Variations of components of the plasminogen activation system with the cell cycle in benign prostate tissue and prostate cancer.

BACKGROUND: Components of the fibrinolytic system are involved in tumor cell invasion and metastasis. Previous investigations suggested a cell cycle-dependent expression of urokinase-type plasminogen activator (u-PA) in epithelial cells. In order to determine a correlation of cell cycle phases with the fibrinolytic system, we investigated the expression of u-PA, tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor type 1 (PAI-1) in normal and tumor-containing prostate extracts and analyzed a possible relationship with flow cytometry-determined proliferative activity of the samples. Cell cycle phases were correlated with fibrinolytic parameters in prostate tissue. METHODS: Samples were obtained from patients undergoing radical prostatectomy for prostate cancer and separated into two portions for DNA analysis and the detection of u-PA, t-PA, and PAI-1. Flow cytometric analysis was performed according to the Vindelov technique. The concentrations of u-PA, t-PA, and PAI-1 were determined from tissue extracts after homogenization by an enzyme-linked immunosorbent assay (ELISA) technique. RESULTS: Correlations of u-PA and t-PA expression with the frequency of G0/G1, S, G2M, S-phase fraction (SPF), and proliferation index (PI) for normal prostate and prostate cancer revealed no significant correlation. The only significant finding was observed in normal tissue revealing a positive correlation between PAI-1 expression and G0/G1 and a negative correlation with S-phase, SPF, and PI. No dependence of PAI-1 expression on different cell phases was found in prostate cancer. Furthermore, no significant correlation of u-PA, t-PA, and PAI-1 with cell cycles in organ-confined ( or = pT3a) tumors was found. No significant correlation in prostate cancer of components of the fibrinolytic system differentiated according to tumor grade or perineural tumor infiltration and cell cycle analysis was found. Only in highly differentiated G1 (Gleason 2-4) cancer, u-PA had a significant positive correlation with G2M-phase. CONCLUSION: Absence of a correlation between levels of components of the fibrinolytic system and cell cycle phases suggests that the reported association between increases of some of these components and aggressive biological behavior of prostate cancer is secondary to non-cell cycle-related mechanisms.

Interaction of monocytes from patients with psoriatic arthritis with cultured microvascular endothelial cells.

The aim of this study was to investigate the interaction of monocytes of the peripheral blood of patients with psoriatic arthritis with cultured human dermal microvascular endothelial cells (HDMEC) compared to monocytes from control persons. The surface expression of adhesion molecules (ADM) and other cell surface molecules in psoriatic arthritis and control monocytes was investigated by quantitative flow cytometry. The receptor densities of these molecules were determined in terms of monoclonal antibody (mAb) binding sites. Cocultivation experiments including peripheral blood mononuclear cells and HDMEC were performed to determine the adhesion to and transmigration through activated or resting endothelial cell monolayers. In order to achieve optimal responses of cellular functions, activation for adhesion experiments was induced by lipopolysaccharide (LPS), while in transmigration experiments the endothelial cells were activated by TNF-alpha. For transendothelial migration studies HDMEC cultivated on collagen gels were used. In the supernatants of cocultivated cells the cytokines IL-6 and IL-8 were determined by ELISA. A significantly reduced expression of CD11b in nonactivated psoriatic arthritis peripheral blood monocytes compared to control monocytes was verified (mean number of adhesion molecules/cell: 33,756 +/- 10,138 vs 61,023 +/- 6925). In agreement with these findings, adhesion to, as well as transendothelial migration through, activated HDMEC was found to be significantly reduced in psoriatic arthritis monocytes. Transendothelial migration engendered an enrichment of monocytes in the migrated cell fraction for both control and psoriatic arthritis peripheral blood mononuclear cells. The activation of HDMEC by LPS induced a highly significantly enhanced cytokine release for IL-6 and IL-8, irrespective of the origin of monocytes (psoriatic arthritis vs. controls). However, IL-8 production in the supernatants of nonactivated monocytes/HDMEC cocultures was significantly reduced in the case of monocytes from psoriatic arthritis patients (6650 +/- 2489.32 pg/ml) vs 9280.00 +/- 3209.51 pg/ml in control patients. Impaired adhesion as well as transendothelial migration of monocytes derived from peripheral blood of psoriatic arthritis patients can be explained by the reduced expression of adhesion molecules MAC-1 (CD11b/CD18) at the surface of monocytes. The reduced IL-8 production also corresponds to a diminished cellular interaction under nonflow conditions. These results support the view that there are systemic immunological alterations in psoriatic arthritis patients.

Effect of first treatment with aminobisphosphonates pamidronate and ibandronate on circulating lymphocyte subpopulations.

Up to 60% of patients receiving their first infusion of the bisphosphonate pamidronate experience an acute-phase reaction. In this study, we used flow cytometry to determine the effects of pamidronate treatment on circulating lymphocyte subpopulations, and we investigated whether pamidronate and ibandronate treatment affect lymphocyte subpopulations differently. Twenty patients received a pamidronate infusion, 20 patients received intravenously injected ibandronate, and 10 controls received a clodronate infusion. Pamidronate treatment was followed by a significant increase in median body temperature at the 10-hour measurement and a significant decrease in counts of circulating lymphocytes, natural killer cells, T cells, and CD4+ and CD8+ T-cell subsets. Ibandronate treatment did not affect median body temperature, and it was associated at the 10-hour measurement with maximum increases in total lymphocyte count, B cells, T cells, and CD4+ and CD8+ T-cell subsets. Thus, there is a substantial difference in the hematologic response to initial treatments with pamidronate and ibandronate. Clodronate treatment did not induce changes in body temperature or significantly affect the number of circulating T cells and NK cells. The reduction in lymphocyte subsets after initial pamidronate therapy might be mediated by the release of tumor necrosis factor alpha, whose source in the acute-phase reaction could be T cells.

Analysis of fibrinolytic proteins in relation to DNA ploidy in prostate cancer.

The tissue concentrations of urokinase-type plasminogen activator (u-PA), urokinase-type plasminogen activator receptor (u-PAR), plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were investigated by an ELISA technique in normal and malignant samples of the prostate from 24 patients undergoing radical prostatectomy for organ-confined prostate cancer. The median concentration of u-PA was significantly higher in cancerous than in normal prostate tissue (p = 0.006). No significant increase of u-PAR, PAI-1 and t-PA was found in cancer tissue in comparison with the benign samples (p > 0.05). Assessment of the relationship between fibrinolytic proteins and DNA ploidy revealed an increased u-PA, u-PAR and PAI-1 in diploid prostate cancer as compared with the normal controls. However, in aneuploid cancer u-PA remained high but u-PAR and PAI-1 were decreased. This led to a higher local concentration of u-PA in aneuploid samples than in normal prostate and in diploid prostate cancer. No alteration of median t-PA was found in benign prostate or in diploid or aneuploid prostate cancer. The altered expression of u-PA, u-PAR and PAI-1 in diploid and aneuploid prostate cancer suggests a possible role of fibrinolytic proteins in the different biologic behavior of tumors, and may be one explanation for the higher metastatic potential of aneuploid tumors.

Precision and accuracy of monocyte counting. Comparison of two hematology analyzers, the manual differential and flow cytometry.

The Coulter STKS (Coulter, Hialeah, FL), the Abbott CD3500 (Abbott Diagnostics, Abbott Park, IL), a 400-cell manual differential, and flow cytometry using double-staining with fluorescence-labelled monoclonal antibodies (CD45-FITC and CD14-PE) on a Coulter Epics Profile II were evaluated for precision and accuracy in relative monocyte counting. STKS, CD3500, and Profile II achieved a precision analogous to a 3,542-, 1,835-, and 11,998-cell differential, respectively, demonstrating the superiority of automated methods. Analysis of 156 normal and abnormal samples revealed that the mean relative monocyte counts of the manual differential, CD3500 and Profile II were not significantly different. Only the STKS results showed a positive bias (0.79% +/- 1.65), which was increased in lymphocytic samples. Linear regression between the Profile II as independent viable, and the other techniques yielded acceptable correlation coefficients (STKS: 0.861, CD3500: 0.844, manual differential:0.833). Profile II results were also compared to those of a Becton Dickinson FACScan (Becton Dickinson, Mountain View, CA), which yielded an excellent correlation (r = 0.991) but a slightly smaller relative monocyte count (bias-0.39% +/- 0.60) of the latter. On the basis of these data, the authors recommend the use of monoclonal antibodies as a new reference method, but also indicate the need for further methodological investigations.

Standardization of the flow cytometric determination of HLA class I antigens, 'platelet-specific' glycoproteins and activation markers.

Flow cytometry (FC) provides a reproducible investigation of cell surface antigens on platelets. The aim of this study was to elaborate appropriate protocols and to compare them with other techniques that have already been published. (1) Venipuncture with tubes containing citrate was better for the preservation of the antigenicity than using ACD tubes. The isolated platelets could not be completely distinguished from detritus and protein aggregates. Therefore a platelet concentration between 10(7) and 10(8)/ml measurement buffer was necessary to obtain a sufficient resolution by FC. (2) Isolation methods using either differential centrifugation or diluted Ficoll-Hypaque as a flotation medium provided platelets of equal purity. The method with Ficoll-Hypaque resulted in a higher number of isolated platelets than differential centrifugation. The demonstration of platelets and their antigens in whole blood without isolation gave good results provided the platelets were not activated. Activation of platelets with 1 NIH-U thrombin/l resulted in the loss of a part of the highly activated platelets because of their aggregation. (3) Comparing different concentrations of paraformaldehyde in PBS, fixation with 1% for 15 min provided the best antigen preservation for most of the antigens investigated. Isolation induced platelet activation. In order to avoid this effect, the whole anticoagulated blood was fixed with 1% paraformaldehyde for 15 min immediately after venipuncture. Then the platelets were isolated using diluted Ficoll-Hypaque. In this way, systemic activation of platelets can be detected with antibodies against glycoproteins which are translocated from the alpha-granules or lysosomes to the cell membrane. These activation markers can be determined on immediately fixed platelets (already in the whole blood) without any interference due to unspecific activation caused by the isolation procedure. (4) Platelet treatment with citric acid at pH 3, in order to remove the antigenicity of HLA-class I molecules, was sensitive to immediate fixation with paraformaldehyde in the whole blood. Fixation after isolating the platelets made it possible to demonstrate antigen stripping, and the free heavy chain, devoid of the beta 2-microglobulin, could be clearly demonstrated. (5) Using standardization beads, the average number of antigenic sites per platelet could be determined for the investigated specificities. It was shown that antibodies which have been directly conjugated or biotinylated and combined with streptavidin-phycoerythrin yielded similar results in terms of the number of antigenic binding sites while unconjugated antibodies in combination with FITC-conjugated anti-mouse-IgG led to overestimation of antigenic binding sites.

[Report of initial experiences in establishing a regional multicenter evaluation of lymphocyte typing with flow cytometry]. / Bericht über erste Erfahrungen bei der Etablierung eines regionalen Rundversuches von Lymphozytentypisierung mittels Flowzytometrie.

Participation in a multicenter evaluation of flowcytometric investigations is the only possibility of providing external quality control. Up to now nine different laboratories have taken part in this program, measuring two samples of fresh EDTA blood according to a recommended antibody panel. The choice of antibodies was intended to reach a standardized immune status. Three different types of flowcytometers were used. All results were expressed in lymphocyte subset percentages and in absolute values. The coefficient of variation of each lymphocyte subpopulation was used for interpretation of the results of all working groups, as well as the control measurement. The results of the control measurement already showed that there was a difference between the obtainable coefficient of variation for the different subpopulations. In the case of CD3, CD4 and CD8 positive lymphocytes the relative percentages of the total group gave values far below 10%. The coefficients of variation of the activated T-lymphocytes or the cytotoxic T-cells, as well as of the absolute values were definitely higher. Already after the second multicenter evaluation the achieved results showed positive aspects such as improvements of the outcome, advancement of the user contacts, cooperation and impulses by discussing interesting problems.

[4-years experience with quality control of decentralized reflectometry blood glucose measurement in the Vienna-Lainz hospital]. / 4 Jahre Erfahrung mit der Qualitätskontrolle der dezentralen reflektometrischen Blutzuckermessung im Krankenhaus der Stadt Wien-Lainz.

Since 1987 a quality control programme for ward-based blood glucose assays has been established in Lainz Hospital, Vienna. In 1991 following a decision to standardise the type of reflectometer used in the ward, 62 Reflolux IIS reflectance meters (Boehringer Mannheim) were assessed in a quality control consisting of 4 ring trials a year (blood glucose determination in two "unknown" samples) and in daily measurements of control solutions of known glucose concentrations. Experience with this quality control procedure is reported in this presentation of the relevant results of the ring trials. The most frequent errors in performing the test are discussed and the importance of instructing and training the nursing staff in the proper use and maintenance of the meters is pointed out.