Udo Kellner

Expression of human beta-defensins 1 and 2 in kidneys with chronic bacterial infection

BackgroundConstitutive expression and localization of antimicrobial human β-defensin-1 (HBD-1) in human kidneys as a potential mechanism of antimicrobial defense has been previously reported. Inducible expression of human β-defensin-2 (HBD-2) has been described in various epithelial organs but not for the urogenital tract.MethodsWe investigated the gene- and protein expression of HBD-1 and HBD-2 by reverse transcriptase-polymerase chain reaction, and immunohistochemistry in 15 normal human kidney samples and 15 renal tissues with chronic bacterial infection. Additionally, cell culture experiments were performed to study HBD gene expression by real-time RT-PCR in response to inflammatory cytokines TNFα and IL-1β as well as lipopolysaccharide from Gram-negative bacteria.ResultsConstitutive HBD-1 gene- and protein expression was detected in normal renal tissue and kidneys with chronic infection. As a novel finding, inducible HBD-2 gene- and protein expression was demonstrated in tubulus epithelia with chronic infection but not in normal renal tissue. In pyelonephritic kidneys HBD-1 and HBD-2 expression showed a similar pattern of localizaton in distal tubules, loops of Henle and in collecting ducts of the kidney. Furthermore, real-time RT-PCR of kidney derived cell lines stimulated with inflammatory agents TNF-α, IL-1β and LPS revealed a strong increase in relative HBD-2 transcription level and also a slight increase in relative HBD-1 transcription level.ConclusionsUpregulated HBD-2 expression in renal tubulus epithelium indicates a role of a wider range of human defensins for antimicrobial host defense in the urogenital tract than previously recognized.

Up-regulation of cathepsin X in Helicobacter pylori gastritis and gastric cancer

Recently, we identified increased cathepsin X expression in H. pylori‐infected gastric mucosa. Here, we describe further up‐regulation in gastric cancer and report on the role of inflammatory cytokines required for cathepsin X up‐regulation in H. pylori‐infected gastric mucosa, as well as on consequences for cellular invasion. Biopsy specimens were taken from the antrum, corpus and cardia of H. pylori‐infected and non‐infected patients. Gastric cancer samples were obtained from patients undergoing gastric surgery. Cathepsin X was detected in gastric mucosa by quantitative real‐time RT‐PCR, western blotting and immunohistochemistry. Induction of cathepsin X expression in epithelial and inflammatory cells caused by H. pylori infection was tested in in vitro contact and non‐contact co‐cultures of AGS cells and monocytic cells. Patients with H. pylori gastritis showed significantly higher cathepsin X mRNA (2.5‐fold) and protein (1.6‐fold) expression than H. pylori‐negative patients. Cathepsin X was also up‐regulated in gastric cancer (3–12‐fold) compared to non‐neoplastic mucosa. Cathepsin X was predominantly expressed by macrophages in the mucosal stroma and in glands of the antral mucosa. In addition, tumour cells stained for cathepsin X in 26 (68%) patients with gastric carcinoma. In general, staining was significantly more common (20 vs. 6 patients) and more intense (3.55 vs. 0.83) in intestinal type gastric cancer than in the diffuse type. In vitro cell culture experiments revealed that intercellular signalling between pathogenicity island (PAI)‐positive H. pylori‐infected epithelial cells and macrophages via soluble factors in the culture medium seems to be responsible for increased expression of cathepsin X in monocytes. Using antisense oligonucleotides, cathepsin X up‐regulation was directly associated with higher invasiveness in vitro. Although no correlation of cathepsin X expression and TNM stage was found, our study demonstrates that cathepsin X plays a role not only in the chronic inflammation of gastric mucosa but also in the tumourigenesis of gastric cancer. Copyright

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

Parallel screening for ALK, MET and ROS1 alterations in non-small cell lung cancer with implications for daily routine testing

OBJECTIVES ALK, MET and ROS1 are prognostic and predictive markers in NSCLC, which need to be implemented in daily routine. To evaluate different detection approaches and scoring systems for optimal stratification of patients eligible for mutation testing in the future, we screened a large and unselected cohort of NSCLCs for all three alterations. MATERIAL AND METHODS Using tissue microarrays, 473 surgically resected NSCLCs were tested for ALK and MET expression by IHC and genomic alterations in the ALK, MET and ROS1 gene by FISH. For MET IHC, two different criteria (MetMAb and H-score), for MET FISH, three different scoring systems (UCCC, Cappuzzo, PathVysion) were investigated. RESULTS ALK and ROS1 positivity was seen in 2.6% and 1.3% of all ADCs, respectively, but not in pure SCCs. One ROS1 translocated tumor showed additional ROS1 amplification. MET IHC+/FISH+ cases were found in both histological subtypes (8.6% in all NSCLCs; 10.6% in ADCs; 5.0% in SCCs) and were associated with pleural invasion, lymphatic vessel invasion and lymph node metastasis. MET altered ADCs more frequently showed a papillary growth pattern. Whereas ALK testing revealed homogenous results in IHC and FISH, we saw discordant results for MET in about 10% of cases. Both METIHC scoring systems revealed almost identical results. We did not encounter any combined FISH positivity for ALK, MET or ROS1. However, three ALK positive cases harbored MET overexpression. CONCLUSION In daily routine, IHC could support FISH in the identification of ALK altered NSCLCs. Further research is needed to assess the role of discordant MET results by means of IHC and FISH as well as the relevance of tumors with an increased ROS1 gene copy number.

ALK-FISH borderline cases in non-small cell lung cancer: Implications for diagnostics and clinical decision making

BACKGROUND Fluorescence in-situ hybridization (FISH) for the detection of ALK-rearrangements in non-small cell lung cancer (NSCLC) is based on at first sight clear cut-off criteria (≥15% of tumor cells) for split signals (SS) and single red signals (SRS). However, NSCLC with SS-counts around the cut-off may cause interpretation problems. MATERIAL AND METHODS Tissue microarrays containing 753 surgically resected NSCLCs were independently tested for ALK-alterations by FISH and immunohistochemistry (IHC). Our analysis focused on samples with SS/SRS in the range between 10% and 20% (ALK-FISH borderline group). To better understand the role of these samples in routine diagnostics, we performed statistical analyses to systematically estimate the probability of ALK-FISH-misclassification (false negative or positive) for different numbers of evaluated tumor cell nuclei (30, 50, 100, and 200). RESULTS 94.3% (710/753) of the cases were classified as unequivocally (<10% or ≥20%) ALK-FISH-negative (93%; 700/753) or positive (1.3%; 10/753) and showed concordant IHC results. 5.7% (43/753) of the samples showed SS/SRS between 10% and 20% of the tumor cells. Out of these, 7% (3/43; ALK-FISH: 14%, 18% and 20%) were positive by ALK-IHC, while 93% (40/43) had no detectable expression of the ALK-protein. Statistical analysis showed that ALK-FISH misclassifications occur frequently for samples with rearrangements between 10% and 20% if ALK-characterization is based on a sharp cut-off point (15%). If results in this interval are defined as equivocal (borderline), statistical sampling-related ALK-FISH misclassifications will occur in less than 1% of the cases if 100 tumor cells are evaluated. CONCLUSION While ALK status can be determined robustly for the majority of NSCLC by FISH our analysis showed that ∼6% of the cases belong to a borderline group for which ALK-FISH evaluation has only limited reliability due to statistical sampling effects. These cases should be considered equivocal and therapy decisions should include additional tests and clinical considerations.

Characterization of novel anti-cathepsin W antibodies and cellular distribution of cathepsin W in the gastrointestinal tract

Abstract Human cathepsin W (lymphopain) is a cysteine protease that is restrictively expressed in cytotoxic cells, in particular NK cells. Several anticathepsin W monoclonal antibodies were tested with respect to their capability to detect cathepsin W by Western blot analysis and immunohistochemistry. Subsequently, the distribution of cathepsin Wexpressing cells was studied in gastrointestinal tissue specimens using the antibody CW-401B1. All cathepsin Wpositive cells had a lymphocyte phenotype. Notably, samples from patients suffering from chronic inflammatory bowel disease (Crohns disease, CD; ulcerative coliltis, UC) or autoimmune gastritis revealed variable amounts of cathepsin Wexpressing cells. The relative portion of cathepsin Wpositive cells among the infiltrating leukocytes (determined by CD45) differed remarkably. In autoimmune gastritis, cathepsin Wexpressing cells made up for 65% of all CD45+ cells, whereas the corresponding values for CD and UC were 11% and 6%, respectively. These differences imply a distinct involvement of cytotoxic cells expressing cathepsin W in the pathogenesis among these diseases. Furthermore, it was tested whether the proinflammatory cytokines TNFα and IFNγ can regulate cathepsin W gene expression in NK-92 cells. Both proinflammatory cytokines had only little effect on the cathepsin W gene expression of these cells.

Multicenter Evaluation of a Novel Automated Rapid Detection System of BRAF Status in Formalin-Fixed, Paraffin-Embedded Tissues

The mutated BRAF oncogene represents a therapeutic target in malignant melanoma. Because BRAF mutations are also involved in the pathogenesis of other human malignancies, the use of specific BRAF inhibitors might also be extended to other diseases in the future. A prerequisite for the clinical application of BRAF inhibitors is the reliable detection of activating BRAF mutations in routine histopathological samples. In a multicenter approach, we evaluated a novel and fully automated PCR-based system (Idylla) capable of detecting BRAF V600 mutations in formalin-fixed, paraffin-embedded tissue within 90 minutes with high sensitivity. We analyzed a total of 436 samples with the Idylla system. Valid results were obtained in 421 cases (96.56%). Its performance was compared with conventional methods (pyrosequencing or Sanger sequencing). Concordant results were obtained in 406 cases (96.90%). Reanalysis of eight discordant samples by next-generation sequencing and/or pyrosequencing with newly extracted DNA and the BRAF RGQ Kit confirmed the Idylla result in seven cases, resulting in an overall agreement of 98.57%. In conclusion, the Idylla system is a highly reliable and sensitive platform for detection of BRAF V600 mutations in formalin-fixed, paraffin-embedded material, providing an efficient alternative to conventional diagnostic methods, particularly for routine diagnostics laboratories with limited experience in molecular pathology.

Comparative Genomic Hybridization Synchronous Occurrence of Focal Nodular Hyperplasia and Hepatocellular Carcinoma in the Same Liver Is Not Based on Common Chromosomal Aberrations

Occasionally hepatocellular carcinomas (HCCs) occur synchronously with or within focal nodular hyperplasias (FNHs), raising the question of a putative causal relationship. In the present study, we used comparative genomic hybridization to investigate the occurrence of genomic aberrations in FNHs, which might lead to hepatocarcinogenesis. Tissue samples from FNHs and nonlesional liver tissue were obtained from 7 women. None of the patients had a chronic diffuse liver disease. A synchronous HCC not spatially related to FNH was present in 1 patient. Two patients had received oral contraceptives. Genomic aberrations were found in only 1 FNH. No aberration was found in the FNH occurring synchronously with HCC, but the HCC included gains at chromosomes 1q, 5, 12, and 19q and losses at 4p, 7q22-q35, 9p, 17p, 21q, and 22q. No aberrations were found in nonneoplastic liver tissues. Our findings support the notion that FNH is not a preneoplastic lesion for the occurrence of HCC in humans and that the synchronous occurrence of FNH and HCC is coincidental in our case.

Comparative Genomic Hybridization

Occasionally hepatocellular carcinomas (HCCs) occur synchronously with or within focal nodular hyperplasias (FNHs), raising the question of a putative causal relationship. In the present study, we used comparative genomic hybridization to investigate the occurrence of genomic aberrations in FNHs, which might lead to hepatocarcinogenesis. Tissue samples from FNHs and nonlesional liver tissue were obtained from 7 women. None of the patients had a chronic diffuse liver disease. A synchronous HCC not spatially related to FNH was present in 1 patient. Two patients had received oral contraceptives. Genomic aberrations were found in only 1 FNH. No aberration was found in the FNH occurring synchronously with HCC, but the HCC included gains at chromosomes 1q, 5, 12, and 19q and losses at 4p, 7q22–q35, 9p, 17p, 21q, and 22q. No aberrations were found in nonneoplastic liver tissues. Our findings support the notion that FNH is not a preneoplastic lesion for the occurrence of HCC in humans and that the synchronous occurrence of FNH and HCC is coincidental in our case.

Ektopes Pankreasgewebe im Duodenum – mögliche Begleitursache für symptomatische chronische Pankreatitis

Ek- oder heterotopes Pankreasgewebe ist nicht selten ein Zufallsbefund, da es kaum spezifische Beschwerden verursacht. Pankreaserkrankungen wie z.B. eine Pankreatitis mit entsprechendem Therapiebedarf finden sich jedoch häufig auch in ektopem Pankreasgewebe. Anhand eines Fallberichts wird ein 47-jähriger Patient beschrieben, der seit Jahren an einer chronischen alkoholinduzierten Pankreatitis leidet. Nachdem die medikamentöse Therapie (Enzymsubstitution und Analgetikagabe) ohne Erfolg blieb, wurde der Patient indikationsgemäβ einer kephalen Pankreatoduodenektomie nach Kausch-Whipple unterzogen, welche zur Beschwerdefreiheit führte. Die histologische Aufarbeitung des Präparats zeigte ektopisches Pankreasgewebe in einer duodenalen Duplikatur, das mit für die vorherigen persistierenden Beschwerden verantwortlich war.