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Biomedical subjects

B Werle

Publications and source records attributed to B Werle.

18 recordsLinked to original sources
PubMed2004

Evolution of hepatitis B viral load and viral genome sequence during adefovir dipivoxil therapy.

Phase II and III clinical trials of adefovir dipivoxil (ADV) for the treatment of chronic hepatitis B have shown that this hepadnavirus polymerase inhibitor is well tolerated and effectively suppresses hepatitis B virus (HBV) replication. We therefore analysed the evolution of viral load and the emergence of HBV polymerase mutants in a 22-patient subgroup from a phase III clinical trial of ADV for the treatment of HBeAg-positive chronic hepatitis B. HBV DNA serum titres were quantified using a real-time polymerase chain reaction (PCR) assay with molecular hybridization probes. Emergence of polymerase mutants was assessed by direct sequencing of the viral reverse transcriptase domain after PCR amplification of HBV DNA isolated from serum. Our results indicated that ADV therapy effectively suppressed HBV replication in these patients (median serum HBV decrease at week 48 of treatment = 4.3 log10 copies/mL). The initial drop of HBV DNA titres in serum at week 12 of ADV therapy seemed to be predictive of subsequent HBe seroconversion (P = 0.059). Neither viral breakthrough nor the selection of drug resistant mutants were observed during the study period. Our results showed that ADV administration for 48-72 weeks effectively suppresses HBV replication without the emergence of resistant viral mutants.

Adenine↗
PubMed2001

Cathepsin S in tumours, regional lymph nodes and sera of patients with lung cancer: relation to prognosis.

Cysteine proteinase cathepsin S (Cat S) is expressed mainly in lymphatic tissues and has been characterised as a key enzyme in major histocompatibility complex class II (MHC-II) mediated antigen presentation. Cat S has been measured in tissue cytosols of lung parenchyma, lung tumours and lymph nodes and in sera of patients with lung tumours and of healthy controls, by specific enzyme-linked immunosorbent assay (ELISA). A difference in Cat S level was found between tumour and adjacent control tissue cytosols of 60 lung cancer patients (median 4.3 vs. 2.8 ng mg(-1) protein). In lymph nodes obtained from 24 patients of the same group, the level of Cat S was significantly higher than in tumours or lung parenchyma (P< 0.001). Additionally, significantly higher levels were found in non-infiltrated than in infiltrated lymph nodes (median 16.6 vs 7.5 ng mg(-1) protein). Patients with low levels of Cat S in tumours and lung parenchyma exhibited a significantly higher risk of death than those with high levels of Cat S (P = 0.025 - tumours; P = 0.02 - parenchyma). Immunohistochemical analysis (IHA) of lung parenchyma revealed a staining reaction in alveolar type II cells, macrophages and bronchial epithelial cells. In regional lymph node tissue, strong staining of Cat S was found in lymphocytes and histiocytes. Nevertheless, Cat S was detected also in tumour cells, independently of their origin. Our results provide evidence that Cat S may be involved in malignant progression. Its role, however, differs from that of the related Cats B and L and could be associated with the immune response rather than with remodelling of extracellular matrix.

Antibody Specificity↗
PubMed2001

Cathepsin B/cystatin C complex levels in sera from patients with lung and colorectal cancer.

A sandwich-type ELISA has been developed for quantification of the complex between the cysteine proteinase cathepsin B (CB) and its reversible tight-binding inhibitor cystatin C (CC) in normal and pathological sera. The assay is based on a combination of catching Ab (3E1), raised against CB, and a horseradish peroxidase-labelled detection Ab (1A2), raised against CC. Only the CB/CC complex is able to evoke a signal in this assay. The detection limit of the assay was 15.5 nM and the working range between 31.3-200 nM. The within and between-run coefficients of variance (CV) varied from 4.7% to 9.4% and 11% to 12.8%, respectively, demonstrating satisfactory reproducibility of the method. The concentration of the CB/CC complex was determined in sera from 90 healthy controls, 32 patients with non-cancerous lung diseases, 148 patients with lung and 32 patients with colorectal cancer. The CB/CC complex was significantly less abundant in sera of patients bearing malignant lung tumours than in those with non-cancerous lung diseases or healthy controls (p<0.001). In colorectal cancer sera its level was significantly lower in advanced stages C and D than in early Dukes' stages A and B (p=0.02). Our results show that the increased levels of CB in malignant sera are not impaired effectively by CC and support the hypothesis of hindered inhibitory capability during cancer progression.

Case-Control Studies↗
PubMed2000

Cathepsin B in infiltrated lymph nodes is of prognostic significance for patients with nonsmall cell lung carcinoma.

BACKGROUND: Tumor cells require specific proteolytic enzymes for invasion and metastasis, including lysosomal peptidases--cathepsins. Cathepsin B is a lysosomal cysteine peptidase, which appears to play a major role in invasion and metastasis of human tumors. In this study, the authors focused on the possible role of cathepsin B in lymphogenic metastasis by investigating the enzyme localization and its activity in lung tumors and corresponding tumor-infiltrated lymph nodes. METHODS: Cathepsin B activity was determined in lung tumors, lung parenchyma, and tumor cell-infiltrated and noninfiltrated regional lymph nodes of the same patient. The authors investigated 35 cancer patients suffering from nonsmall cell lung carcinoma. Cathepsin B throughout activity was measured by cleavage of the fluorogenic substrate Z-Arg-Arg-AMC at pH 6.0. RESULTS: The median specific cathepsin B activity was highest in tumors, followed by the infiltrated lymph nodes, noninfiltrated lymph nodes, and lung parenchyma. The authors showed a significant 1.8-fold increase in cathepsin B activity in tumor-infiltrated lymph nodes compared with noninfiltrated regional lymph nodes and a 4.5-fold increase in lung tumor tissue compared with lung parenchyma. High cathepsin B activity, both in tumors and tumor cell-infiltrated lymph nodes, indicated poor prognosis for overall survival. Immunohistochemical analysis showed the presence of cathepsin B in histiocytes and tumor cells but not in lymphocytes of lymph node tissue. CONCLUSIONS: The authors' findings on higher cathepsin B levels in tumor cell-infiltrated lymph nodes show that increased level of cathepsin B activity is characteristic of the invasive tumor cell phenotype. This corroborates the hypothesis, that tumor cell associated cathepsin B may play a role in lymphogenic metastasis. The authors' results support the use of lymph node associated cathepsin B as a prognostic factor for survival of patients with lung carcinoma.

Adult↗
PubMed2000

Cysteine proteinase cathepsin H in tumours and sera of lung cancer patients: relation to prognosis and cigarette smoking.

In order to evaluate the role of cysteine peptidase cathepsin H (Cath H) in human lung cancer its protein levels were determined in 148 pairs of lung tumour tissue and adjacent non-tumourous lung parenchyma using the enzyme-linked immunosorbent assay technique. Additionally, Cath H levels were determined in sera of 171 patients with malignant tumours, 34 patients with benign lung diseases and 47 healthy controls. The median level of Cath H in tumour tissue was 0.64 times that in the corresponding lung parenchyma. Relating tumour levels with histological type we found higher Cath H levels in small-cell and adenocarcinomas and lower levels in squamous cell carcinoma, large-cell carcinoma and secondary tumours. A significant difference in Cath H level between lung tumour tissue and non-tumourous lung parenchyma was associated with the group of cigarette smokers (156 vs 263 ng mg(-1) protein, P < 0.001). For this group of patients Cath H tumour levels correlated with the survival rate, while for the entire patient population this was not the case. Smokers with high tumour levels of Cath H experienced poor survival. Cath H was significantly higher in sera of patients with malignant and benign lung diseases than in control sera (P < 0.001). The increase was significant for all histological types, being the highest in small-cell and squamous cell carcinomas. Our study reveals that in lung tumours there is different behaviour of Cath H compared with other cysteine peptidases, e.g. cathepsin B and cathepsin L. Variations between tissue and serum levels of Cath H indicate either reduced expression or enhanced secretion of this enzyme in lung tumours.

Carcinoma, Non-Small-Cell Lung↗
PubMed1999

Dose-dependent effects of IL-12 treatment to immune response induced after immunization with a recombinant respiratory syncytial virus (RSV) fusion protein fragment.

The amino acid (aa) sequence 190-289 of the RSV fusion (F) glycoprotein expressed in insect cells (bF(190-289)) has been shown to partially protect BALB/c mice and to prime for a Th2 cell response. We evaluated the effects of IL-12 treatment during antigen priming of bF(190-289) on immune response and protective efficacy. Low doses of IL-12 (10 ng) reduced IL-4 and IL-5 secretion (but did not affect IL-10 production) and decreased inflammatory signs whereas high doses of IL-12 had no effects. In addition, IL-12 treatment did not improve resistance to RSV replication. These results suggest that IL-12 treatment attenuates Th2 response and Th2 associated pulmonary inflammatory response in a dose-dependent manner, without improving protective efficacy.

Animals↗
PubMed1999

Immunochemical analysis of cathepsin B in lung tumours: an independent prognostic factor for squamous cell carcinoma patients.

In order to evaluate the possible role of the proteolytic enzyme cathepsin B (cath B) in human non-small cell lung cancer (NSCLC) we examined cath B concentrations (cath B(C)) and activities (cath B(A)) in homogenates of 127 pairs of lung tumour tissues and corresponding non-tumourous lung parenchyma. Total cath B activity (cath B(AT)) and enzymatic activity of the fraction of cath B, which is stable and active at pH 7.5 (cath B(A7.5)) were determined by a fluorogenic assay using synthetic substrate Z-Arg-Arg-AMC. The immunostaining pattern of cath B was determined in 239 lung tumour tissue sections, showing the presence of the enzyme in tumour cells (cath B(T-I)) and in tumour-associated histiocytes (cath B(H-I)). The median levels of cath B(AT), cath B(A7.5) and cath B(C) were 5.6-, 3.2- and 9.1-fold higher (P < 0.001), respectively, in tumour tissue than in non-tumourous lung parenchyma. Out of 131 tissue sections from patients with squamous cell carcinoma (SCC), 59.5% immunostained positively for cath B, while among the 108 adenocarcinoma (AC) patients 48.2% of tumours showed a positive reaction. There was a strong relationship between the levels of cath B(AT), cath B(A7.5), cath B(C) and cath B(T-I) in the primary tumours and the presence of lymph node metastases. Significant correlation with overall survival was observed for cath B(T-I) and cath B(A7.5) (P < 0.01 and P < 0.05, respectively) in patients suffering from SCC. In these patients positive cath B in tumour cells (cath B(T-I)) and negative cath B in histiocytes (cath B(H-I)) indicated significantly shorter survival rate compared with patients with negative cath B(T-I) and positive cath B(H-I) (P < 0.0001). In contrast, in AC patients, both, positive cath B(T-I) and positive cath B(H-I), indicated poor survival probability (P < 0.014). From these results we conclude that the proteolytic enzyme cath B is an independent prognostic factor for overall survival of patients suffering from SCC of the lung.

Adenocarcinoma↗
PubMed1999

Fluorometric microassays for the determination of cathepsin L and cathepsin S activities in tissue extracts.

We established a continuous semi-microassay, and for large-scale studies both a stopped and a continuous microtiter plate assay for the fluorometric determination of cathepsin L and cathepsin S activities in body fluids, tissues or cell extracts in the presence of cathepsin B. For the detection of enzymatic activities we used the synthetic substrate Z-Phe-Arg-AMC, and for discrimination between cathepsin L, S and cathepsin B the specific inhibitor CA-074 for blocking interfering cathepsin B activities was applied. Furthermore, we took advantage of the stability of cathepsin S at pH 7.5 for further differentiation between cathepsin L and cathepsin S activities. The kinetic assays were characterized in terms of imprecision, analytical sensitivity, accuracy and substrate concentration. The within-run coefficients of variation were found to be 4.9%-7.2% for the continuous semi-microassay, 10.3%-11.7% for the stopped, and 4.5%-11.8% for the continuous microtiter plate assay. The between-days coefficients of variation for the continuous semi-microassay were 8.1%-8.9%, while for the stopped and continuous microtiter plate assays the coefficients were 11.2%-13.5% and 5.8%-12.2%, respectively. Compared to the continuous semi-microassay, the stopped and the continuous microtiter plate assays showed 3-fold and 11-fold higher sensitivity, respectively. Comparison between the continuous enzyme activity assays at substrate concentrations of 40 microM and 200 microM demonstrated a significant correlation of r = 0.97 and r = 0.99, respectively. The newly developed microtiter plate assay will allow efficient, sensitive and high precision determination of cathepsin L and cathepsin S activities in large-scale studies of cysteine-cathepsin dependent diseases.

Adult↗
PubMed1998

Immune response to baculovirus expressed protein fragment amino acids 190-289 of respiratory syncytial virus (RSV) fusion protein.

At least two neutralizing epitopes have been identified in the amino acid (aa) sequence 190-289 of the RSV fusion protein. The authors expressed this region in insect cells (bF190-289) and compared the immune response to bF190-289 with that induced by baculovirus expressed full-length fusion protein (bF). As with bF, mice primed with bF190-289 produced exclusively antibodies of IgG1 isotype, generated neutralizing antibodies, reduced significantly the virus titer (about a half log10 reduction) after RSV challenge and induced a Helper T (Th) 2 cell response in mediastinal lymph node cells (MLNC) restimulated in vitro. Thus, the aa sequence 190-289 represents a major immunogenic region of the RSV fusion protein.

Amino Acid Sequence↗
PubMed1997

Expression of cysteine protease inhibitors stefin A, stefin B, and cystatin C in human lung tumor tissue.

In human lung tumor tissue specimen (n = 73) concentrations of stefins A and B were found to be increased 2.0-fold (p < 0.01) and 1.3-fold (p < 0.01), respectively, as compared to matched normal tissue. Stefin A and B concentrations were higher in primary tumors than in secondary tumors, i.e. metastases from other organs to the lung (p < 0.01; p < 0.05, respectively). Cystatin C concentrations were rather low and did not differ between tumor and normal tissue. Both concentrations of stefins did not correlate with TNM stages. Stefin A was higher in squamous cell carcinoma than in adenocarcinoma (p < 0.01), while stefin B did not show such a difference. At investigation of a relationship between survival probability of patients with primary tumors it was found that increased stefin B concentrations and total cysteine-protease-inhibitory activities but not stefin A concentrations were positively correlated with survival probability. It is concluded that stefins A and B are major contributors to the cysteine protease inhibitory activity in primary lung tumors. Stefin B proved to be a prognostic factor, especially in squamous cell carcinoma.

Adolescent↗
PubMed1997

Cathepsin B fraction active at physiological pH of 7.5 is of prognostic significance in squamous cell carcinoma of human lung.

In this study we examined both the pH dependence of cathepsin B (cath B) activity and its stability at physiological pH of 7.5 in lung tumours and normal lung tissue by means of fluorogenic assays with Z-Arg-Arg-AMC as specific substrate. Specificity was verified with the cath B blocking inhibitors E-64 and CA-074. With respect to pH dependence of activity, we found a deviation from a normal-shaped pH- activity curve. Besides the typical activity peak at pH 6.0, there were shoulders at pH 4.5-5.5 and at pH 7.0-7.5. This heterogeneity was found in both tumour and normal tissue. To test the stability of cath B at physiological pH of 7.5, homogenates were kept at pH 7.5 for 60 min. Altogether, 82-100% of residual cath B activity was found at pH 5.0-5.5, whereas activity in the range between 5.5 and 7.4 dropped drastically to 26-42%. At pH 7.5, there was still 20-34% residual cath B activity detectable. To test the hypothesis whether the cath B fraction active at pH 7.5 is more abundant in tumour tissues compared with the normal counterparts, we determined this fraction in 91 pairs of lung tumour and normal lung tissue. We found a 2.3-fold increase of median cath B fraction active at pH 7.5 in tumour tissue, although this fraction represented only a small part (about 16%) of the native, acidic (pH 6.0) cath B activity. However, in contrast to native cath B at 6.0, the cath B fraction active at pH 7.5 was related to post-operative probability of survival in curatively operated patients, since activity values higher than 292 (muEU mg(-1) protein) were significantly associated with poor prognosis in patients with squamous cell carcinomas (n = 33, P= 0.04). It is concluded that in lung tumour and in normal lung tissue, cath B activity can be divided into at least three fractions with stability optima at different pH values, indicating various forms of cath B. The cath B fraction active at pH 7.5 provides prognostic information in patients with squamous cell carcinoma.

Adenocarcinoma↗
PubMed1995

Assessment of cathepsin L activity by use of the inhibitor CA-074 compared to cathepsin B activity in human lung tumor tissue.

In a series of pairs of lung tumor tissue and non-tumor lung parenchyma from 50 patients, the activity of cathepsin L was measured with Z-Phe-Arg-AMC using the inhibitor CA-074 to delimitate from cathepsin B activity also present in the tissue extracts. Cathepsin B was assessed in the same samples with its specific substrate Z-Arg-Arg-AMC. It was found that in tumor tissue the median activities of cathepsin L and cathepsin B were increased 1.6-fold and 4.9-fold, respectively. The levels of activity of both enzymes did not correlate with TNM stages nor with cell differentiation of bronchial carcinomas. Cathepsin L activity was found to be insignificantly higher in adenocarcinoma compared to squamous cell carcinoma, while cathepsin B activity did not vary across the histologies. The activities of both enzymes were low in pulmonary carcinoids, which are known to be low-grade malignant neoplasms. The amount of cathepsin B activity exceeded by far that of cathepsin L activity as proven by measurement with Z-Phe-Arg-AMC in the presence of the inhibitor Z-Phe-Phe-CHN2:95-98% of cathepsin B activity vs 2-5% of cathepsin L activity were determined. By SDS-PAGE separation and immunoblot analysis, it could be demonstrated that significant amount of cathepsin L is complexed with the cysteine proteinase inhibitor kininogen. This explains the rather low cathepsin L activity values in the tissue extracts.

Adenocarcinoma↗
PubMedDate not supplied

Cysteine proteinases and their inhibitors in extracellular fluids: markers for diagnosis and prognosis in cancer.

Cathepsins B, H and L have been shown to participate in processes of tumor growth, vascularization, invasion and metastasis. Their levels in tumor tissue extracts can provide useful clinical information to predict disease-free and overall survival in breast, lung, colorectal, brain and head and neck cancer patients. Recently we have found that both cysteine cathepsins and their endogenous protein inhibitors stefins and cystatin C can also predict prognosis when measured extracellularly. In melanoma and colorectal cancer patients high serum levels of cathepsins B and H correlated with shorter survival. Similarly, increased extracellular levels of stefins A and B and cystatin C correlated significantly with high risk of adverse outcome in cancer patients. However, the cathepsin B/cystatin C complex was found to be less abundant in sera of patients with malignant tumors than in those with benign diseases or in healthy controls, suggesting an imbalance between the enzyme and its inhibitor in cancer patients.

Biomarkers, Tumor↗
PubMedDate not supplied

Cathepsin B in tumors, normal tissue and isolated cells from the human lung.

Human lung tumors of different histologic cell types and adjacent normal lung parenchyma, purified lung parenchyma, purified lung macrophages and three human lung-derived cell lines were investigated in an attempt to identify tumor specific premature and mature cathepsin B species. By polyacrylamide gel electrophoresis and immunoblotting techniques with specific antibodies we detected mature cathepsin B forms with molecular masses of 31 kDa and 32 kDa in tissues. Reductive conditions revealed in these populations single and double chain 31/32 kDa forms. The latter were recognized by their heavy part, the 26/27 kDa molecule forms. Qualitative differences in the pattern of cathepsin B species between tumor and corresponding normal material or between different histologies of lung tumors were not found. However, tumor material is considerably richer in cathepsin B activity than normal material. Isolated alveolar macrophage populations and established cell lines showed the same cathepsin B pattern as tissues. All these cells released cathepsin B proforms of 44 to 46 kDa into the culture medium, indicating that the release of pro-cathepsin B cannot be considered a tumor-specific mechanism. The secreted proforms were sensitive to in vitro activation by pepsin.

Cathepsin B↗
PubMedDate not supplied

Prognostic value of increased lung tumor tissue cathepsin B.

In the study of 65 matched pairs of lung tumor tissue and normal lung parenchyma, cathepsin B (CB) activity was found to be increased about 4.6-fold, when regarding median levels. CB activity was found to be insignificantly higher in adenocarcinoma compared to the other histologic cell types. CB levels did not correlate with tumor stages (TNM) nor with cell differentiation, while increased CB activity was related to shorter survival rates of the patients. It is concluded that lung tumor CB is of some prognostic value for the outcome of the malignant disease.

Adult↗
PubMedDate not supplied

Charge polymorphism in human lung cell pro-cathepsin B.

Secreted pro-cathepsin B of HS-24 human lung-tumour cells, human alveolar macrophages and Wi-38 human lung-fibroblast cells was pre-purified by ion exchange chromatography and investigated by 2D gel electrophoresis. Four (Wi-38), six (HS-24) and ten (alveolar macrophages) polypeptides differing in charge, but with the same molecular mass of 45 kDa, were found. The isoelectrical points of these polypeptides ranged from 5.43 to 6.57. Deglycosylation reduced the mass (7 kDa) but did not change the charge pattern. This investigation established cell type-specific patterns of secreted pro-cathepsin B-forms, but only parts of these may be cell type-specific forms depending on differentiated expression of mRNA and post-translational modification.

Carcinoma, Squamous Cell↗