Daniel Neureiter

Apoptotic cell death is not a widespread phenomenon in normal aging and osteoarthritic human articular knee cartilage: A study of proliferation, programmed cell death (apoptosis), and viability of chondrocytes in normal and osteoarthritic human knee cartilage

OBJECTIVE Chondrocytes are crucial for adequate matrix balance and function. Cell proliferation and, recently, extensive apoptotic cell death have been reported in osteoarthritic (OA) cartilage. Apoptotic cell death would be an obvious central factor in the initiation and progression of OA, since there is no potential for replacing articular chondrocytes in the adult. Therefore, we studied the occurrence of apoptotic cell disintegration and cell proliferation in OA and normal articular cartilage obtained from the knees of adult donors of all ages. METHODS Following immunostaining for cellular proteins as well as staining for nuclear DNA, we performed triple-channel confocal laser scanning microscopy on thick cartilage slices to evaluate lacunar emptying and cell viability. Cell proliferation and apoptotic cell death were evaluated morphologically, by immunodetection of the proliferation-associated Ki-67 antigen, and by the TUNEL reaction. RESULTS With the exception of the calcified layer, we were not able to detect any major (apoptotic or nonapoptotic) cell disintegration in normal young or aged articular knee cartilage. Single apoptotic cells were detected in OA articular knee cartilage. A significant increase in lacunar emptying was observed in late-stage specimens with higher Mankin scores compared with age-matched normal control cartilage specimens, but not in low-grade lesions. A significant (but lesser) increase in empty lacunae was also observed with age in normal cartilage. Cell proliferation was rarely detected in OA cartilage samples and was not detected at all in normal cartilage samples. CONCLUSION Our results confirm the findings of previous studies showing that cell proliferation occurs in OA cartilage. They also show that, contrary to previous suggestions, apoptotic cell death is not a widespread phenomenon in aging or OA cartilage.

Extracellular matrix composition and gene expression in collagenous colitis.

BACKGROUND & AIMS Collagenous colitis is a rare diarrheal disease of unknown pathophysiology that is histologically defined by subepithelial bandlike structures. The objective of this study was to elucidate the biochemical composition and the origin of the bandlike structures in collagenous colitis. METHODS Immunohistochemical and in situ hybridization analyses were performed on endoscopic specimens using specific antibodies and riboprobes for collagen types I, III, IV, and VI and for the glycoprotein tenascin. RESULTS In collagenous colitis, the mucosal matrix with the exception of the bands retained a normal architecture and extracellular matrix composition. The bands stained most prominently for type VI collagen and tenascin. Less abundant staining for both proteins was also found in the subepithelial matrix of the normal mucosa. In situ hybridization showed no significant increase in collagen type VI messenger RNA expression in cells around and entrapped in the bands in collagenous colitis compared with normal specimens. CONCLUSIONS The results support the suggestion that collagenous colitis is a localized alteration of the extracellular matrix, which involves the pericryptal-subepithelial myofibroblast sheath. The data suggest that reduced matrix degradation and not overactivation of matrix synthesis may be the reason for the subepithelial accumulation of matrix proteins.

Eosinophil infiltration and degranulation in oesophageal mucosa from adult patients with eosinophilic oesophagitis: a retrospective and comparative study on pathological biopsy

Aim: To examine eosinophil infiltration and degranulation in 50 oesophageal biopsy specimens from 30 patients (21 men, 9 women; mean 39 years) with eosinophilic oesophagitis, by haematoxylin and eosin staining and immunohistochemistry. Methods: Immunohistochemistry was carried out using a monoclonal antibody for human eosinophilic major basic protein (MBP). Eosinophils were counted in three high power fields (×40) and degranulation, as quantified by extracellular MBP immunostaining, was scored on a scale of 1–4. Morphological changes (basal cell hyperplasia, elongation of papillae and dilatation of intercellular spaces) were scored on a 1–4 scale on sections stained with haematoxylin and eosin. Results: Numbers of intraepithelial eosinophils were significantly higher with MBP immunostaining than with haematoxylin and eosin staining (mean 109.6 v 80.6; p<0.001), whereas numbers of eosinophils were considerably correlated (r = 0.794). Eosinophil degranulation was higher in the distal oesophagus. Additionally, basic morphological changes were markedly associated with eosinophil infiltration. Extracellular deposition of eosinophil-MBP and eosinophil infiltration in subepithelial connective tissue, present in the biopsy specimens, were detected by immunohistochemistry. Conclusion: Numbers of eosinophils and degranulation are underestimated by haematoxylin and eosin staining. Immunohistochemistry detected up to two times more eosinophils than routine haematoxylin and eosin staining. Moreover, eosinophil-MBP immunoreactivity in extracellular regions indicates the release of toxic eosinophil granule proteins and gives further evidence for a causative role of eosinophils with regard to structural changes in eosinophilic oesophagitis. Immunohistochemistry may serve as a useful diagnostic tool to support the morphological differential diagnosis of eosinophilic oesophagitis and gastro-oesophageal reflux disease.

Severe disturbance of the distribution and expression of type VI collagen chains in osteoarthritic articular cartilage

OBJECTIVE The aim of this study was to evaluate the messenger RNA (mRNA) expression and distribution of the major pericellular type VI collagen in normal and osteoarthritic (OA) cartilage. METHODS Conventional and confocal laser scanning immunohistochemistry, as well as in situ hybridization experiments, were performed for all 3 collagen type VI chains in sections of normal and OA articular cartilage. RESULTS Normal adult articular chondrocytes were surrounded by a type VI collagen-positive pericellular matrix and showed significant levels of mRNA expression for all 3 type VI collagen chains. In OA cartilage, the expression and overall distribution of type VI collagen was largely increased in the lower middle and upper deep zones. In contrast, the upper zones showed a significant loss of pericellular type VI collagen staining. CONCLUSION Our results suggest that there is a significant basic turnover of type VI collagen in normal articular cartilage. In OA cartilage, the chondrocytes of the lower middle and upper deep zones account for a net increase in type VI collagen synthesis. The loss of type VI collagen staining in the upper zones is most likely the result of increased protein degradation rather than reduced synthetic activity.

Experimental endoscopic submucosal dissection training in a porcine model: Learning experience of skilled western endoscopists

Background:  Endoscopic submucosal dissection (ESD) demands a new level of endoscopic skill in Europe. A 2‐day workshop was set up for trainees to carry out five ESD each in order to obtain the skill level required to perform ESD in the stomach or rectum. This study describes: (i) the workshop setup; (ii) the participants performance; and (iii) the training effect on post‐workshop clinical ESD performance.

Apoptosis, proliferation and differentiation patterns are influenced by Zebularine and SAHA in pancreatic cancer models

Objective. Pancreatic cancer continues to be an urgent clinical problem. We used the novel DNA methyltransferase inhibitor Zebularine and the histone deacetylase inhibitor SAHA to investigate the epigenetic influence on viability and differentiation of the pancreatic cancer cell lines YAP C, DAN G and Panc-89 in vitro and in vivo. Material and methods. Cell vitality, proliferation and expression of PDX-1, cytokeratin 7 and 20, chromogranin A, vimentin, bax and bcl-2 were determined on the protein and mRNA level in vitro and in a subcutaneous xenograft model. Results. A time- and dose-dependent increase of apoptosis, paralleled by decreased proliferation, was observed after incubation with single agents or a combination therapy with lower concentrations. This was associated with up-regulation of pro-apoptotic bax and a phenotypic stabilization by the enhanced expression of cytokeratin 7. In vivo, growth of xenografts was delayed with the most pronounced effect in Panc-89 after 1 week of daily intraperitoneal injections of Zebularine paralleled with CK7 up-regulation and down-regulation of dedifferentiation markers. Conclusions. Epigenetic modulation via inhibition of DNA methyltransferase and histone deacetylase induces apoptosis in human pancreatic cancer cells in vitro and delays xenograft growth in vivo, which is associated with a morphological/molecular phenotypic stabilization. These compounds may therefore be suitable as adjunctive therapeutic agents in the treatment of pancreatic cancer.

Atherosclerosis and Vascular Calcification in Chronic Renal Failure

Cardiovascular complications are a major clinical problem in patients with chronic kidney disease and end-stage renal failure; cardiac death accounts for approximately 40–50% of all deaths in these patients. Death from cardiovascular causes is up to 20 times more common in uremic patients than in the general population with the risk being even higher than in patients with diabetes mellitus. A high rate of myocardial infarction and excessive cardiac mortality have repeatedly been documented in patients with kidney disease and renal failure. Not only is the prevalence of myocardial infarction high, but also the case fatality rate is significantly higher in uremic patients with and without diabetes, respectively, compared to nonuremic patients. This is of particular interest since the prevalence of coronary atheroma in uremic patients was shown to be approximately 30% by autopsy and coronary angiography studies. Thus, coronary factors, i.e. atherosclerosis, and non-coronary factors may play an important role in the genesis of cardiac complications in the renal patient. In addition, renal failure recently has also be identified as a predictor of mortality in different stages of peripheral vascular disease. In particular, marked differences in the pathogenesis, morphology and course of atherosclerosis and arteriosclerosis under the conditions of renal failure have been documented. Among others increased plaque formation and particularly higher proportion and intensity of vascular calcification have been found in clinical and autopsy studies. In addition to the so-called classical or traditional risk factors, an important role for nonclassical risk factors such as microinflammation, hyperphosphatemia and oxidative stress has been documented in patients with renal failure and is discussed in detail.

Inhibition of heme oxygenase 1 expression by small interfering RNA decreases orthotopic tumor growth in livers of mice

Endogenous overexpression of the antiapoptotic protein heme oxygenase 1 (HO‐1) has been shown to occur in various cancer diseases and might contribute to cancer progression. We compared the expression levels of HO‐1 in human liver to expression levels in hepatocellular carcinoma (HCC), as well as the effect of HO‐1 inhibition by small interfering RNA (siRNA) on cellular survival and apoptosis in the mouse hepatoma cell lines Hepa129 and Hepa1‐6 and on orthotopic tumor growth in immune‐competent C3H/HeN mice. Our results show that HO‐1 is frequently overexpressed in human HCC. Downmodulation of HO‐1 by siRNA resulted in increased cellular damage and apoptosis, reduced proliferation, reduced growth of orthotopic HCC and reduced angiogenesis. Livers and kidneys of treated animals did not reveal signs of damage by this treatment. In conclusion, a specific knockdown of HO‐1 might represent a novel therapeutic approach in HCC therapy.

Clinical significance of histone deacetylases 1, 2, 3, and 7: HDAC2 is an independent predictor of survival in HCC

Histone deacetylases (HDAC) are responsible for the transcriptional control of genes through chromatin remodeling and control tumor suppressor genes. In several tumors, their expression has been linked to clinicopathological factors and patient survival. This study investigates HDACs 1, 2, 3, and 7 expressions in hepatocellular carcinoma (HCC) and their correlation with clinical data and patient survival. Tissue microarrays of 170 surgically resected primary HCCs and adjacent uninvolved tissue were evaluated immunohistochemically for the expression of HDACs 1, 2, 3, 7, and Ki-67 and were analyzed with respect to clinicopathological data and patient survival. HDACs 1, 2, 3, and Ki-67 were expressed significantly higher in cancer cells compared to normal tissue (HDAC1: p = 0.034, HDACs 2 and 3 and Ki-67: p < 0.001), while HDAC7 expression did not differ between HCC and non-cancerous liver tissue. In tumor tissue HDACs 1–3 expression levels showed high concordance with each other, Ki-67 and tumor grade (p < 0.001). High HDAC2 expression was associated with poor survival in low-grade and early-stage tumors (p < 0.05). The expression of the HDACs 1, 2, and 3 (but not HDAC7) isoenzymes correlates with clinicopathological factors, and HDAC2 expression has an impact on patient survival.

The pan-deacetylase inhibitor panobinostat inhibits growth of hepatocellular carcinoma models by alternative pathways of apoptosis.

Inhibition of deacetylases represents a new treatment option for human cancer diseases. We applied the novel and potent pan-deacetylase inhibitor panobinostat (LBH589) to human hepatocellular carcinoma models and investigated by which pathways tumor cell survival is influenced. HepG2 (p53wt) and Hep3B (p53null) responded to panobinostat treatment with a reduction of cell proliferation and a significant increase in apoptotic cell death at low micromolar concentrations. Apoptosis was neither mediated by the extrinsic nor the intrinsic pathway but quantitative RT-PCR showed an upregulation of CHOP, a marker of the unfolded protein response and endoplasmic reticulum stress with subsequent activation of caspase 12. Dependent on the p53 status, a transcriptional upregulation of p21cip1/waf1, an increased phosphorylation of H2AX, and an activation of the MAPK pathway were observed. In a subcutaneous xenograft model, daily i.p. injections of 10 mg/kg panobinostat lead to a significant growth delay with prolonged overall survival, mediated by reduced tumor cell proliferation, increased apoptosis and reduced angiogenesis in tumor xenografts. Panobinostat increased the acetylation of histones H3 and H4. Panobinostat is a well tolerated new treatment option for HCC that activates alternative pathways of apoptosis, also in p53-deficient tumors.