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Hereditary protein C deficiency is demonstrated by lowered protein C plasma levels in a patient and at least one first-degree relative. This approach is insufficient in some cases owing to overlapping protein C levels in carriers and non-carriers of a protein C gene mutation. The protein C/S ratio is a simple and more accurate tool to detect carriers. In four families, 62% of 29 carriers, compared with none of 39 non-carriers, were protein C deficient. Sensitivity and specificity of the protein C/S ratio were 90% and 92% respectively. The protein C/S ratio seems to be more accurate than the measurement of protein C levels alone to identify carriers of a protein C gene mutation.

The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), a M, 38,000 transmembrane antigen also known as 17-1A or Ep-CAM, is commonly used for targeted immunotherapy of carcinomas because it is strongly expressed by most carcinomas. EGP-2 is, however, also expressed in most normal epithelia, To evaluate anti-EGP-2-directed treatment-associated effects on tumors and on EGP-2-positive normal tissue, we generated EGP-2-expressing transgenic mice. A 55-kb DNA fragment consisting of the 14-kb genomic coding sequence of the human EGP-2 gene with similar to 10-kb-upstream and similar to 31-kb-downstream sequences was isolated and used to direct EGP-2 expression in an epithelium-specific manner. In the EGP-2 transgenic mice, EGP-2 appeared to be specifically expressed in all of those epithelial tissues that also express EGP-2 in humans, whereas all of the other tissues were negative. The specific in vivo localization of the i.v. administered anti-EGP-2 monoclonal antibody MOC31 was studied in EGP-2-positive and -negative tumors induced s.c, in this EGP-2 transgenic mouse model. Immunohistochemical analysis showed specific localization of MOC31 in the EGP-2-positive tumors but not in the EGP-2-negative tumors. No anti-EGP-2 monoclonal antibody localization was observed in any of the EGP-2-positive normal mt,use tissues, which indicated a limited in vivo accessibility. In conclusion, an EGP-2 transgenic mouse model has been generated that expresses the EGP-2 antigen as in humans and, therefore, can serve as a model to evaluate the efficacy and safety of a variety of anti-EGP-2-based immunotherapeutic modalities in both tumors and normal tissue.

Objective Anti-endothelial cell antibodies in serum of patients with different inflammatory diseases can be detected by, a whole cell enzyme-linked immunosorbant assay using primary cultures of human umbilical vein endothelial cells. To avoid repeated isolation, it would be of great value if an immortal endothelial cell line could be used to perform anti-endothelial cell antibody assays. Methods In this study endothelial cells from human umbilical and iliac veins and arteries were transfected with a plasmid containing the Simian Virus 40 large T-antigen. Endothelial cell line(s) derived from this procedure were compared with human umbilical vein endothelial cells in the anti-endothelial cell antibody assay. Results After transfection, clones of homologous cell populations showed an extended lifespan, before entering a period of crisis. In one human umbilical vein endothelial cell clone a subpopulation of cells escaped crisis and became immortal (EVLC2). Telomerase was activated in this endothelial cell line, resulting in maintenance of the telomere length. There,vas a significant correlation between anti-endothelial cell antibody testing on human umbilical vein endothelial cells and on the cell line EVLC2. Conclusion The Simian Virus 40 large T-antigen immortalized human umbilical vein endothelial cell line EVLC2 may be useful for the detection of anti-endothelial cell antibodies.

Endothelial cell integrity (coverage and quality) of large donor vessels is important because these vessels are used for vascular reconstructions in solid-organ transplantation. Disruption of the endothelial cell monolayer will initiate blood coagulation and may lead to thrombosis of large vessels, often resulting in the loss of the transplanted organ. Iliac arteries and veins, removed from 10 heart-beating multi-organ donors at the end of the donor procedure. were analyzed using scanning electron microscopy at three different rime points of preservation. Endothelial cell coverage and quality were determined immediately after removal from the donor, after 10 h (time of transplantation) and 7 d storage in 'University of Wisconsin' cold preservation solution (UW). Endothelial cell coverage decreased during the preservation of arteries, but was maintained in veins. Storage of the veins for 7 d in plastic bags showed a decreased endothelial cell coverage compared to storage in glass vials. Early removal of the blood vessels and proper storage, free floating and in clean UW, may improve maintenance of the endothelial cell integrity. These findings may be important in order to reduce the risk of thrombosis and, consequently, organ failure after transplantation. Furthermore, vessels with maintained endothelial cell integrity after 7 d may be used for in vitro research.

Background Telomerase activation is thought to be essential for the immortality of cancer cells. It may be a prognostic factor in small volume well differentiated prostate cancers and hence a guide for the aggressiveness of the approach. The length of the chromosome tips (telomeres) are maintained by a specific enzyme (telomerase) independently of the normal cell division cycle. Although telomerase is not expressed in most normal human tissues, it is expressed in most human tumours. For the detection of telomerase in small prostate needle biopsy samples a recently developed telomeric repeat amplification protocol (TRAP) assay was used. The aim of the present study was: to measure telomerase activity in human prostate samples, and to evaluate the applicability of this assay on specimens from a prostate biopsy. Materials and methods From 36 patients referred for lower urinary tract symptoms (LUTS) or suspicion of having prostate cancer a total of 288 prostate biopsy samples were obtained (8 in each patient). When the digital rectal examination was abnormal and/or when the PSA level was elevated in L.U.T.S., or asymptomatic patients' tissue samples were obtained by transrectal ultrasound (TRUS) guided biopsies. Samples were tested for telomerase activity by a modified TRAP and forwarded for histology. Results In 19 out of 36 patients prostate cancer was diagnosed on histology. In 11 of these 19 tumours substantial telomerase activity was detected, whereas only very low telomerase activity existed in 2 of 17 samples from benign prostatic hypertrophy (BPH) patients. In this small series the relative telomerase activity in prostate cancer correlated with histopathological grade. Conclusions Our results show the applicability of a TRAP assay to measure telomerase activity in small needle biopsied prostate samples. In poorly differentiated and metastatic cancer we observed that levels of telomerase activity were high. To establish accuracy and to distinguish the 'relative good from the ugly' further study is needed.

The UBEIL gene isolated from the chromosome 3p21 region has an extremely reduced level of mRNA in lung cancer. Sequence analysis showed a 45% homology to the human ubiquitin-activating enzyme El at the amino acid level. To further characterize the protein product, we generated UBEIL protein-specific antibodies. Immunoblot analysis revealed a full-length gene product of approximately 112 kDa. Assessment of the level and distribution pattern of the UBEIL protein in normal and tumor tissue using the generated antibodies showed that the UBEIL protein was present in normal lung cells and non-lung cancer cell lines, but was undetectable in all 14 human lung cancer cell lines analyzed. This difference in expression of the UBEIL protein between normal lung tissue and lung tumor-derived cell lines suggests a possible involvement of an El-like protein in the origin and/or progression of lung tumors. (C) 2000 Wiley-Liss, Inc.

Autocrine and paracrine production of interleukin-6 (IL-6) is considered to be involved in the ongoing proliferation of ovarian-cancer cells. In view of the variability in IL-6 expression between various ovarian-cancer cells, we questioned whether differences in IL-6-gene regulation might be observed in ovarian tumor cells with and without IL-6 expression. The CAOV-3 cell line spontaneously secreted IL-6, which was enhanced by tumor necrosis factor-alpha (877 +/- 89 vs. 8,452 +/- 1,762 pg/ml, x +/- sd, p

Purpose: To evaluate the diagnostic value of semiquantitative telomerase activity assessment in cervical scrapings together with human papillomavirus (HPV) typing for detection of (pre)neoplastic cervical lesions and to compare telomerase activity in cervical scrapings and frozen specimens from the same patients. Patients and Methods: A cross-sectional study was performed in 161 patients referred for an abnormal cervical cytology report. In cervical scrapings, telomerase activity was determined by modified telomere repeat amplification protocol (TRAP) assay and HPV typing by polymerase chain reaction (PCR) with general and type-specific primers. Final diagnosis was made by pathologic examination of biopsy and/or loop excision specimens. Results: Telomerase activity was detectable in assessable scrapings fram one of nine (11%) patients without cervical intraepitheleal neoplasia (CIN), in three of 26 (12%)with CIN I, eight of 35 (22%) with CIN II, 18 of 62 (29%) with CIN III, and four of 13 (31%) with cancer. Sensitivity and negative predictive value of the TRAP assay for CIN II/III and cancer lesions were 25% and 28%, respectively, while specificity for no CIN or CIN I was 89%. In representative frozen sections, frequency of detectable telomerase activity was related to grade of CIN/cancer; none of 21 normal cervices, none of two CIN I, two of 12 (17%) CIN II, 10 of 31 (32%) CIN III, and 18 of 21 (86%) cervical cancer lesions were telomerase-positive (P Conclusion: telomerase activity is more frequent in higher grade CIN/cervical cancer lesions. Telomerase activity assessment in cervical scrapings has a low sensitivity for CIN II/III and/or cervical cancer and does not appear to be useful in primary screening for cervical cancer. However, increased telomerase activity in frozen CIN sections may be a possible marker of progressive disease. (C) 1998 by American Society of Clinical Oncology.

Brain tissue of rats pretreated with methylprednisolone or with the 21-aminosteroid U74389F, and that of untreated control rats, was assessed for the expression of Annexin-l (Anx-1) and the transcription of its mRNA. For this purpose Anx-1 cDNA was amplified and simultaneously a T7-RNA-polymerase promotor was incorporated into the cDNA using Polymerase Chain Reaction (PCR). Then digoxigenin-ll-UTP was incorporated into the transcribed cRNA with T7-RNA-polymerase. With this probe ill situ hybridization was carried out in sections of the brain. The probe was visualized by an immunoassay using an anti-digoxigenin antibody conjugate. Anx-1 protein was assessed by means of immunohistochemistry using a polyclonal antibody. The various brain areas of the control animals showed an appreciable amount of Anx-1 at mRNA or protein level; on the other hand, the animals which had been pretreated with either steroid, showed a more intense Anx-1 mRNA signal than the controls in many areas. In the pretreated animals Anx-1 immunostaining was unchanged in cortex, basal ganglia, amygdala and septum, but more intense in hippocampus, hypothalamus and thalamus. In ependyma, choroid plexus, meninges, and vascular walls there was no Anx-1 mRNA transcription detectable. An opposite profile was shown by the Anx-1 immunoreactivity, the protein was present in control animals as well as the steroid-pretreated animals, suggesting that here the protein was either from systemic origin, or has diffused from adjacent structures. The results indicate that Anx-1 mRNA transcription is upregulated by either steroid, and that in the untreated animals there is a resting level of Anx-1 mRNA transcription, presumably reflecting physiological influences on Anx-1 expression.

The specificity of random primer R143 for Aspergillus fumigatus DNA was determined in order to test its usefulness in establishing the presence of A. fumigatus DNA in fungal cultures. When PCR reaction products of these cultures were compared with those of 21 other bacterial and fungal DNA samples, R143 proved to produce a 1346 bp band with only A. fumigatus. This band has been sequenced completely and the EcoRI restriction site was used for subsequent confirmation of PCR products. The specificity for A. fumigatus DNA was also confirmed by Southern blotting. Comparison of morphological typing of Aspergillus species in cultures with PCR using R143 on DNA isolated from these cultures showed concordance in 22 of 24 cases. In two cases there was discordance: both times PCR results showed correctly the presence of A. fumigatus, initially not detected by culture. R143 is an A. fumigatus specific random primer, with potential for use in detection of A. fumigatus DNA in clinical specimens.

There is still no satisfactory explanation for the low catalytic activity of tissue factor (TF)/factor VII(a) complexes towards coagulation factor X, as found on the apical surface side of cell layers. It has been hypothesized that TF exists in a latent form. Layers of cultured human smooth muscle cells, constitutively expressing TF, were immunogold-labeled for TF in situ and processed for electron microscopy. We showed that, besides internalization and accumulation in lysosomal-like structures, TF remained associated with noncoated, flask-shaped microinvaginations of the plasma membrane. These invaginations were identified as caveolae. In regions in which intercellular contacts were interrupted, more TF-positive caveolae were observed. Enzymatically detached smooth muscle cells exhibited a similar enlargement of caveolar structures. Concomitantly, an increase of catalytic activity of apically formed TF/VIIa complexes towards factor X was found on the suspended cells. We speculate that caveolae-associated TF may function as a latent pool of procoagulant activity, which can rapidly be activated at sites in which vessel wall integrity is lost. (C) 1996 by The American Society of Hematology.

A discrepancy exists between basal tissue factor (TF) expression found in endothelial cell cultures and the failure to detect TF in unpertubated endothelial cells in vivo. We demonstrated that basal TF expression in endothelial cell cultures originated from contaminating cells. These cells were ultrastructurally and flowcytometrically identified as smooth muscle cells. The cell cultures had been obtained from collagenase-treated human umbilical cord vessels. Histologic studies revealed that after collagenase treatment the basement membrane was digested and underlying structures were disrupted at some areas of the vein. We selected chymotrypsin as an alternative for the isolation of endothelial cells. Using chymotrypsin, the endothelial lining was selectively lost leaving the basement membrane undisturbed. Furthermore, use of chymotrypsin instead of collagenase minimized the level of basal TF activity. Taken together, we demonstrated that basal TF expression in endothelial cell cultures is caused by contaminating smooth muscle cells. This contamination can strongly be reduced using chymotrypsin instead of collagenase for isolation of endothelial cells.