Werner Ebert

Anti-Pr3: serological and immunochemical identification of a new anti-Pr subspecificity.

Abstract. A monoclonal IgM (x) anti‐Pr cold agglutinin occurring after a rubella infection is shown to have the ‘new’ anti‐Pr subspecificity anti‐Pr3. Pr3 determinants are found on cat and sheep erythrocytes which lack Pr1 and Pr2 determinants. By carbodiimide treatment of human erythrocyte glycoproteins, which causes intramolecular coupling of N‐acetylneuraminic acid carboxyl groups and nucleophilic centers of the glycoprotein backbone, Pr3 antigen activity is strongly increased, while Pr1 and Pr2 determinants are inactivated.

Expression of cysteine proteinase 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.

Serological identification of the new cold agglutinin specificity anti-Gd.

Abstract. The specificity anti‐Gd of human cold autoagglutinins is characterized using untreated and enzyme‐treated human red blood cells. Gd determinants of human RBC are resistant to proteases, but are inactivated by neuraminidase (RDE). In contrast, I/i determinants are not inactivated by proteases or RDE, while Pr1–3 determinants are inactivated by proteases and RDE, and Pra determinants are resistant to RDE, but are inactivated by proteases.

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

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.

Demonstration of Low-Titer Anti-Pr Cold Agglutinins

Abstract. Hemagglutination tests with protease‐ and neuraminidase(RDE)‐treated red cells are essential to demonstrate anti‐Pr cold agglutinins, since RDE and proteases inactivate the Pr antigens in contrast to the I/i antigens. RDE treatment of red cells, however, reveals the T antigen which reacts with anti‐T present in anti‐Pr sera. Furthermore, anti‐I, also present in anti‐Pr sera, shows increased reactions with RDE‐ and protease‐treated red cells. Therefore, T‐anti‐T and I‐anti‐I reactions mask the loss of anti‐Pr activity against RDE‐ and protease‐treated red cells when low‐titer anti‐Pr sera are studied. Absorption of anti‐Pr sera with RDE‐treated red cells removes anti‐T and anti‐I, leaving anti‐Pr cold agglutinins which only react with untreated red cells, not with RDE‐ or protease‐treated red cells. Anti‐I contaminating anti‐Pr in warm eluates from untreated red cells or stroma can be also removed by absorption with enzyme‐treated red cells. By eliminating T‐anti‐T interference from sera, it was possible to show that all Pr determinants known are determined by N‐acetylneuraminic acid.

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

Abstract 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 semimicroassay 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 μM and 200μM 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.

Cystatins C, E/M and F in human pleural fluids of patients with neoplastic and inflammatory lung disorders

Abstract Secretory type 2 cystatins, like cystatins C, E/M and F, are thought to be involved in many pathobiological processes, including vascular amyloidosis, rheumatoid arthritis, Alzheimers disease, osteoporosis, viral and bacterial infections, inflammatory disorders and tumour invasion and metastasis. In order to define the levels of cystatins C, E/M, and F in pleural effusions and to investigate whether these cystatins correlate with diagnostic parameters of pleural and lung diseases, we determined their concentrations in 160 pleural effusions. The median concentration of cystatin C in pleural effusions was 1437 ug/l (95.8 nM), ranging between 18-3967 ug/l. Cystatin C did neither correlate with malignant nor with benign diseases. The concentration of cystatin E/M was significantly higher in effusions of primary pleural tumours (mesotheliomas) compared to secondary pleural tumours and benign diseases. Furthermore, there was a significant correlation between the concentration of cystatin E/M of mesotheliomas and the pleural fluid tumour cell count and of cystatin C. The median values of cystatin F were significantly increased in parapneumonic/ empyema thoracis pleural effusions and tuberculous pleurisy compared to malignant pleural effusions, respectively. The concentration of cystatin F in benign effusions correlated significantly with diagnostic parameters and inflammation (total protein; lactate dehydrogenase; C-reactive protein). Finally, only in the group of parapneumonic/empyema thoracis was there a significant correlation between cystatin F and the neutrophil count. In conclusion, pleural effusions of different origin contain high levels of cystatin C, perhaps constituting the major part of an inhibitor reservoir. The level of cystatin E/M appears to be significantly associated with primary pleural tumours and cystatin F correlates with inflammatory processes of lung disorders.

I-, MN- and Pr1/Pr2-activity of human erythrocyte glycoprotein fractions obtained by ficin treatment.

Abstract. Human erythrocyte glycoproteins obtained by the phenol/saline extraction procedure are split using ficin. Ficin treatment results in diffusable and non‐diffusable glycopeptides. The nondialysable material is separated on sephadex G 50 columns in 4 fractions with different antigen activities. The main I activity is found in fraction 1 (neuraminic acid (NANA) content 21%). Quantitative differences concerning the MN activity are observed in the fractions 1–3. While fraction 2 (NANA content 22%) is Pr1active but Pr2‐inactive, fraction 3 (NANA content 20%) is Pr1‐inactive but Pr2‐active. Therefore, the NANA determinating Pr1 is not involved in the Pr2 antigenicity. NANA‐free fraction 4 is serologically inactive.

Technical performance and diagnostic utility of the new elecsys® neuron-specific enolase enzyme immunoassay

Abstract This international multicenter study was designed to evaluate the technical performance of the new double-monoclonal, single-step Elecsys neuron-specific enolase (NSE) enzyme immunoassay (EIA) and to assess its utility as a sensitive and specific test for the diagnosis of small-cell lung cancer (SCLC). Intra- and inter-assay coefficients of variation, determined in five control or serum specimens in six laboratories, ranged from 0.7 to 5.3 (inter-laboratory median: 1.3%) and from 1.3 to 8.5 (inter-laboratory median: 3.4%), respectively. Laboratory-to-laboratory comparability was excellent with respect to recovery and inter-assay coefficients of variation. The test was linear between 0.0 and 320 ng/ml (highest measured concentration). There was a significant correlation between NSE concentrations measured using the Elecsys NSE and the established Cobas Core NSE EIA II in all subjects (n = 723) and in patients with lung cancer (n = 333). However, NSE concentrations were systematically lower (approximately 9%) with the Elecsys NSE than with the comparison test. Based on a specificity of 95% in comparison with the group suffering from benign lung diseases (n = 183), the cut-off value for the discrimination between malignant and benign conditions was set at 21.6 ng/ml. NSE was raised in 73.4% of SCLC patients (n = 188) and was significantly higher (p < 0.01) in extensive (87.8%) as opposed to limited disease (56.7%). NSE was also elevated in 16.0% of the cases with nonsmall cell lung cancer (NSCLC, n = 374). It is concluded that the Elecsys NSE EIA is a reliable and accurate diagnostic procedure for the measurement of NSE in serum samples. The special merits of this new assay are the wide measuring range (according to manufacturers declaration up to 370 ng/ml) and a short incubation time of 18 min.

Elastinolytic activity of alveolar macrophages in smoking-associated pulmonary emphysema

Current concepts of pathomechanisms leading to acquired emphysema suggest that alveolar macrophages (AM) activated by cigarette smoking may cause an elastase/antielastase imbalance localized to the microenvironment formed by phagocytes and lung tissue. A functional cell assay was used to evaluate the cell-associated elastinolytic activity of AM. AM were obtained by bronchoalveolar lavage from patients with emphysema and from patients with non obstructive chronic pulmonary diseases (non-COPD) and cultured under serum-free conditions in direct contact with 3H-labeled elastin particles. Elastinolytic activity was calculated from the released radioactivity in culture supernatants and expressed as micrograms of 3H-elastin degraded × 10−5 AM × 72 h−1. AM of patients with emphysema had significantly higher elastinolytic activity compared to that of non-COPD patients (median: 10.8 versus 4.1 μg; P < 0.01). Further differentiation of patients revealed the lowest median activity in sarcoidosis (2.3 μg). In respect to smoking habits there was a major difference between smokers with and those without emphysema; AM of smokers with emphysema degraded more than twice the amount of elastin than smokers in the non-COPD group (median:11 versus 3.9 μg, P = 0.01). From these data we conclude that AM-derived elastinolytic proteases may be involved in the destruction of lung elastin, which is thought to be the key event occurring in the pathogenesis of pulmonary emphysema.