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Biomedical subjects

J Kos

Publications and source records attributed to J Kos.

At least 37 records · Page 2Linked to original sources
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↗
PubMed2000

Biological repair of thyroid cartilage defects by osteogenic protein-1 (bone morphogenetic protein-7) in dog.

The efficacy of human recombinant osteogenic protein-1 (OP-1; bone morphogenetic protein-7) in regeneration of dog larynx was examined by treating thyroid cartilage defects (1.5 cm2) in dogs with thyroid allografts covered with host perichondrium or fascia. Prior to implantation allografts were frozen, thawed and demineralized. The treatment groups were as follows: I--Allograft control implant (n = 3); II--Implants coated with 500 micrograms OP-1 (n = 4); III--Implants coated with 100 micrograms OP-1 (n = 3); IV--Implants coated with 500 micrograms OP-1 and covered with neck fascia (n = 3); and V--Implants extracted with 1 M NaCl and guanidine hydrochloride, and coated with 500 micrograms OP-1 (n = 4). Dogs were sacrificed four months following surgery. Each larynx was removed, carefully dissected and a three-dimensional reconstruction of the defect area was performed on serial sections. The results revealed that the implants of control dogs remained intact with no apparent reduction in size and new tissue formation. OP-1 enriched thyroid allografts, dose dependently induced bone, cartilage and ligament-like structures comprising up to 80% of the total regenerated defect area. Boundaries of the defects healed by formation of new bone when bone resided within the old thyroid cartilage layers. Old cartilage not containing bone within its layers healed by complete integration with newly formed cartilage. Both new bone and cartilage were embedded into layers of new ligament-like tissue which expressed specific morphologic and molecular markers. The three newly formed tissues were tightly connected into a "bone-cartilage-ligament continuum" of tissues, suggesting that OP-1 served as a multiple tissue morphogen in this specific microenvironment.

Animals↗
PubMed2000

Molecular cloning and chimerisation of CDI 315B monoclonal antibody.

A chimeric mouse-human antibody has been created that recognizes an antigen found on breast cancer cells and melanoma cells. Immunoglobulin constant domains of mouse monoclonal antibody CDI 315B Cgamma1 and CK, were substituted by the human Cgamma1 and Ckappa. The CDI 315B variable heavy and light chain regions were PCR amplified from hybridoma RNA and sequenced. Mouse variable VH and VL regions were joint to human IgG1 and kappa constant regions and subcloned into pcDNA3 expression vectors. The Sp2/0 murine myeloma cells were transfected with expression vectors pcDNA3L and pcDNA3H and the reactivity of chimeric antibodies was tested by indirect ELISA using B16F1 murine melanoma cells as well as MCF7 human breast cancer cells, as antigen.

Amino Acid Sequence↗
PubMed2000

Cathepsin B and its inhibitor stefin A in brain tumors.

Cysteine protease cathepsin B (CatB) and its endogenous inhibitor stefin A (StA) play an important role in tumor progression. Increase of CatB expression and lower levels of its inhibitors were associated with tumor malignancy in brain tumors. In this study of 100 patients, CatB was localized by immunostaining to both, tumor and endothelial cells of primary brain tissue. Significant correlation with poor prognosis was found by univariate Cox's regression model. Intense overall immunostaining and immunostaining in endothelial cells alone were prognostic for survival (p=0.003 in both). When comparing CatB expression at mRNA level, we found considerable differences between center and periphery of a tumor as well as between different tumor samples. StA mRNA was only detected in benign, but not in malignant tissues. We suggest that screening of cysteine-protease genes expression can be applied in clinical prognosis of brain tumors.

Adult↗
PubMed2000

Cysteine proteinase inhibitors stefin A, stefin B, and cystatin C in sera from patients with colorectal cancer: relation to prognosis.

The levels of cysteine proteinase inhibitors stefin A, stefin B, and cystatin C were determined using ELISAs in sera obtained preoperatively from 345 patients with colorectal cancer and in control sera from 125 healthy blood donors. The levels of stefin A and cystatin C were found to be moderately increased in patient sera (1.4-fold and 1.6-fold, respectively; P < 0.0001), whereas the level of stefin B remained statistically unchanged when compared with controls. The medians were 4.3 ng/ml versus 3.2 ng/ml for stefin A, 1.2 ng/ml versus 1.7 ng/ml for stefin B, and 679 ng/ml versus 425 ng/ml for cystatin C. In patient sera, a weak correlation of cystatin C with age (r = 0.34; P < 0.001) and gender (P = 0.01) was found. Stefin A and cystatin C levels were independent of Dukes' stage, whereas stefin B correlated significantly with Dukes' stage, its level being the highest in stage D (P < 0.007). Stefin B and cystatin C correlated with survival, whereas stefin A was not a significant prognostic factor in this study. Using medians as cutoff values, patients with high levels of stefin B and patients with high levels of cystatin C exhibited a significantly higher risk of death than those with low levels of inhibitors (hazard ratio = 1.6; 95% confidence interval, 1.2-2.2; P = 0.002 for stefin B; hazard ratio = 1.3; 95% confidence interval, 1.0-1.8; P = 0.04 for cystatin C). Our results reveal a correlation between high levels of extracellular cysteine proteinase inhibitors and short survival in patients with colorectal cancer, and the data thus support previous studies suggesting a contributing role of protease inhibitors in the progression of cancer.

Adult↗
PubMed2000

Cathepsin B, a prognostic indicator in lymph node-negative breast carcinoma patients: comparison with cathepsin D, cathepsin L, and other clinical indicators.

New prognosticators are needed for breast cancer patients after the initial surgical treatment to make therapeutic decisions that ultimately will affect their DFS. These consist of specific proteolytic enzymes including lysosomal endopeptidases. In this study, the activity and protein concentrations of cathepsins (Cats) D, B, and L were measured in 282 invasive breast tumor cytosols. These potential biological prognostic indicators were compared with other histopathological parameters, such as tumor size, lymph node involvement, tumor-node-metastasis stage, histological grade, DNA analysis, and steroid receptors. CatD protein concentration correlated with lymph node involvement. CatB and CatL levels correlated significantly with Scarf-Bloom-Richardson histological grade and were also higher in estrogen-negative tumors, and CatB was higher in larger tumors. As prognostic markers, CatB concentration was significant for increased risk for recurrence in the entire patient population and specifically also in lymph node-negative patients as follows: high CatB concentration (above 371 micrograms/g) in tumor cytosols was significant (P < 0.00) for high risk of recurrence but was of only borderline prognostic significance (P < 0.06) for overall survival of all patients. In lymph node-negative patients, CatB (above 240 micrograms/g, P < 0.003) was highly significant for recurrence-free survival, followed by CatL (above 20 micrograms/g, P < 0.049) and CatD (above 45 nmol/g, P < 0.044) concentrations. For overall survival of node-negative patients, only CatB was a significant (P < 0.014) prognosticator. We conclude that CatB is useful as a prognostic indicator in lymph node-negative patients. This suggests that selective adjuvant therapy should be applied in this lower risk group of patients when high levels of CatB are determined.

Adult↗
PubMed2000

Prognostic significance of cysteine proteinases cathepsins B and L and their endogenous inhibitors stefins A and B in patients with squamous cell carcinoma of the head and neck.

Cysteine proteinases cathepsins (Cats) B and L and their endogenous inhibitors stefins (Stefs) A and B are implicated in the processes of local and metastatic tumor spread. They were identified as potential prognosticators in various malignant diseases, particularly in breast cancer. The aim of the present study was to determine the concentrations of Cats B and L and Stefs A and B in the tumor and adjacent normal tissue samples collected from 49 patients (the present group) with squamous cell carcinoma of the head and neck (SCCHN), using quantitative immunosorbent assays (ELISA; KRKA d.d., Novo mesto, Slovenia). Their clinical significance was compared with that from a previous study (the reference group, 45 patients; Budihna et al., Biol. Chem. Hoppe-Seyler, 377: 385-390, 1996). The follow-up of patients from the latter report was updated for this purpose. In the present group, significantly higher concentrations of Cat B (P < 0.0001), Cat L (P < 0.0001) and Stef A (P = 0.006) were found in tumors compared with concentrations in their normal tissue counterparts. Cat concentrations in normal laryngeal tissue were significantly/marginally elevated compared with nonlaryngeal tissue (Cat B, P = 0.02; Cat L, P = 0.06). The tumor concentration of Cat L was found to correlate with pT classification (P = 0.005) and tumor-node-metastasis stage (P = 0.05), whereas the concentrations of Stefs A and B correlated with pN classification (P = 0.007 and P = 0.03, respectively) and tumor-node-metastasis stage of the disease (P = 0.02 and P = 0.03, respectively). There was no statistically significant difference between low and high Cat B or Cat L groups, regarding either disease-free survival or disease-specific survival, using a minimum P approach to determine cutoff concentrations. The risk of disease recurrence and SCCHN-related death was significantly higher in patients with low Stef A (P = 0.0006 and P = 0.0005, respectively) and Stef B (P = 0.0009 and P = 0.0007, respectively) tumors, compared with those with high-Stef A and Stef B tumors. These results remained significant even after Ps were adjusted for a possible bias in the estimated effect on survival. The survival analysis in the reference group also confirmed these findings (Stef A: P = 0.0009 and P = 0.002, respectively; Stef B: P = 0.03 and P = 0.009, respectively). To avoid any possible bias arising from the differences between the laboratories that performed the biochemical analysis, the concentrations of both Stefs in the present group and in the reference group were standardized and coupled together to form a uniform group. In univariate survival analysis, standardized values of Stef A and Stef B correlated inversely with the rate of relapse (P = 0.0000) and mortality rate (P = 0.0000). Multivariate regression analysis showed that the standardized value of Stef A is the strongest independent prognostic factor for both disease-free survival and disease-specific survival. These findings show the specific role of Cats B and L and Stefs A and B in the invasive behavior of SCCHN. Furthermore, Stef A proved to be a reliable prognosticator of the risk of relapse and death in patients with this type of cancer.

Adult↗
PubMed2000

Enhanced urinary gelatinase activities (matrix metalloproteinases 2 and 9) are associated with early-stage bladder carcinoma: a comparison with clinically used tumor markers.

Matrix metalloproteinases (MMPs) are involved in tumor growth and metastasis, promoting the migration and invasion of cells. In this study, the amount of MMP-2 and MMP-9 activity was measured in urine from superficial bladder carcinoma patients (pTa, pT1) to evaluate their possible diagnostic value. The active and total amount of MMP-2 and MMP-9, respectively, in urine from tumor patients were compared with the levels in urine from age- and gender-matched healthy volunteers. Both MMP-2 and MMP-9 activity levels were significantly enhanced in urine from patients with high invasive cancers (pT2, PT3), whereas in urine from healthy controls no or very low MMP activities were found. More importantly, a substantial number of urine samples from patients with superficial tumors contained elevated MMP-2 and MMP-9 activities, suggesting that enhanced urinary MMP activity levels, indeed, might be indicative for early-stage bladder cancer. Overall, urinary MMP-2 and MMP-9 activity levels were significantly correlated to each other, with some individual exceptions. A comparison between urinary MMP-9 activity and a recently proposed urinary marker for bladder cancer, NMP-22, showed slightly lower numbers of patients with elevated levels for MMP-9. But because MMP-9 and NMP-22 levels were not correlated, enhanced urinary MMP activity might be useful as a marker for superficial bladder carcinoma like, or especially in combination with, other markers.

Adult↗
PubMed1999

Detection by ELISA of the humoral immune response in rabbits naturally infected with Trichophyton mentagrophytes.

An indirect enzyme linked immunosorbent assay (ELISA) was developed and its diagnostic potential evaluated for rabbits infected by Trichophyton mentagrophytes. Within-run and between-run coefficient of variance varied from 2.3 to 7.7% and from 5.9 to 8.5%, respectively, indicating satisfactory reproducibility of the ELISA. There was no significant cross-reaction with antigens of Microsporum canis, Malassezia pachydermatis and Aspergillus fumigatus. The level of specific IgG to Trichophyton mentagrophytes was measured in sera of 25 11-week-old and 12 younger infected rabbits. There was no significant difference in the IgG level between 12 5-week-old infected rabbits and controls (p = 0.38). The antibody response was higher in 12 7-week-old rabbits compared with controls (p = 0.001). The IgG level in 25 11-week-old rabbits differed from the controls very significantly (p < 0.0001). Increased specific IgG in 11-week-old rabbits exhibited 96% sensitivity and 94% specificity. Predictive values of a positive and a negative test were 96 and 94%, respectively. Western immunoblotting associated three protein bands (21.5, 31, 44 kDa) with Trichophyton mentagrophytes infection.

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↗
PubMed1999

Cathepsin B immunohistochemical staining in tumor and endothelial cells is a new prognostic factor for survival in patients with brain tumors.

The cysteine endopeptidase, cathepsin (Cat) B, and its endogenous inhibitor, stefin A, were found relevant for cancer progression of many neoplasms, including human brain tumors. Histological sections of 100 primary brain tumors, 27 benign and 73 malignant, were stained immunohistochemically for Cat B and stefin A. The immunohistochemical staining of Cat B in tumor cells, endothelial cells, and macrophages was scored separately from 0-12. The score in tumor and endothelial cells was significantly higher in malignant tumors compared with benign tumors (P<0.000). A significant correlation between immunostaining of Cat B (scored together for tumor and endothelial cells) and clinical parameters, such as duration of symptoms, Karnofsky score, psycho-organic symptoms, and histological score was demonstrated. Univariate survival analysis indicated that total Cat B score above 8 was a significant predictor for shorter overall survival (P = 0.003). In glioblastoma multiforme, intense Cat B staining of endothelial cells was a significant predictor for shorter survival (P = 0.003). Stefin A immunostaining was weak and detected only in a few benign and some malignant tumors, suggesting that this inhibitor alone is not sufficient in balancing proteolytic activity of Cat B. We conclude that specific immunostaining of Cat B in tumor and endothelial cells can be used to predict the risk of death in patients with primary tumors of the central nervous system.

Adolescent↗
PubMed1998

Refolding of recombinant sulphonated procathepsin S and of reduced chicken cystatin; implications for renaturation experiments.

Kinetic stopped-flow measurements of refolding of the recombinant sulphonated procathepsin S from 6 M urea are presented. The experiments were performed using intrinsic tryptophan fluorescence and fluorescence of the hydrophobic probe 1-anilino-naphthalene-8-sulfonate (ANS). Initially, (t1/2 = 3 +/- 1 ms) an intermediate with increased ANS fluorescence and protected tryptophan environment is formed. Much later, a slow increase in ANS fluorescence occurs with no accompanying changes in tryptophan fluorescence. The reaction of the slow ANS fluorescence increase correlates with the rate of aggregation as shown by the size exclusion chromatography (SEC). For comparison, the folding reactions of the reduced chicken cystatin were measured, both, by intrinsic tryptophan and extrinsic ANS fluorescence. An early intermediate forms very fast in the refolding of reduced chicken cystatin on 6-fold dilution from 5.7 M GuHCl (t1/2 = 5 +/- 2 ms), similarly to that observed for the sulphonated procathepsin S. ANS fluorescence and tryptophan fluorescence decrease further (t1/2 = 100 +/- 50 ms) leading to a late, 'more structured' intermediate which is prone to dimerization.

Animals↗
PubMed1998

Prognostic significance of cathepsins B and L in primary human breast cancer.

PURPOSE: Evaluation of the clinical significance of cytosolic tumor levels of the lysosomal cysteine proteases cathepsin B (catB) and cathepsin L (catL) in patients with primary breast cancer. PATIENTS AND METHODS: CatB (n = 1,500) and catL (n = 1,391) levels were determined by enzyme-linked immunosorbent assay (ELISA) in cytosols routinely prepared from frozen-tissue samples that were submitted to our laboratory for the assessment of steroid-hormone-receptor status. The median duration of follow-up of patients still alive at the time of analysis was 93 months. RESULTS: Relating catB and catL levels with classical prognostic factors, the proteases were positively correlated with the number of positive lymph nodes (P < .01), and negatively with the level of steroid-hormone receptors (P < .01). We did not find a significant relationship between catB or catL levels with age and menopausal status of the patients or with the size of the primary tumor. The levels of catB and catL were positively correlated with each other and with the rates of relapse and death (all, P < .0001). In multivariate regression analysis for relapse-free survival (RFS) and overall survival (OS), corrected for the contribution of age/menopausal status, tumor size, the number of positive lymph nodes, and steroid-hormone-receptor status, catB and catL were significant predictors of the rates of relapse and death (all, P < .01). No statistically significant interactions of catB or catL with any of the classical prognostic factors or with each other were observed in their associations with the rates of relapse and death. CONCLUSION: CatB and catL levels measured in routinely prepared cytosols are strong parameters to predict the rate of relapse and the length of survival after treatment of the primary breast tumor.

Adult↗
PubMed1998

Cysteine proteinases in cancer progression and their clinical relevance for prognosis.

Lysosomal cysteine proteinases, also known as cysteine cathepsins (Cats), belong to the papain family of proteinases, and share a similar protein structure and mechanism of action. However, subtle structural differences between these cathepsins, e.g. Cats B, H and L, give rise to potentially important variations in substrate specificity and differences in inhibition by their endogenous inhibitors, the cystatins, stefins and kininogens, under physiological and pathological conditions. Alterations in expression of Cat B and Cat L have been observed at various levels in malignant human tumor tissue compared to normal and benign tissue counterparts. We proposed that an imbalance between cathepsins and cystatins, associated with the metastatic tumor cell phenotype, may facilitate tumor cell invasion and metastasis and be responsible for early relapse of the disease after removal of the primary tumor. The results of our initial investigations on cysteine cathepsins and their endogenous inhibitors in human breast, lung and head and neck carcinomas, as well as in body fluids of melanoma and colorectal carcinoma bearing patients, have indeed shown their high prognostic impact for the survival of these patients.

Animals↗
PubMed1998

Prognostic values of cathepsin B and carcinoembryonic antigen in sera of patients with colorectal cancer.

The level of cathepsin B (Cat B) was determined in sera obtained preoperatively from 325 patients with colorectal cancer using an ELISA. Control sera from 90 healthy blood donors were analyzed. The levels of Cat B detected included all forms that were present in the sera, i.e., mature enzyme, precursor molecule, and enzyme-inhibitor complexes. The level of Cat B was significantly increased in sera of patients with colorectal cancer. The median level was 10.7 ng/ml versus 2.1 ng/ml in controls (P < 0.0001). A correlation between Cat B serum level and advanced Dukes' stage (P < 0.003) was found, whereas no associations have been found with age, sex, or level of carcinoembryonic antigen (CEA). In survival analysis, the patients with high serum Cat B experienced significantly lower survival probability. At the optimal cutoff value of 9.4 ng/ml, the relative hazard ratio was 1.8 (95% confidence interval, 1.1-2.8; P = 0.016) in the univariate Cox proportional hazards model. The median observation time was 4.4 years (range, 3.2-5.5 years). In multivariate analysis, Dukes' stage was the strongest prognostic variable, followed by age, whereas serum Cat B and CEA were not significant prognostic factors in this model, in accordance with their association with Dukes' stage. When the data for Cat B and CEA were combined, CEA-positive patients were further separated by Cat B into high- and low-risk groups. Patients with high serum levels of both molecules had significantly shorter survival (relative hazard ratio of 2.2; 95% confidence interval, 1.5-3.2; P < 0.0001), as compared with patients with low levels of both molecules.

Adult↗