Detection of Aspergillus species in blood and bronchoalveolar lavage samples from immunocompromised patients by means of 2-step polymerase chain reaction: clinical results.

Bronchoalveolar lavage (BAL) samples from 67 patients who were at high risk for invasive aspergillosis were examined using a recently developed 2-step polymerase chain reaction (PCR) that detects

Functional activity of HERV-K-T47D-related long terminal repeats.

The human genome contains a family of endogenous retroviruses, HERV-K(HML-4), that comprises the full-length provirus HERV-K-T47D, five related elements, and hundreds of solitary long terminal repeats (LTRs). We here show that HERV-K-T47D-related LTRs are dispersed over all human chromosomes and have arisen after the divergence of Old and New World monkeys. By screening a cDNA library derived from the human mammary carcinoma cell line T47D with a HERV-K-T47D LTR probe, we isolated several clones containing LTR/cellular gene chimeras and assessed the transcriptional activity of these LTRs in transient transfection experiments. All LTRs were able to drive the expression of a reporter gene, thereby displaying distinct activities in different cell lines. We found that sequences located downstream of the LTR-U3 region modulate the level of gene expression. Based on the impact of the R region we distinguished between three different LTR types; the activity of type I LTRs was enhanced in the presence of the LTR-R region in all cell lines tested, whereas a type II LTR was downregulated. Type III LTRs are characterized by lacking or having a varying influence of the R region that was dependent on the cell line used. Finally, our results attribute to LTR-U5-gag sequences a role in determining LTR activity.

Cell type-specific expression and promoter activity of human endogenous retroviral long terminal repeats.

Evolution over millions of years has adapted several thousand copies of retrovirus-like elements and over 10 times as many solitary long terminal repeats (LTRs) to their present location in the human genome. Transcription of these human endogenous retroviruses (HERVs) has been detected in various cells and tissues, and in some cases their transcriptional control elements have been recruited by cellular genes. We used a retroviral pol-specific expression array to obtain a HERV transcription profile in a variety of human cells such as epidermal keratinocytes, liver cells, kidney cells, pancreatic cells, lymphocytes, and lung fibroblasts. This rapid screening test revealed a distinct HERV pol-expression pattern in each cell type tested so far. About 40 different U3/R regulatory sequences from the HERV-H and HERV-W families were then amplified from actively transcribed 3'HERV LTRs of various cell lines and tissues. Their promoter activities were compared with LTR sequences of other known HERV families in 12 human cell lines using a transient luciferase reporter system. Expression of the isolated HERV LTRs varied significantly in these cell lines, in some cases showing strict cell type specificity. These results suggest that endogenous retroviral LTRs may be a valuable source of transcriptional regulatory elements for the construction of targeted retroviral expression vectors.

Large-scale serial analysis of gene expression reveals genes differentially expressed in ovarian cancer.

Difficulties in the detection, diagnosis, and treatment of ovarian cancer result in an overall low survival rate of women with this disease. A better understanding of the pathways involved in ovarian tumorigenesis will likely provide new targets for early and effective intervention. Here, we have used serial analysis of gene expression (SAGE) to generate global gene expression profiles from various ovarian cell lines and tissues, including primary cancers, ovarian surface epithelia cells, and cystadenoma cells. The profiles were used to compare overall patterns of gene expression and to identify differentially expressed genes. We have sequenced a total of 385,000 tags, yielding >56,000 genes expressed in 10 different libraries derived from ovarian tissues. In general, ovarian cancer cell lines showed relatively high levels of similarity to libraries from other cancer cell lines, regardless of the tissue of origin (ovarian or colon), indicating that these lines had lost many of their tissue-specific expression patterns. In contrast, immortalized ovarian surface epithelia and ovarian cystadenoma cells showed much higher similarity to primary ovarian carcinomas than to primary colon carcinomas. Primary tissue specimens therefore appeared to be a better model for gene expression analyses. Using the expression profiles described above and stringent selection criteria, we have identified a number of genes highly differentially expressed between nontransformed ovarian epithelia and ovarian carcinomas. Some of the genes identified are already known to be overexpressed in ovarian cancer, but several represent novel candidates. Many of the genes up-regulated in ovarian cancer represent surface or secreted proteins such as claudin-3 and -4, HE4, mucin-1, epithelial cellular adhesion molecule, and mesothelin. Interestingly, both apolipoprotein E (ApoE) and ApoJ, two proteins involved in lipid homeostasis, are among the genes highly up-regulated in ovarian cancer. Selected serial analysis of gene expression results were further validated through immunohistochemical analysis of ApoJ, claudin-3, claudin-4, and epithelial cellular adhesion molecule in archival material. These experiments provided additional evidence of the relevance of our findings in vivo. The publicly available expression data reported here should stimulate and aid further research in the field of ovarian cancer.

HERV-K-T47D-Related long terminal repeats mediate polyadenylation of cellular transcripts.

The human genome harbors thousands of long terminal repeats (LTRs) that are derived from endogenous retroviruses and contain elements able to regulate the expression of neighboring cellular genes. We have investigated the ability of human endogenous retroviral (HERV)-K LTRs to provide transcriptional processing signals for nonviral sequences. Four chimeric cDNA clones isolated from a cDNA library derived from the human cell line T47D were found to be polyadenylated within an HERV-K-T47D-related LTR. Two transcripts containing an as yet unknown cellular sequence were probably derived from the same genomic locus but their 3' ends were processed at different positions of the LTR. Structural analysis of the polyadenylation site suggests RNA stem-loop structures similar to the HTLV-1 Rex responsive element that bring the two remote AAUAAA and GU-rich elements into the spatial juxtaposition necessary for correct 3' end processing. The cellular part of the third chimeric clone shows significant homology to an exon of the human tyrosine phosphatase 1 gene, although oriented in the antisense direction compared to the adjacent LTR. Furthermore, we found that the 3' untranslated region of the human transmembrane tyrosine kinase gene FLT4 is probably derived from a partial HERV-K-T47D LTR sequence. Taken together, our data suggest that LTRs of the HERV-K-T47D family display biological function by mediating polyadenylation of cellular sequences.

Rapid identification of all known retroviral reverse transcriptase sequences with a novel versatile detection assay.

We have developed a highly sensitive, universal assay that allows detection as well as identification of all known retroviral reverse transcriptase (RT)-related nucleic acids in a biological sample by a single two-step experiment. The assay combines polymerase chain reaction (PCR) and reverse dot-blot hybridization (RDBH), using an array of immobilized synthetic retrovirus-specific oligonucleotides and two sets of mixed oligo primers (MOPs). These primers were derived from highly conserved motifs found in all known reverse transcriptase genes. The PCR/RDBH assay was used for qualitative analyses of human endogenous retrovirus (HERV) transcription in peripheral blood mononuclear cells (PBMCs) and in particles released by the human mammary carcinoma-derived cell line T47D. Sensitivity was further demonstrated by detection of down to 10 copies of pig endogenous retrovirus (PERV) DNA in human cDNA samples. Therefore, this assay is particularly useful for the identification of retroviral sequences in xenografts as well as in recipients of xenografted tissues and organs. Moreover, it is a valuable tool to detect retroviral transcripts and particles in cell cultures used for production of therapeutic polypeptides. The assay is further suitable for monitoring vector preparation used in human gene therapy to exclude transfer of copackaged endogenous retroviruses into target cells.

HERV-IP-T47D, a novel type C-related human endogenous retroviral sequence derived from T47D particles.

A new type C retrovirus-related endogenous pol sequence (ERV-FTD) found to be occasionally copackaged in retrovirus-like particles released by the human mammary carcinoma cell line T47D was used to screen a human genomic library (Seifarth W, Skladny H, Krieg-Schneider F, Reichert A, Hehlmann R, and Leib-Mösch C: J Virol 1995;69:6408-6416). The DNA sequence of one full-length clone now reveals a human endogenous proviral sequence (HERV) of 4190 bp in length comprising a 5' LTR (489 bp) and regions with 37 and 74% overall amino acid homology to RTVL-Ia gag and pol genes, respectively. About 35 related elements were found to be distributed on all human chromosomes except 16, 17, and Y. Sequence comparisons with Mo-MuLV and various type C-related HERVs suggest that despite a proline primer-binding site this novel HERV element, now named HERV-IP-T47D, can be assigned to one family together with known HERV-I elements. Phylogenetic analyses of 5 proviral and 25 solitary LTR sequences confirmed the existence of two distinct but closely related subgroups of the HERV-IP superfamily in the primate genome. In contrast to most known HERV-families, the evolutionary age of HERV-IP elements dates back prior to the divergence of New and Old World monkeys. Despite their old age, members of the HERV-IP family are still transcriptionally active and were found to be highly expressed in specific human tissues such as liver and kidney.

Systemic infections with Candida sp. and Aspergillus sp. in immunocompromised patients with hematological malignancies: current serological and molecular diagnostic methods.

Within recent years, novel serological and molecular methods have been developed to improve the early diagnosis of invasive fungal infections especially in patients with malignant hematological diseases being at highest risk of developing these life-threatening infections. Early diagnosis is essential for adequate therapeutic management, which, however, often remains difficult since the diagnostic tools clinically used either lack specificity or sensitivity, or both at worst. The clinical value, the advantages and remaining problems of recently developed, more sensitive and specific serological assays and molecular methods are reviewed.

Specific detection of Aspergillus species in blood and bronchoalveolar lavage samples of immunocompromised patients by two-step PCR.

The increasing incidence of aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the currently limited diagnostic tools. We developed a two-step PCR assay that specifically amplifies a region of the 18S rRNA gene that is highly conserved in Aspergillus species. A number of primers with the least homology to equivalent human or Candida gene sequences were screened for the pairs that gave the highest sensitivity and specificity. No cross-reaction with the wide range of fungal and bacterial pathogens so far tested was observed. This assay allows direct and rapid detection of down to 10 fg of Aspergillus DNA corresponding to 1 to 5 CFU per ml of blood. A total of 315 blood and bronchoalveolar lavage samples from 140 subjects, including 93 patients at risk for invasive fungal disease, were screened. The result was a 100% correlation between positive histology, culture, or high-resolution computed tomography findings and PCR results. The test specificity was 89%. Our data point to the considerable potential clinical value of this simple, specific, rapid, and inexpensive PCR assay for improving the means of early diagnosis of systemic aspergillosis in high-risk patients.

Proviral structure, chromosomal location, and expression of HERV-K-T47D, a novel human endogenous retrovirus derived from T47D particles.

We previously described that type B retrovirus-like particles released from the human mammary carcinoma cell line T47D are pseudotypes and package retroviral RNA of different origins (W. Seifarth, H. Skladny, F. Krieg-Schneider, A. Reichert, R. Hehlmann, and C. Leib-Mösch, J. Virol. 69:6408-6416, 1995). One preferentially packaged retroviral sequence, ERV-MLN, has now been used to isolate the corresponding full-length provirus from a human genomic library. The 9,315-bp proviral genome comprises a complete retroviral structure except for a 3' long terminal repeat (LTR) truncation. A lysine tRNA primer-binding site and phylogenetic analyses assign this human endogenous retroviral element, now called HERV-K-T47D, to the HML-4 subgroup of the HERV-K superfamily. The gag, prt, pol, and env genes exhibit 40 to 60% amino acid identity to HERV-K10. HERV-K-T47D is located on human chromosome 10, with five closely related elements on chromosomes 8, 9, 15, 16, and 19 and several hundred HERV-K-T47D-related solitary LTRs dispersed over the human genome. HERV-K-T47D-related sequences are detected in the genomes of higher primates and Old World monkeys but not in those of New World monkeys. High HERV-K-T47D transcription levels were observed in human placenta tissue, whereas transcription in T47D cells was strictly steroid dependent.

Secondary H/D isotope effect on hydrogen-bonded hydroxyl groups as a tool for recognizing distance constraints in conformational analysis of oligosaccharides.

An 'isotopomer-selected NOE' (ISNOE) method for the unequivocal identification of mutually hydrogen-bond-linked hydroxyl groups is described. It relies on the fact that the OH group's signal patterns obtained for a partially deuterated sample originate from both isotopomers of the 'partner' hydroxyl, whereas a NOE for this group can originate from cross-relaxation with the protio isotopomer of this hydroxyl only. Hence, the isotopically shifted component of this group's signal does not appear in a ROE difference spectrum obtained with selective excitation of the 'partner' hydroxyl. This method is also applicable in those cases when only one of two mutually hydrogen-bonded groups exhibits resolvable isotope shifts. Furthermore, it is shown that isotope shifts may occur even for pairs of OH groups that are not mutually hydrogen-bonded, if these participate in hydrogen bonds with other hydroxyls and thereby affect conformational equilibria. The ISNOE experiment enables one to distinguish between these two sources of isotope shifts. Since the O[Symbol: see text][Symbol: see text][Symbol: see text]O distance for hydrogen-bonded hydroxyls in sugars is known to lie between 2.7 and 3.0 A , the hydrogen bonds established by ISNOE can be used in conformational analysis as reliable, motionally non-averaged distance constraints for the conformations containing these bonds.

High-level expression of the retinoic acid receptor beta gene in normal cells of the uterine cervix is regulated by the retinoic acid receptor alpha and is abnormally down-regulated in cervical carcinoma cells.

Retinoic acid (RA) is essential for regulation of epithelial cell differentiation. The intracellular effects of RA are mediated by RA-binding nuclear receptors, including the RA receptors (RARs) alpha, beta, and gamma. The ligand-activated receptors induce the transcription of target genes by binding to RA-responsive elements in the promoter regions. One target gene is the RAR beta gene, which encodes a potential tumor suppressor. Loss of RA inducibility of RAR beta gene expression is assumed to play a role in the development of several types of human carcinomas, including carcinomas of the uterine cervix. We have analyzed RAR beta gene expression in normal cervical cells and in cervical carcinoma cell lines. The results show that the RAR beta mRNA levels are high and RA inducible in the primary keratinocytes, whereas they are low and not inducible or only slightly inducible by RA in all of the cervical carcinoma cell lines analyzed. The basal and the RA-induced RAR beta mRNA levels tend to increase with senescence of the normal cells. Fusion of primary ectocervical keratinocytes with HeLa cervical carcinoma cells revealed that the characteristics of RAR beta gene expression of the normal cells are dominant over that of the tumor cells. Using synthetic retinoids with receptor-preferential agonist activities and a RAR alpha-specific antagonist, we show that RAR alpha is the major endogenous RAR subtype for induction of RA-dependent RAR beta gene expression. Taken together, our results indicate that abnormal downregulation of RAR beta gene expression may be an important step in the multifactorial process of cervical carcinogenesis.

Different ligand responsiveness of human retinoic-acid-receptor beta-gene transcription in tumorigenic and non-tumorigenic cervical-carcinoma-derived cell lines is mediated through a large retinoic-acid-response domain.

The retinoic-acid-receptor beta gene (RAR-beta) encodes a suspected tumor suppressor for several types of human carcinomas. RAR-beta transcription is induced by retinoic acid (RA) through retinoid receptors which bind as heterodimers of a RA-activated RA receptor (RAR) and a retinoid X receptor to the RA-responsive element in the RAR-beta promoter region (beta RARE). RA inducibility of RAR-beta gene expression is often lost or reduced in human carcinoma cells. As previously shown, the RAR-beta gene is highly RA-inducible in nontumorigenic HeLa x fibroblast hybrid cells, but neither in HeLa cervical carcinoma cells nor in a tumorigenic hybrid segregant line. We report here that severe reduction of RA-induced RAR-beta mRNA levels is a general feature of tumorigenic HeLa x fibroblast segregants. To study the molecular basis of differential RA inducibility, we have performed transient transfection assays in HeLa and nontumorigenic 444 hybrid cells using reporter constructs with different 5' and internal deletions of the RAR-beta transcription-control region. Remarkably, maximal RA inducibility in 444 cells required the integrity of the complete RAR-beta upstream region. In HeLa cells, all reporter constructs showed only low RA inducibility levels. The differential RA inducibility in 444 and HeLa cells could be conferred by the RAR-beta upstream region, but not by subfragments of it, on a heterologous RA-responsive promoter. The data indicate that maximal RA inducibility of RAR-beta gene transcription in nontumorigenic 444 cells depends on the cooperation of the beta RARE with additional upstream elements. All elements together constitute a large RA response domain as the higher-order transcription control unit. The communication between the upstream elements and the beta RARE seems to be disturbed in HeLa cells. Similar defects may be responsible for the loss of RA responsiveness of RAR-beta gene expression in other human tumors.

Retinoic acid-mediated repression of human papillomavirus 18 transcription and different ligand regulation of the retinoic acid receptor beta gene in non-tumorigenic and tumorigenic HeLa hybrid cells.

Human papillomavirus type 18 (HPV18) belongs to the group of genital papillomaviruses involved in the development of cervical carcinomas. Since retinoic acid (RA) is a key regulator of epithelial cell differentiation and a growth inhibitor in vitro of HPV18-positive HeLa cervical carcinoma cells, we have used HeLa and HeLa hybrid cells in order to analyse the effects of RA on expression of the HPV18 E6 and E7 oncogenes and of the cellular RA receptor genes RAR-beta and -gamma. We show here that RA down-regulates HPV18 mRNA levels apparently due to transcriptional repression. Transient cotransfection assays indicated that RARs negatively regulate the HPV18 upstream regulatory region and that the central enhancer can confer RA-dependent repression on a heterologous promoter. RA treatment resulted in induction of RAR-beta mRNA levels in non-tumorigenic HeLa hybrid cells, but not in tumorigenic hybrid segregants nor in HeLa cells. No alterations of the RAR-beta gene or of the HeLa RAR-beta promoter could be revealed by Southern and DNA sequence analysis, respectively. As determined by transient transfection assays, however, the RAR-beta control region was activated by RA more strongly in non-tumorigenic hybrid cells than in HeLa cells, thus indicating differences in trans-acting regulatory factors. Our data suggest that the RARs are potential negative regulators of HPV18 E6 and E7 gene expression, and that dysregulation of the RAR-beta gene either causatively contributes to or is an indicator of tumorigenicity in HeLa and HeLa hybrid cells.