Heike Bantel

Detection of apoptotic caspase activation in sera from patients with chronic HCV infection is associated with fibrotic liver injury

Chronic hepatitis C virus (HCV) infection is characterized by inflammatory liver damage and is associated with a high risk of development of cirrhosis and hepatocellular carcinoma. Although histological examination of liver biopsies is currently the gold standard for the detection of early liver damage, there is a strong need for better noninvasive methods. We recently demonstrated that the proapoptotic activation of caspases is considerably enhanced in histological sections from HCV‐infected liver tissue, suggesting an important role of apoptosis in liver damage. Here, we investigated whether caspase activation is detectable also in sera from patients with chronic HCV infection. Using a novel enzyme‐linked immunosorbent assay that selectively recognizes a proteolytic neoepitope of the caspase substrate cytokeratin‐18, we demonstrate that caspase activity is markedly increased in the sera of HCV patients. Interestingly, while 27% of patients with chronic HCV infection showed normal aminotransferase levels despite inflammatory and fibrotic liver damage, more than 50% of those patients exhibited already elevated serum caspase activity. Moreover, 30% of patients with normal aminotransferase but elevated caspase activity revealed higher stages of fibrosis. In conclusion, compared with conventional surrogate markers such as aminotransferases, detection of caspase activity in serum might be a more sensitive method of detecting early liver injury. Thus, measurement of caspase activity might provide a novel diagnostic tool, especially for patients with normal aminotransferases but otherwise undiagnosed histologically active hepatitis and progressive fibrosis. (HEPATOLOGY 2004;40:1078–1087.)

alpha-Toxin is a mediator of Staphylococcus aureus-induced cell death and activates caspases via the intrinsic death pathway independently of death receptor signaling

Infections with Staphylococcus aureus, a common inducer of septic and toxic shock, often result in tissue damage and death of various cell types. Although S. aureus was suggested to induce apoptosis, the underlying signal transduction pathways remained elusive. We show that caspase activation and DNA fragmentation were induced not only when Jurkat T cells were infected with intact bacteria, but also after treatment with supernatants of various S. aureus strains. We also demonstrate that S. aureus–induced cell death and caspase activation were mediated by α-toxin, a major cytotoxin of S. aureus, since both events were abrogated by two different anti–α-toxin antibodies and could not be induced with supernatants of an α-toxin–deficient S. aureus strain. Furthermore, α-toxin–induced caspase activation in CD95-resistant Jurkat sublines lacking CD95, Fas-activated death domain, or caspase-8 but not in cells stably expressing the antiapoptotic protein Bcl-2. Together with our finding that α-toxin induces cytochrome c release in intact cells and, interestingly, also from isolated mitochondria in a Bcl-2-controlled manner, our results demonstrate that S. aureus α-toxin triggers caspase activation via the intrinsic death pathway independently of death receptors. Hence, our findings clearly define a signaling pathway used in S. aureus–induced cytotoxicity and may provide a molecular rationale for future therapeutic interventions in bacterial infections.

Proteins induced by telomere dysfunction and DNA damage represent biomarkers of human aging and disease.

Telomere dysfunction limits the proliferative capacity of human cells by activation of DNA damage responses, inducing senescence or apoptosis. In humans, telomere shortening occurs in the vast majority of tissues during aging, and telomere shortening is accelerated in chronic diseases that increase the rate of cell turnover. Yet, the functional role of telomere dysfunction and DNA damage in human aging and diseases remains under debate. Here, we identified marker proteins (i.e., CRAMP, stathmin, EF-1α, and chitinase) that are secreted from telomere-dysfunctional bone-marrow cells of late generation telomerase knockout mice (G4mTerc−/−). The expression levels of these proteins increase in blood and in various tissues of aging G4mTerc−/− mice but not in aging mice with long telomere reserves. Orthologs of these proteins are up-regulated in late-passage presenescent human fibroblasts and in early passage human cells in response to γ-irradiation. The study shows that the expression level of these marker proteins increases in the blood plasma of aging humans and shows a further increase in geriatric patients with aging-associated diseases. Moreover, there was a significant increase in the expression of the biomarkers in the blood plasma of patients with chronic diseases that are associated with increased rates of cell turnover and telomere shortening, such as cirrhosis and myelodysplastic syndromes (MDS). Analysis of blinded test samples validated the effectiveness of the biomarkers to discriminate between young and old, and between disease groups (MDS, cirrhosis) and healthy controls. These results support the concept that telomere dysfunction and DNA damage are interconnected pathways that are activated during human aging and disease.

Mesalazine inhibits activation of transcription factor NF-κB in inflamed mucosa of patients with ulcerative colitis

OBJECTIVES:The salicylate mesalazine is commonly used for the treatment of inflammatory bowel diseases, yet its precise mechanism of action is unknown. Because transcription factor NF-κB plays an important role in inflammatory bowel diseases, we investigated the effects of mesalazine therapy on NF-κB activation in patients with ulcerative colitis.METHODS:A total of 20 patients with moderately active ulcerative colitis received mesalazine for 8 wk. Biopsies were taken before and after drug administration and analyzed for NF-κB activation using an antibody specific for active NF-κB.RESULTS:In biopsies of active ulcerative colitis but not in noninflamed mucosa, activation of NF-κB was detected predominantly in macrophages. Mesalazine therapy resulted in a strong abrogation of NF-κB activation in situ.CONCLUSIONS:Our results suggest that the therapeutic properties of mesalazine rely at least in part on the inhibition of NF-κB activation, resulting in the suppression of proinflammatory gene expression in the inflamed mucosa.

Apoptosis in hepatitis C virus infection

AbstractInfection with hepatitis C virus (HCV) is characterized by inflammatory liver damage and a long viral persistence associated with an increased risk of developing hepatocellular carcinoma. Both in liver damage and in oncogenesis a disturbance of apoptosis has been implicated, although the underlying mechanisms in these apparently opposite processes are incompletely understood. HCV-triggered liver injury is mediated mainly by host immune mechanisms and eventually by direct cytopathic effects of HCV. Recent data shows that caspase activation, either triggered by death ligands, other cytokines, granzyme B or HCV proteins, is considerably upregulated in HCV-infected liver. Interestingly, caspase activation appears to correlate closely with the inflammatory response. Data about the role of single HCV proteins, either in cultured cells or transgenic animals models, however, are contradictory, as both pro- and anti-apoptotic effects have been observed. Nevertheless, apoptosis induction upon HCV infection may critically contribute to liver damage, while inhibition of apoptosis may result in HCV persistence and development of hepatocellular carcinoma.

Increased hepatotoxicity of tumor necrosis factor-related apoptosis-inducing ligand in diseased human liver

Tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) induces apoptosis in tumor cells but not in most normal cells and has therefore been proposed as a promising antitumor agent. Recent experiments suggested that isolated primary human hepatocytes but not monkey liver cells are susceptible to certain TRAIL agonists, raising concerns about the use of TRAIL in cancer treatment. Whether TRAIL indeed exerts hepatotoxicity in vivo and how this is influenced by chemotherapeutic drugs or liver disease are completely unknown. Employing different forms of recombinant TRAIL, we found that the cytokine can induce proapoptotic caspase activity in isolated human hepatocytes. However in marked contrast, these different TRAIL preparations induced little or no cytotoxicity when incubated with tissue explants of fresh healthy liver, an experimental model that may more faithfully mimic the in vivo situation. In healthy liver, TRAIL induced apoptosis only when combined with histone deacetylase inhibitors. Strikingly, however, TRAIL alone triggered massive apoptosis accompanied by caspase activation in tissue explants from patients with liver steatosis or hepatitis C viral infection. This enhanced sensitivity of diseased liver was associated with an increased expression of TRAIL receptors and up‐regulation of proapoptotic Bcl‐2 proteins. Conclusion: These results suggest that clinical trials should be performed with great caution when TRAIL is combined with chemotherapy or administered to patients with inflammatory liver diseases. (HEPATOLOGY 2007.)

Prospective biopsy-controlled evaluation of cell death biomarkers for prediction of liver fibrosis and nonalcoholic steatohepatitis

Fibrosis and steatosis are major histopathological alterations in chronic liver diseases. Despite various shortcomings, disease severity is generally determined by liver biopsy, emphasizing the need for simple noninvasive methods for assessing disease activity. Because hepatocyte cell death is considered a crucial pathogenic factor, we prospectively evaluated the utility of serum biomarkers of cell death to predict different stages of fibrosis and steatosis in 121 patients with chronic liver disease. We compared the M30 enzyme‐linked immunosorbent assay (ELISA), which detects a caspase‐cleaved cytokeratin‐18 (CK‐18) fragment and thereby apoptotic cell death, with the M65 ELISA, which detects both caspase‐cleaved and uncleaved CK‐18 and thereby overall cell death. Both biomarkers significantly discriminated patients with different fibrosis stages from healthy controls. However, whereas both markers differentiated low or moderate from advanced fibrosis, only the M65 antigen could discriminate even lower stages of fibrosis. The M65 assay also performed better in distinguishing low (≤10%) and higher (>10%) grades of steatosis. In a subgroup of patients, we evaluated the biomarkers for their power to predict nonalcoholic steatohepatitis (NASH). Importantly, both markers accurately differentiated healthy controls or simple steatosis from NASH. However, only serum levels of M65 antigen could differentiate simple steatosis from healthy controls. Conclusion: Cell death biomarkers are potentially useful to predict fibrosis, steatosis, or NASH. Compared with the widely used apoptosis marker M30, the M65 assay had a better diagnostic performance and even differentiated between lower fibrosis stages as well as between healthy individuals and patients with simple steatosis. (HEPATOLOGY 2012)

Staphylococcus aureus alpha-toxin-induced cell death: predominant necrosis despite apoptotic caspase activation

AbstractRecent data suggest that α-toxin, the major hemolysin of Staphylococcus aureus, induces cell death via the classical apoptotic pathway. Here we demonstrate, however, that although zVAD-fmk or overexpression of Bcl-2 completely abrogated caspase activation and internucleosomal DNA fragmentation, they did not significantly affect α-toxin-induced death of Jurkat T or MCF-7 breast carcinoma cells. Caspase inhibition had also no effect on α-toxin-induced lactate dehydrogenase release and ATP depletion. Furthermore, whereas early assessment of apoptosis induction by CD95 resulted solely in the generation of cells positive for active caspases that were, however, not yet permeable for propidium iodide, a substantial proportion of α-toxin-treated cells were positive for both active caspases and PI. Finally, electron microscopy demonstrated that even in the presence of active caspases, α-toxin-treated cells displayed a necrotic morphology characterized by cell swelling and cytoplasmic vacuolation. Together, our data suggest that α-toxin-induced cell death proceeds even in the presence of activated caspases, at least partially, in a caspase-independent, necrotic-like manner.

Critical role of NF-κB and stress-activated protein kinases in steroid unresponsiveness

Glucocorticoid resistance is a serious clinical problem in chronic inflammatory diseases, because many patients with rheumatoid arthritis, asthma, or Crohns disease fail to respond to steroid treatment. The molecular mechanisms underlying this unresponsiveness, however, are completely unknown. The effects of steroids are largely mediated by the interference of the glucocorticoid receptor (GR) with proinflammatory transcription factors. In the present study, we therefore investigated the activation of the transcription factors nuclear factor‐κB (NF‐κB), activator protein‐1 (AP‐1), and the upstream kinases p38 and c‐Jun N‐terminal kinase (JNK) in steroid‐sensitive and steroid‐resistant patients with Crohns disease. We demonstrated that steroid‐sensitive and steroid‐resistant patients reveal a remarkably different cellular activation pattern of these proinflammatory mediators. In steroid‐sensitive patients, activation of NF‐κB, AP‐1, p38, and JNK was mainly found in lamina propria macrophages. In contrast, steroid‐ resistant patients revealed activation of all these mediators mostly in epithelial cells. The functional interference of the proinflammatory mediators with the glucocorticoid response was supported by reporter gene assays. Expression of NF‐κB and, interestingly, also JNK1 and p38 inhibited the activity of the GR. Thus, our results suggest that steroid resistance is associated with increased epithelial activation of stress‐activated protein kinases and NF‐κB, which might inhibit the anti‐inflammatory action of a limited number of GRs.

Clinical feasibility of liver elastography by acoustic radiation force impulse imaging (ARFI)

BACKGROUND Transient elastography is increasingly used for assessment of liver fibrosis. Acoustic radiation force impulse imaging (ARFI) is a new technology to perform liver elastography. AIMS We evaluated the clinical feasibility, validity and accuracy of the ARFI method and compared it to Fibroscan(®) and liver histology. METHODS Ultrasonographic elastography of the liver using ARFI was performed in 29 patients with liver cirrhosis, 70 patients with liver disease and 23 healthy controls. RESULTS ARFI was feasible in all patients providing a mean propagation velocity of 1.65±0.93 m/s. ARFI results of the right and left liver lobes were comparable (p<0.001). In cirrhotic patients, ARFI gave significantly higher values than in the other patients (p<0.001). Rate of invalid measurements was lower in ARFI than in Fibroscan(®) (p<0.04). Both elastography methods were highly correlated to each other (p<0.001). Furthermore, ARFI correlated to histological grading of liver fibrosis (p<0.001) and to inflammatory activity (p<0.05). Liver steatosis had no statistical influence on ARFI results (p=0.2) in contrast to Fibroscan(®) (p<0.05). CONCLUSIONS The new ultrasonographic method of ARFI elastography allows valid, accurate and flexible evaluation of liver stiffness. It seems more feasible in patients with liver cirrhosis than Fibroscan(®). ARFI elastography of the left liver lobe is also possible. Liver steatosis does not seem to influence ARFI elastography.