Polymerase chain reaction for microsporidian DNA in gastrointestinal biopsy specimens of HIV-infected patients.

OBJECTIVE: To study the accuracy of polymerase chain reaction (PCR) for microsporidian DNA in gastrointestinal biopsy specimens of HIV-infected patients for the diagnosis of intestinal microsporidiosis. SETTING: Infectious disease in- and outpatient clinic of a university hospital in Cologne, Germany. PATIENTS: Forty-six HIV-infected patients with diarrhoea. METHODS: PCR and Southern blot hybridization were performed using DNA extracted from intestinal biopsy specimens with primers and probes from the small subunit rRNA gene of Enterocytozoon bieneusi and Septata intestinalis. Histological examination of intestinal biopsy specimens was performed using a fluorescence technique. Transmission electron microscopy of intestinal biopsy specimens was performed in 13 patients. RESULTS: Amplification and Southern blot hybridization with species-specific primers and probes gave positive results in 10 patients for E. bieneusi, and in 10 patients for S. intestinalis. Overall, five cases of double infection with E. bieneusi and S. intestinalis were seen when both primer pairs and probes were used. Histological examination showed microsporidian spores in all 15 cases, but light microscopy was unable to distinguish between species in almost all cases. CONCLUSIONS: PCR detection of microsporidian DNA in intestinal biopsy specimens can be used reliably for the diagnosis of intestinal microsporidiosis in HIV-infected patients and is also useful for species differentiation between microsporidia. Infections with S. intestinalis and double infections with two types of microsporidia appear to be more common than previously described.

Increased expression of alpha-enolase in c-jun transformed rat fibroblasts without increased activation of plasminogen.

Two-dimensional gel electrophoresis was used to identify polypeptides differentially expressed between normal and c-jun transformed rat fibroblasts. The level of a 49 kDa polypeptide was 3-fold elevated in c-jun transformed cells. Sequence analysis by ion trap mass spectrometry identified the polypeptide as rat alpha-enolase. Enolase functions as a cell surface receptor for plasminogen, suggesting that upregulation may increase plasminogen activation and cell surface proteolysis important for tumor growth. However, no difference was observed between normal and transformed cells in formation of plasmin, suggesting that upregulation of alpha-enolase may contribute to an increased metabolic capacity, but not to increased plasminogen activation.

Napsin A, a member of the aspartic protease family, is abundantly expressed in normal lung and kidney tissue and is expressed in lung adenocarcinomas.

A pair of 35 kDa polypeptides (TAO1/TAO2) are expressed in more than 90% of all primary lung adenocarcinomas but not in other major malignancies. Mass spectrometry of tryptic peptides showed that TAO1/TAO2 is identical to napsin A, a recently described member of the aspartic proteinase family. The site of processing of pronapsin A to the mature form was located. Napsin expression was detected in human lung adenocarcinoma tumors, compatible with the marker nature of TAO1/TAO2 in the diagnosis of primary lung adenocarcinoma. This is important since identification of markers which can distinguish primary lung adenocarcinomas from distant metastases is desirable. Northern blot analysis showed expression of napsin also in normal lung and kidney tissue, and in situ hybridization showed expression in type II alveolar cells of the lung. This protease is concluded to have a specific functional role in the normal alveolar epithelium and is a candidate protease for the proteolytic processing of surfactant precursors.

Limited invasive capacity of plt plus ras transformed rat fibrosarcoma cells effective in experimental metastasis.

Nine cell lines were isolated after cotransfection of rat embryo fibroblasts with polyomavirus large-T (plt) and T24-ras oncogenes. Five of these lines were highly tumorigenic following subcutaneous injection, but differed in their metastatic and in vitro invasive properties. Two cell lines, expressing low levels of ras mRNA, showed low capacity for experimental metastasis. Three cell lines, expressing high levels of ras mRNA, were tumorigenic and showed high capacity for experimental metastasis. High expression of interstitial collagenase, stromelysin and 92 kDa type IV collagenase was observed in the highly metastatic cell lines. Immunochemical analysis revealed that these cell lines expressed apparently wild-type p53 protein. Furthermore, the level of a 43 kDa/pI 5,44 polypeptide was elevated and the levels of a series of 41 to 43 kDa acidic polypeptides were decreased in the metastatic cells. Within this panel of transformed cell lines, high capacity for experimental metastasis did not correlate with high chemoinvasive capacity in the reconstituted basal membrane assay. The limited invasive propensity could not be attributed to low chemotactic or adhesive capacity. We conclude that in vitro invasion does not correlate with experimental metastasis in this model system.

Genotypic and phenotypic characterization of two newly established renal cell carcinoma cell lines.

Two new cell lines from human renal cell carcinoma are reported. Primary cell cultures from 75 consecutive cases of nephrectomy and metastatic surgery due to different stages of RCC during 4 years were studied. Two cell cultures could be propagated for more than 50 passages in vitro. HN4 was derived from a grade III clear cell carcinoma. HN51 originated from a metastatic brain lesion of a clear cell carcinoma grade III. Karyotype analysis of HN4 revealed triploidy with a clonal aberration, der(10)t(3;10)(q13;p12). HN51 also had a triploid pattern with different marker chromosomes but without any clonal aberration. Loss of heterozygosity studies revealed no loss of heterozygosity on 3p or other chromosomal markers in HN4 but LOH was found on one 3p marker and one 14q marker in addition to all 17 p and q markers in HN51. In vitro light microscopy showed distinctly different morphology in the two cell lines although they both had a typical epithelial growth pattern. Doubling times in vitro were low but slightly higher for HN51. Repeated tumorigenenic experiments in athymic mice only gave rise to subcutaneous tumors with HN51. On characterization by 2-dimensional gel electrophoresis, the two cell lines exhibited different polypeptide patterns with higher expression of proliferating cell nuclear antigen in HN51 and higher expression of glutathione-S-transferase in HN4 constituting the most prominent differences.

[Fibrinolytic therapy of deep vein thrombosis]. / Lysetherapie bei tiefen Beinvenenthrombosen.

BACKGROUND: The most important complications of deep vein thrombosis are pulmonary embolism and postthrombotic syndrome. While the medicine of lethal pulmonary embolism is reduced to less than 2% by conventional anticoagulation, fibrinolytic therapy aims at a reduction of the greater than 50% incidence of postthrombotic syndrome. The optimal therapeutic regimen concerning risks and effect has not been established yet. RESULTS: A review of 26 studies involving ultrahigh-dose streptokinase (UHSK), urokinase (UK), and tissue-type plasminogen activator (rt-PA) shows the highest success rate for UHSK (45% complete and 40% parital patency), whereas there are lower rates for UK (25% and 40%) and low-dose locoregionally applied rt-PA (22% and 44%). The studies were not directly comparative, however. Published data concerning complications range from 1.7% mortality for UHSK to 0.9% for UK and 0.0% for rt-PA. Success criteria, however, are varying and not well defined. The influence of fibrinolytic therapy on the incidence of postthrombotic syndrome has not been established prospectively, but a reduction by 40 to 50% can be assumed. Calf vein thromboses are not indication for lytic therapy. In patients with iliacal vein thromboses there is an increased risk of pulmonary embolism using UHSK. CONCLUSIONS: UHSK can be regarded the standard concerning success rate in deep vein thromboses. DATA involving locoregional therapy with rt-PA are inconsistent and worse, but bleeding complications might be less frequent. Large prospective studies evaluating the impact on incidence and severity of the postthrombotic syndromes, which involve a controlled application of compression therapy are needed.

Undifferentiated pelvic adenocarcinomas: diagnostic potential of protein profiling and multivariate analysis.

BACKGROUND AND AIMS: Despite improved techniques, the determination of tumor origin in poorly differentiated adenocarcinomas still remains a challenge for the pathologist. Here we report the use of protein profiling combined with principal component analysis to improve diagnostic decision-making in tumor samples, in which standard pathologic investigations cannot present reliable results. MATERIALS AND METHODS: A poorly differentiated adenocarcinoma of unknown origin located in the pelvis, infiltrating the sigmoid colon as well as the ovary, served as a model to evaluate our proteomic approach. Firstly, we characterized the protein expression profiles from eight advanced colon and seven ovarian adenocarcinomas using two-dimensional gel electrophoresis (2-DE). Qualitative and quantitative patterns were recorded and compared to the tumor of unknown origin. Based on these protein profiles, match sets from the different tumors were created. Finally, a multivariate principal component analysis was applied to the entire 2-DE data to disclose differences in protein patterns between the different tumors. RESULTS: Over 89% of the unknown tumor sample spots could be matched with the colon standard gel, whereas only 63% of the spots could be matched with the ovarian standard. In addition, principal component analysis impressively displayed the clustering of the unknown case within the colon cancer samples, whereas this case did not cluster at all within the group of ovarian adenocarcinomas. CONCLUSION: These results show that 2-DE protein expression profiling combined with principal component analysis is a sensitive method for diagnosing undifferentiated adenocarcinomas of unknown origin. The described approach can contribute greatly to diagnostic decision-making and, with further technical improvements and a higher throughput, become a powerful tool in the armentarium of the pathologist.

TCDD receptor ligands present in extracts of urban air particulate matter induce aryl hydrocarbon hydroxylase activity and cytochrome P-450c gene expression in rat hepatoma cells.

In previous studies we have shown that substances associated with particulates collected from urban air and automobile exhaust bind with high affinity to the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor present in rat liver cytosol. In this study we used a rat hepatoma cell line, H4IIE, to investigate the effect of such substances on an enzyme system, aryl hydrocarbon hydroxylase (AHH), which is regulated via the TCDD receptor. The results demonstrate that AHH activity in the H4IIE cell line can be induced by extracts of particulates collected from urban air and automobile exhausts in a dose-dependent manner, and that the AHH activity is inducible by five polycyclic aromatic hydrocarbons (PAHs) including 1-/3-nitrobenzo[a]pyrene and 6-chlorochrysene, all present in extracts of particulates from urban air and automobile exhausts. The induction of AHH activity is correlated to apparent TCDD receptor affinity for investigated PAHs (r = 0.85) and particulate extracts. Biochemically, treatment of the cells with 5,6-benzoflavone significantly increased the level of cytochrome P-450c but not P-450d as shown by immunoblotting and analysis of mRNA levels. The data indicate that substances present in extracts of urban air particulates can interact with the TCDD receptor in intact cells and cause an accumulation of cytochrome P-450c mRNA leading to an increased synthesis of the gene product and thus an increase in enzyme activity.

Cancer proteomics: from identification of novel markers to creation of artifical learning models for tumor classification.

Studies of global protein expression in human tumors have led to the identification of various polypeptide markers, potentially useful as diagnostic tools. Many changes in gene expression recorded between benign and malignant human tumors are due to post-translational modifications, not detected by analyses of RNA. Proteome analyses have also yielded information about tumor heterogeneity and the degree of relatedness between primary tumors and their metastases. Results from our own studies have shown a similar pattern of changes in protein expression in different epithelial tumors, such as decreases in tropomyosin and cytokeratin expression and increases in proliferating cell nuclear antigen (PCNA) and heat shock protein expression. Such information has been used to create artificial learning models for tumor classification. The artificial learning approach has potential to improve tumor diagnosis and cancer treatment prediction.

Prognostic value of the combined assessment of proliferating cell nuclear antigen immunostaining and nuclear DNA content in invasive human mammary carcinomas.

The expression of the S-phase associated, nuclear protein proliferating cell nuclear antigen (PCNA) was investigated in routinely paraffin-embedded surgical specimens from 209 breast cancer patients. Cytometric DNA assessments were performed on fine-needle aspirates, upon which the primary diagnosis of breast cancer had been based. The mean clinical follow-up was 16 years (range 13-20 years). The percentage of PCNA immunoreactive tumour cells ranged between less than 5 to 60% (mean value 13.34%). There was a direct association between PCNA expression, high histological tumour grade (p < 0.01), and DNA aneuploidy (p = 0.009). In a subgroup of 22 patients with near-diploid DNA distribution patterns the PCNA expression yielded additional prognostic information. Patients with tumours of near-diploid DNA histograms and more than 20% of PCNA immunoreactive neoplastic cells had a significantly worse clinical course, than patients with near-diploid tumours containing less than 20% PCNA immunoreactive cells (p = 0.0001). In contrast, the PCNA immunoreactivity did not yield additional prognostic information for patients with distinctly diploid or highly aneuploid tumour variants. In a multivariate analysis comprising all 209 patients, nodal status (p < 0.01), tumour size (p < 0.01), and DNA ploidy (p < 0.01) were found to have significant prognostic effect. The findings indicate that carcinomas characterised by high proliferative activity and near-diploid DNA distribution patterns can show rapid tumour progression. The combined assessment of the PCNA immunoreactivity and of the nuclear DNA content in routinely processed surgical specimens of breast cancer patients appears to be of prognostic value.

Nonenzymatic extraction of cells from clinical tumor material for analysis of gene expression by two-dimensional polyacrylamide gel electrophoresis.

We have compared different methods of preparation of malignant cells for two-dimensional electrophoresis (2-DE). We found all methods using fresh tissue to be superior compared to methods using frozen tissue. Our results indicate that nonenzymatic methods of preparation of tumor cells, including fine needle aspiration, scraping and squeezing, have advantages over methods using enzymatic extraction of cells. Nonenzymatic methods are rapid, appear to reduce loss of high molecular protein species, and alleviate the necessity of separating viable and nonviable cells by Percoll gradient centrifugation. Using these techniques, high-quality 2-DE maps were derived from tumors of the lung and breast. In the resulting polypeptide patterns, heat shock proteins, non-muscle tropomyosins and intermediate filament were identified. We conclude that nonenzymatic extraction of malignant cells from fresh tumor tissue improves the possibilities that these techniques may be useful in clinical diagnosis.

Genesis of squamous cell lung carcinoma. Sequential changes of proliferation, DNA ploidy, and p53 expression.

Squamous cell lung carcinomas (SCCs) represent a highly malignant group of tumors, and effective treatment is greatly dependent upon early diagnosis. However, objective diagnosis of atypia is difficult and useful markers need to be defined. In this study, genomic instability, cell proliferation, and cellular accumulation of mutant p53, as reflected by DNA aneuploidy, proliferating cell nuclear antigen, and p53 immunoreactivity, respectively, were evaluated in bronchial squamous metaplasia without atypia (n = 4), bronchial squamous metaplasia with low-grade atypia (n = 12), bronchial squamous metaplasia with high-grade atypia (n = 15), early-stage SCC (n = 15), and advanced-stage SCC (n = 33). Our results suggest that hyperproliferation is an early event followed by DNA aneuploidy, which in turn precedes p53 immunoreactivity in the genesis of SCC. We conclude that routine assessment of proliferating cell nuclear antigen, DNA ploidy, and p53 may be valuable for the early diagnosis of SCC.

Two-dimensional polyacrylamide gel electrophoresis of human lung cancer: qualitative aspects of tissue preparation in relation to histopathology.

It is of great importance to identify new objective markers significant for the diagnosis, treatment, and prognosis of human cancers. Cytometric assessment of the DNA content has been shown to be one parameter which correlates excellently with the clinical course of patients with solid cancers of the breast, prostate, and thyroid. However, additional specific marker polypeptides are needed in order to further improve the diagnostic and prognostic sensitivity. Two-dimensional polyacrylamide gel electrophoresis (2-DE) is a unique tool in this field of cancer research, but serious problems concerning intersample variations in the 2-DE pattern must be solved because quantitative variations of potential marker polypeptides are expected to occur at low levels. In this study we examined a modified preparation method for tumor tissues, focusing on the relation between histopathological properties and 2-DE gel quality. We selected a group of human lung cancers sharing similar prognosis; nevertheless, the 2-DE patterns showed significant intersample but low intrasample variations. It is concluded that histopathological features, such as a local homogeneity, and the amounts of connective tissue and serum proteins are critical factors for the successful preparation and high quality of 2-DE-gels.

Characterization of periplasmic Escherichia coli protein expression at high cell densities.

We have used two-dimensional electrophoresis (2-DE) to analyze changes in protein expression profiles during a microbial cultivation process on an industrial scale. An Escherichia coli strain W31 10 containing the gene for recombinant human growth hormone production was used. Samples were taken at time intervals ranging from fast to slow growth rate (late growth phase at high cell density/starvation) and 2-DE analysis combined with image analysis using the PDQuest software showed significant alterations in expression levels of a number of proteins. Twenty-four protein spots were identified using a combination of matching with SWISS-2DPAGE E. coli map, N-terminal sequence analysis and mass spectrometry matrix-assisted laser desorption/ionization (MALDI). Two of the most abundant proteins expressed at late growth phase (pI 5.4/28 kDa and pI 5.5/28 kDa) were subjected to N-terminal sequence analysis after electrotransfer of the proteins from a preparative 2-DE gel to polyvinylidene difluoride (PVDF) membrane. Sequence tags of five amino acids in combination with approximate pI and Mr identified both proteins as deoxyribose phosphate aldolase (gene name deoC). In addition, both spots were subjected to tryptic in-gel digestion and analyzed using MALDI. Peptide mass fingerprints from both spots showed similar MALDI spectra and 10 of 10 tryptic fragments confirmed the identity as deoC. The identification of the acidic variant of deoC on 2-DE gels and the observation of this variant as induced during late growth phase is novel.

Amino acid sequence of the tryptic peptide containing the catalytic-site thiol group of bovine liver uridine diphosphate glucose dehydrogenase.

The catalytic-site thiol groups of UDP-glucose dehydrogenase from bovine liver were carboxymethylated with iodo[2-14C]acetate or with iodoacetamidofluorescein. After the residual thiol groups were carboxymethylated with iodoacetate, the proteins were digested with trypsin. The 14C-labelled peptide from the carboxymethylated enzyme was purified to homogeneity by successive thick-layer chromatography on silica gel, paper electrophoresis and chromatography, and column chromatography on Bio-Gel P-6. Homogeneous fluoresceincarboxamidomethylated peptide was prepared from a tryptic digest of fluoresceincarboxamidomethylated enzyme by specific adsorption--desorption from Sephadex G-25. The sequences of either peptide determined by the manual Edman dansyl procedure is: Ala-Ser-Val-Gly-Phe-Gly-Gly-Ser-Cys-Phe-Glx-Glx-Gly-Lys.

Two-dimensional gel analysis of protein expression in ovarian tumors shows a low degree of intratumoral heterogeneity.

The process of tumor progression leads to the emergence of multiple clones, and to the development of tumor heterogeneity. One approach to the study of the extent of such heterogeneity is to examine the expression of marker proteins in different tumor areas. Two-dimensional gel electrophoresis (2-DE) is a powerful tool for such studies, since the expression of a large number of polypeptide markers can be evaluated. In the present study, tumor cells were prepared from human ovarian tumors and analyzed by 2-DE and PDQUEST. As judged from the analysis of two different areas in each of nine ovarian tumors, the intratumoral variation in protein expression was low. In contrast, large differences were observed when the protein profiles of different tumors were compared. The differences in gene expression between pairs of malignant carcinomas were slightly larger than the differences observed between pairs of benign tumors. We conclude that 2-DE analysis of intratumoral heterogeneity in ovarian cancer tissue indicates a low degree of heterogeneity.

Down-regulation of tropomyosin-2 expression in c-Jun-transformed rat fibroblasts involves induction of a MEK1-dependent autocrine loop.

Overexpression of the c-Jun transcription factor in rodent fibroblasts may result in cell transformation or in apoptosis. The mechanisms whereby c-Jun induces transformation are unknown. We show here that the expression of high-molecular weight tropomyosin-2 (TM-2) is down-regulated in c-jun-transformed FR3T3 rat fibroblasts. However, down-regulation did not seem to be a direct effect of c-Jun on TM-2 gene expression. Thus, TM down-regulation in c-jun-transformed cells was alleviated by inhibitors of Ras (BZA-5B) or MEK1 (PD98059). Furthermore, medium conditioned by c-jun-transformed cells induced TM-2 down-regulation in untransformed cells by a mechanism requiring MEK1. Consistent with a central role for the MEK/ERK, but not SEK/JNK, pathway for TM down-regulation, constitutively active mutants of Raf induced TM down-regulation, whereas constitutively active Rac did not. We also show that anchorage-independent growth of c-jun-transformed cells requires MEK1. These findings suggest that indirect induction of the MEK/ERK pathway is central to c-Jun-induced transformation of rat fibroblasts.

Polypeptide expression in prostate hyperplasia and prostate adenocarcinoma.

Cells were collected from prostate hyperplasias (n = 6) and prostate carcinomas (n = 6) and subjected to two-dimensional gel electrophoresis (2-DE). The resulting polypeptide patterns were analysed with the PDQUEST computer software. Malignant tumors showed significant increases in the level of expression of proliferating cell nuclear antigen (PCNA), calreticulin, HSP 90 and pHSP 60, oncoprotein 18(v), elongation factor 2, glutathione-S-transferase pi (GST-pi), superoxide dismutase and triose phosphate isomerase. In addition, decreases in the levels of tropomyosin-1 and 2 and cytokeratin 18 were observed in prostate carcinomas compared to prostate hyperplasias. This pattern of alterations is similar to that observed in other carcinomas in our previous studies. All malignant tumors showed simultaneous alterations in 5 or more of 9 markers studied, whereas only one case of benign hyperplasia showed alterations in 5 markers. The EST-data base for prostate tumors available from NCI (CGAP) was searched for the expression of the mRNAs corresponding to proteins identified in our gels. Large differences in the relative expression of mRNAs and proteins were observed. Our data show alterations in the pattem of polypeptide expression in prostate carcinomas which are similar to those observed in other carcinomas.

Analysis of polypeptide expression in benign and malignant human breast lesions: down-regulation of cytokeratins.

Malignant progression of tumour cells is caused by the accumulation of genetic defects, which when combined will generate a large phenotypic diversity. Simultaneous quantitation of a large number of gene products in tumour cells is desirable, but difficult to achieve. We have here quantitated the levels of a number of abundant polypeptides in human breast carcinoma cells using two-dimensional gel electrophoresis (2-DE; PDQUEST). For this purpose, tumour cells were prepared from the tissue of 17 breast carcinomas. Fibroadenoma tissue was used as reference for benign cells. An increase of the spot density of the PCNA polypeptide was observed in rapidly proliferating tumour cells, confirming the validity of the procedures used. In the set of 24 polypeptide spots with known identity, decreases in cytokeratin and tropomyosin levels were observed. The levels of all cytokeratin forms resolved (CK7, CK8, CK15 and CK18) were significantly lower in carcinomas than in fibroadenomas. The levels of tropomyosin 2 and 3 were lower in carcinomas than in fibroadenomas. In contrast, the levels of some members of the stress protein family (pHSP60, HSP90 and calreticulin) were higher in carcinomas. Furthermore, changes in the expression of lactate dehydrogenase and GT-pi, but not in nm23, were observed. We conclude that simultaneous analysis of multiple polypeptides in human carcinomas can be achieved by 2-DE and may be useful in prognostic studies, and that malignant progression of breast carcinomas results in the decreased expression of cytokeratin polypeptides. This phenomenon must be considered in studies where cytokeratins are used as markers to identify the epithelial cell compartment in breast carcinomas.