distance of 15 km within three weeks while a further ten rats run a total of 30 km within six weeks. Thirteen Wistar rats without running exercises served as controls. Complete knee joint sections of all rats were evaluated histologically using MANKINs grading system with categorization of the findings into non, mild moderate, and severe osteoarthritis. In addition, immunoreactivity of the chondrocytes to MMP-3 as an important cartilage degrading enzyme in OA was assessed by immunostaining with monoclonal MMP-3 IgG antibodies. Histological assessment of the knee joint sections revealed a significant increase in osteoarthritic changes with higher running load. While in rats with 15 km running all but two knee joints showed mild OA, moderate OA was the predominant finding in rats with 30 km running. In contrast, no OA was found in the controls. Immunostaining for MMP-3 revealed a significant increase in immunoreactivity of the chondrocytes to MMP-3 with higher running load, indicating a running load-depending production of this cartilage-degrading enzyme in the course of increasing OA. Compared to 47.4% immunoreactive chondrocytes to MMP-3 in the controls, this ratio rose to 70.4% in rats with 15 km running and even up to 89.9% in rats with 30 km running. In conclusion, in Wistar rats, excessive running load leads to marked, running distance-depending osteoarthritic changes which are caused, at least in part, by an increase in MMP-3 production rising with greater running distance. Within this exercise model of OA, intracranial self-stimulation is an effective method to motivate Wistar rats to extremely excessive running in a running wheel. This model offers a wide range of further approaches to studying different processes of the development of OA.

The Number of Lymph Node Metastases in Gastric Cancer Correlates with the Angiotensin I–Converting Enzyme Gene Insertion/Deletion Polymorphism

Purpose: In the present study, we aimed to substantiate the putative significance of angiotensin I–converting enzyme (ACE) on gastric cancer biology by investigating the influence of its gene polymorphism on gastric cancer progression. Experimental Design: Genomic DNA was purified from peripheral blood mononuclear cells or tissue specimens. Amplified ACE gene fragments were separated on agarose gels. D or I alleles were identified by the presence of 190- or 490-bp fragments, respectively. Local expression of ACE was investigated by immunohistochemistry. Results: Twenty-four of 113 (21%) gastric cancer patients had the II, 57 (51%) the ID, and 32 (28%) the DD genotype. The distribution of the ACE genotypes did not differ significantly from the control group of 189 patients without gastric cancer. However, the ACE genotypes correlated with the number of lymph node metastases and the Unio Internationale Contra Cancrum (UICC) tumor stage. Patients with the II genotype had a highly significantly smaller number of lymph node metastases (P < 0.001) and a significantly lower UICC tumor stage (P = 0.01) than patients with the DD genotype. No correlation was found between tumor type, tumor location, local tumor growth, distant metastases, and the ACE genotype. The expression of ACE in gastric cancer was investigated by immunohistochemistry in 100 of 113 patients. ACE was expressed by endothelial cells in all (100%) specimens and by tumor cells in 56 (56%) specimens. Conclusions: Our study shows that ACE is expressed locally in gastric cancer and that the gene polymorphism influences metastatic behavior.

Analysis of mutant P53 protein in osteosarcomas and other malignant and benign lesions of bone

SummaryAlterations of tumour suppressor genes are considered crucial steps in the development of human cancers. Expressions of p53 protein, a product of the tumour suppressor gene altered most commonly in human cancers examined so far, were investigated immunohistochemically in 18 osteosarcomas and 40 other malignant and benign lesions of bone. A monoclonal antibody clone PAb240, which recognizes a common conformational epitope of mutant p53 proteins, stained nuclei of tumour cells in 12 of 18 osteosarcomas (67%). Six tumours (33%) particularly showed positive immunoreactions in more than half of the tumour cells. PAb240 also stained tumour cells in a small number of other malignant bone tumours, such as malignant fibrous histiocytoma, chondrosarcoma, and Ewings sarcomas. Furthermore, a small number of cells of giant-cell tumours were positively stained. In contrast, PAb240 was completely negative in 21 benign bone tumours and reactive lesions examined. Another monoclonal antibody clone PAb1801, which reacts with both wild- and mutant-type p53 protein, reacted in nuclei of tumour cells of 7 osteosarcomas (39%). Most of those also reacted with PAb240. PAb1801 was expressed much more frequently in other malignant bone tumours and giant-cell tumours. In addition, PAb1801 showed intranuclear positive reactions in tumour cells of a benign chondroblastoma, and reactive cells such as actively proliferating preosteoblasts in a myositis ossificans and osteoclast-like giant cells in a giantcell tumour. The immunoelectron-microscopic observation that p53 protein was localized in euchromatic areas of nuclei of osteosarcoma cells supported the specificity of immunoreaction for p53 protein, indicating an active role of p53 protein in the regulation of DNA synthesis and transcription. These findings suggest that point mutation of the p53 gene is frequently involved in the development of osteosarcomas. PAb240 may be a useful tool not only in screening point mutations of the p53 gene in osteosarcomas but also in the differential diagnosis between osteosarcomas and reactive bone-forming lesions. Expressions of mutant p53 protein were not correlated with any clinical or pathological factors examined, although the results should be confirmed in studies of a large number of osteosarcomas.

Tumor heterogeneity in osteosarcoma as identified by flow cytometry

Measurements of the cellular DNA content by flow cytometry were carried out in 25 untreated osteosarcomas to identify the frequency of DNA aneuploidies and heterogeneous DNA stemlines in relation to histopathology. Analyzing multiple specimens from each single tumor (2–16; median, 4), highly malignant osteosarcomas were found to express DNA aneuploidies in 18 of 21 cases (86%) with multiple aneuploid DNA stemlines in 10 cases (48%). In three paraosteal osteosarcomas, no DNA aneuploidy was detected and a significantly lower proportion of cells in S‐phase was observed as compared to the highly malignant osteosarcomas (mean 8.6% vs. 18.8%; P <0.05). Like in the paraosteal osteosarcomas, no DNA aneuploidy and a low fraction of cells in S‐phase was found in the predominant cell population of one of the very rare sclerosing small cell osteosarcomas, which also revealed a second DNA stemline with a DNA index of 2.0. These results demonstrate a high degree of DNA stemline heterogeneity in highly malignant osteosarcomas. The data emphasize the usefulness of DNA measurements for the characterization of bone tumors and indicate the possibility of discriminating highly malignant from low‐grade osteosarcomas. Cancer 59:324–328, 1987.

Cathepsin L antisense oligonucleotides in a human osteosarcoma cell line: effects on the invasive phenotype.

Alterations in cathepsin L expression and trafficking have been associated with the progression and metastasis of several tumor entities. In the present study, we examined the effects of various cathepsin L antisense (as) phosphorothioate oligonucleotides on both the expression of cathepsin L and the invasive potential of the human osteosarcoma cell line MNNG/HOS. Seven oligonucleotides of 20-bp length each and one random control oligonucleotide were chosen to block cathepsin L expression. Northern blot analysis demonstrated a significant reduction in cathepsin L mRNA expression by the six antisense oligonucleotides at a concentration of 10 μM. Cathepsin L protein expression was reduced significantly (50–85%) by the antisense oligonucleotides, as compared with the controls. Adhesion to matrices of collagen I and matrigel was not affected. In in vitro motility and invasion assays performed in uncoated and precoated transwell chambers, the ability of cells to migrate through the filters was inhibited by 35–75% using antisense oligonucleotides. The random control did not show any inhibitory effect. These data demonstrate that in MNNG/HOS cells cathepsin L influences cellular malignancy by promoting migration and basement membrane degradation. Cancer Gene Therapy (2001) 8, 522–528

The Angiotensin I–Converting Enzyme Gene Insertion/Deletion Polymorphism Is Linked to Early Gastric Cancer

The insertion/deletion polymorphism of the angiotensin I–converting enzyme (ACE) gene has recently been linked to the pathogenesis and progression of human cancers. Using genomic DNA from 88 patients with early gastric cancer confined either to mucosa (pT1a) or submucosa (pT1b), we assessed the insertion (I) and deletion (D) polymorphism by PCR analysis and compared it with a large noncancer control population (n = 145). In the noncancer control group, the II genotype was observed in 33 (23%) individuals, whereas the ID and DD genotypes were found in 72 (50%) and 40 (27%) individuals, respectively. Interestingly, in the cancer group, we found the II genotype in six (7%) patients and the ID genotype in 46 (52%) individuals, whereas the DD genotype was observed in 36 (41%) individuals (P = 0.0034). Accordingly, the odds ratio for the II genotype was 0.20 [95% confidence interval (95% CI), 0.08-0.54; P = 0.009] and 0.55 for the ID/II genotype (95% CI, 0.31-0.96; P = 0.044) using the high-activity genotype DD as the reference category. No correlation was found among tumor type, tumor stage, the presence of Helicobacter pylori, and the ACE genotype. Our study provides further evidence that the ACE insertion/deletion gene polymorphism may be linked to the development of early gastric cancer. (Cancer Epidemiol Biomarkers Prev 2005;14(12):2987–9)