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PubMed1980

Patterns of viral DNA integration in cells transformed by wild type or DNA-binding protein mutants of adenovirus type 5 and effect of chemical carcinogens on integration.

The integration pattern of viral DNA was studied in a number of cell lines transformed by wild-type adenovirus type 5 (Ad5 WT) and two mutants of the DNA-binding protein gene, H5ts125 and H5ts107. The effect of chemical carcinogens on the integration of viral DNA was also investigated. Liquid hybridization (C(0)t) analyses showed that rat embryo cells transformed by Ad5 WT usually contained only the left-hand end of the viral genome, whereas cell lines transformed by H5ts125 or H5ts107 at either the semipermissive (36 degrees C) or nonpermissive (39.5 degrees C) temperature often contained one to five copies of all or most of the entire adenovirus genome. The arrangement of the integrated adenovirus DNA sequences was determined by cleavage of transformed cell DNA with restriction endonucleases XbaI, EcoRI, or HindIII followed by transfer of separated fragments to nitrocellulose paper and hybridization according to the technique of E. M. Southern (J. Mol. Biol. 98: 503-517, 1975). It was found that the adenovirus genome is integrated as a linear sequence covalently linked to host cell DNA; that the viral DNA is integrated into different host DNA sequences in each cell line studied; that in cell lines that contain multiple copies of the Ad5 genome the viral DNA sequences can be integrated in a single set of host cell DNA sequences and not as concatemers; and that chemical carcinogens do not alter the extent or pattern of viral DNA integration.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2001

Cell-matrix contact structures.

Cell-extracellular matrix contacts are points on cell surfaces where adhesion receptors tether cells to matrix and are linked intracellularly to cytoskeletal components. These structures integrate cell organisation within tissues, support cell motility and specialised activities of differentiated cells, and transduce extracellular signals. Current characterisations of matrix contacts are based on morphological and biochemical criteria, yet the levels of definition of different contact types are very varied. Some contacts are surprisingly little-studied given their likely importance in vivo. Here, I describe the general features of matrix contacts, review the functions and molecular composition of major types of transient and stable matrix contacts, and discuss the information that is emerging on contact integration and dynamics in single cells.

Animals↗
PubMed2004

Micro- and nanofluidics for DNA analysis.

Miniaturization to the micrometer and nanometer scale opens up the possibility to probe biology on a length scale where fundamental biological processes take place, such as the epigenetic and genetic control of single cells. To study single cells the necessary devices need to be integrated on a single chip; and, to access the relevant length scales, the devices need to be designed with feature sizes of a few nanometers up to several micrometers. We will give a few examples from the literature and from our own research in the field of miniaturized chip-based devices for DNA analysis, including dielectrophoresis for purification of DNA, artificial gel structures for rapid DNA separation, and nanofluidic channels for direct visualization of single DNA molecules.

Cell Separation↗
PubMed2026

Optimizing NGN2 Dosage Enhances the Neuronal Enrichment of iPSC-Derived Neuronal Cultures.

Proteomic analysis of highly neuron-enriched iPSC-derived cultures can provide valuable insights into the pathogenesis of neurological disease by minimizing confounding signals from non-neuronal populations. However, single-cell iPSC clones with stable NGN2 integration at the AAVS1 locus exhibit spontaneous loss of the mCherry reporter. In addition, following NGN2 induction, cultures frequently contain proliferative progenitor cells that compromise neuronal physical integrity. Here, we show that increased DNA methylation of the EF1-α promoter is associated with mCherry silencing. Importantly, reporter silencing does not affect iNeuron derivation, as evidenced by two lines of evidence. First, single-cell proteomic analysis indicated that mCherry silencing does not drive detectable proteomic divergence in undifferentiated iPSCs. Second, bulk proteomics and immunofluorescence analyses indicated that iPSC-derived neuronal cultures, whether expressing or lacking mCherry, resemble cortical glutamatergic neurons. Instead, the primary confounding factor in iNeuron generation was suboptimal neuronal conversion, which led to cell aggregates comprised of actively proliferating progenitor cells and astrocytes as the culture developed. We found that a 4-day period of NGN2 induction substantially increases neuronal maturation and the expression of neuronal-specific markers. Moreover, transient inhibition of Notch signaling is consistent with a reduction in progenitor cells. Finally, we observed that monoallelic NGN2 integration, rather than biallelic integration, preferentially increases expression of the CNS neuronal marker GPM6A over PNS markers (PRPH, POU4F1, ILS1).

Basic Helix-Loop-Helix Proteins↗
PubMed1989

Genetic recombination of human immunodeficiency virus.

We investigated genetic recombination of the human immunodeficiency virus (HIV) in a tissue culture system. A clonal cell line expressing a single integrated HIV provirus with a termination codon affecting pol gene expression was transfected with different defective mutants derived from an infectious molecular clone of HIV. Replication-competent viral particles were recovered, passaged, and plaque purified. Restriction analyses of the proviral DNA corresponding to several of these viruses indicated that their emergence was the result of genetic recombination.

Cells, Cultured↗
PubMed2005

[Detection of IFN-gamma level in single CD8+ T cell].

AIM: To establish a new method capillary electrophoretic immunoassay laser-induced fluorescence (CEIA-LIF), to detect IFN-gamma level in single CD8(+) T cell. METHODS: CD8(+) T cells were isolated from peripheral blood of two patients with severe aplastic anemia(SAA) and one normal person by Ficoll-Hypaque gradient centrifugation and then were purified by immunomagnetic microbead separation. Then purified CD8(+) T cells were incubated with digitonin for 15 min followed by FITC-anti-IFN-gamma mAb for 20 min. The single cell was detected continuously by CEIA-LIF. The feasibility of the method was confirmed by inverted microscopy and laser scanning confocal fluorescence microscopy. RESULTS: The IFN-gamma content in purified CD8(+) T cells was detected under the condition of cell-membrane integrity. The IFN-gamma level in single CD8(+) T cell from 2 SAA patients was (151.53+/-28.92)zmol and (223.72+/-45.23)zmol, respectively, and much higher that from normal control (47.47+/-17.97)zmol ( P=0.001). CONCLUSION: It is feasible to quantitate IFN-gamma in single CD8(+) T cell by CEIA-LIF. CEIA-LIF might be useful in the clinical detection of intracellular cytokines.

Antigen-Antibody Complex↗
PubMed1991

Human adenovirus type 9-induced rat mammary tumors.

Following subcutaneous inoculation of newborn Wistar-Furth rats with human adenovirus type 9 (Ad9), 16 of 16 female and 0 of 11 male rats developed mammary tumors. Tumor-positive animals usually developed tumors in multiple glands. Histopathological analyses indicated that three general categories of tumor could be identified. Mammary fibroadenomas were the most common tumor type encountered, but phyllodeslike tumors and solid sarcomas were also frequently found. In situ hybridization and immunohistochemical techniques established that benign fibroadenomas were derived from mammary fibroblasts (collagen type I- and vimentin-positive cells) and that malignant tumors were derived from myoepithelial cells (collagen type IV-, vimentin-, and muscle-specific actin-positive cells). The fact that mammary tumors were limited to female rats suggested that female hormones are essential for tumor growth and development. In this regard, ovariectomy of Ad9-infected female rats prevented tumor development, while subsequent diethylstilbestrol (DES) treatment elicited tumor formation. In addition, Ad9-infected and castrated male rats which received DES also developed mammary tumors. Established male mammary tumors regressed when DES treatment was stopped and reappeared after DES treatment was resumed. Together, these results indicate that estrogen is required for both initiation and maintenance of Ad9-induced mammary tumors. Southern blot analysis of high-molecular-weight tumor DNA showed that mammary tumor cells contained single or multiple integrated copies of the entire Ad9 genome. RNase protection experiments established that estrogen receptor as well as Ad9 E1a and E4 mRNAs were expressed in mammary tumors, but Ad9 E3 and, surprisingly, E1b mRNAs were not expressed at detectable levels.

Adenoviruses, Human↗
PubMed1988

Integration and loss of a single v-Ki-ras gene affects tumorigenic potential of human osteosarcoma cells.

The human osteosarcoma cell line Te85 clone F-5 is not tumorigenic in vivo. Its transformation with Kirsten murine sarcoma virus (KiMSV) (KHOS) confers full malignant properties and stable non-tumorigenic revertants of this KHOS cell line have been obtained. Here we show that integration and expression of a single copy of the KiMSV proviral DNA, which is totally lost in the HOS 240S revertant, is responsible for the acquisition of tumorigenicity. Cytogenetic analysis and the absence of a residual LTR copy in the revertant cellular genome suggest that the loss of KiMSV provirus is caused either by chromosomal segregation or by recombination not involving the LTR. In addition analysis of the expression of ras proteins revealed no changes in the pattern of c-ras products and the expression of v-ras only in the KHOS cells. All these data suggest that Te85 and HOS 240S cell lines could represent a human alternative recipient system to rodent cells in studies with oncogenes.

Cell Line↗
PubMed1997

Conformational integrity and ligand binding properties of a single chain T-cell receptor expressed in Escherichia coli.

We recently showed that a soluble, heterodimeric murine D10 T-cell receptor (TCR) (Valpha2Calpha, Vbeta8.2Cbeta) expressed in insect cells binds both Vbeta8.2-specific bacterial superantigen staphylococcal enterotoxin C2 (SEC2) and a soluble, heterodimeric major histocompatibility complex class II I-Ak.conalbumin peptide complex with a low micromolar affinity. To define further the structural requirements for the TCR/ligand interactions, we have produced in Escherichia coli a soluble, functional D10 single chain (sc) TCR molecule in which the Valpha and Vbeta domains are connected by a flexible peptide linker. Purified and refolded D10 scTCR bound to SEC2 and murine major histocompatibility complex class II I-Ak.conalbumin peptide complex with thermodynamic and kinetic binding constants similar to those measured for the baculovirus-derived heterodimeric D10 TCR suggesting that neither the TCR constant domains nor potential N- or O-linked carbohydrate moieties are necessary for ligand recognition and for expression and proper folding of the D10 scTCR. Purified D10 scTCR remained soluble at concentrations up to 1 mM. Circular dichroism and NMR spectroscopy indicated that D10 scTCR is stabilized predominantly by beta-sheet secondary structure, consistent with its native-like conformation. Because of its limited size, high solubility, and structural integrity, purified D10 scTCR appears to be suitable for structural studies by multidimensional NMR spectroscopy.

Amino Acid Sequence↗
PubMed2005

Determination of reactive oxygen species in single human erythrocytes using microfluidic chip electrophoresis.

Reactive oxygen species (ROS) are known to not only mediate the damage of cellular constituents but also to regulate cellular signaling. Analysis of ROS is essential if we wish to understand the mechanisms of cellular alterations. In this paper, a microfluidic chip-based approach to the determination of ROS in single erythrocyte was developed by using a simple crossed-channel glass chip with integrated operational functions, including cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser-induced fluorescence (LIF) detection. Non-fluorescent dihydrorhodamine 123 (DHR 123), which can be oxidized intracellularly by ROS to the fluorescent rhodamine 123 (Rh 123), was used as the fluorogenic reagent. The effect of pH on the migration time of Rh 123 and detection sensitivity was discussed. The present method minimized dilution of intracellular ROS during reaction with DHR 123 and determination. As a result, an extremely low detection limit of 0.8 amol has been achieved. The time required for complete analysis of one human erythrocyte was less than 3 min. A migration time precision of 4.1% RSD was obtained for six consecutively-injected cells. Upon stimulation with 4 mmol/l H2O2 for 10 min, the intracellular ROS concentration was found to increase on average by about a factor of 8.4.

Calibration↗
PubMed2003

Temporal integration by calcium dynamics in a model neuron.

The calculation and memory of position variables by temporal integration of velocity signals is essential for posture, the vestibulo-ocular reflex (VOR) and navigation. Integrator neurons exhibit persistent firing at multiple rates, which represent the values of memorized position variables. A widespread hypothesis is that temporal integration is the outcome of reverberating feedback loops within recurrent networks, but this hypothesis has not been proven experimentally. Here we present a single-cell model of a neural integrator. The nonlinear dynamics of calcium gives rise to propagating calcium wave-fronts along dendritic processes. The wave-front velocity is modulated by synaptic inputs such that the front location covaries with the temporal sum of its previous inputs. Calcium-dependent currents convert this information into concomitant persistent firing. Calcium dynamics in single neurons could thus be the physiological basis of the graded persistent activity and temporal integration observed in neurons during analog memory tasks.

Action Potentials↗
PubMed1987

Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination.

The phenomenon of homologous recombination, which allows specific gene conversion and gene insertion, can be a powerful system for the study of eukaryotic cell biology. Data are presented demonstrating that integration of a transfected plasmid by homologous recombination occurs in the motile eukaryotic cell Dictyostelium discoideum. A plasmid carrying a G418 resistance gene and the amino terminal half of the myosin heavy chain gene was used to transfect Dictyostelium. A large fraction of the resultant G418-resistant cells had the plasmid integrated into the single genomic copy of the heavy chain gene. These cells, which fail to express the native myosin but express the myosin fragment, are defective in cytokinesis and become large and multinucleate. In spite of the absence of native myosin, these cells, termed hmm cells, exhibit many forms of cell movement, including membrane ruffling, phagocytosis, and chemotaxis. The hmm cells can aggregate but are blocked at a later stage in the Dictyostelium developmental cycle. The hmm cells revert to the wild-type phenotype. Reversion of the hmm phenotype is due to excision and loss of the transforming plasmid. The revertant cells express native myosin, are G418 sensitive, and have a normal developmental cycle. These results constitute genetic proof that the intact myosin molecule is required for cytokinesis and not for karyokinesis.

Cell Division↗
PubMed2026

Genomic characterization of a hypervirulent Aeromonas veronii NN0115 from Nile tilapia and head kidney transcriptome of infected fish reveals B-cell-dominated immune response with specific immunoglobulin downregulation.

Aeromonas veronii is a pathogen of multiple fish species, yet systematic understanding of its infection in Nile tilapia (Oreochromis niloticus) remains limited. A dominant strain, NN0115, was isolated from a natural outbreak and identified as A. veronii by 16S rRNA and whole-genome average nucleotide identity (ANI, 96.33%). Experimental infection revealed high virulence (LD50 = 3.41 × 106 CFU/mL, equivalent to 8.53 × 104 CFU/fish). The genome is 4.58 Mb (58.57% GC) and encodes 4216 proteins. Virulence factor analysis identified 1253 genes, dominated by motility-related (264) and immune modulation (208) factors. Genomic island GI2 harbors 7 virulence genes and two dual-function resistance-virulence genes. The strain is resistant to 9 of 25 agents tested but carries three RND efflux pump genes whose predicted resistance was not phenotypically observed. The head kidney transcriptome of tilapia at 24 h post-bacterial infection identified 773 differentially expressed genes; among them, 57 were immunoglobulin (Ig) genes, and 56 were down-regulated. Integration of published single-cell transcriptomic data showed that non-Ig B-cell marker genes were down-regulated by 32%, whereas Ig genes were reduced by 63%, indicating selective transcriptional suppression of Ig genes rather than a general decrease in B-cell transcriptional activity. Together, this study provides a comprehensive characterization of a highly virulent A. veronii from Nile tilapia and reveals that selective downregulation of B-cell Ig genes is the dominant transcriptional feature of the host head kidney response.

Animals↗
PubMed2026

CERTOMICS: trusted single-cell multiomics pipeline for high-resolution profiling of adoptive cellular immunotherapies.

SUMMARY: Adoptive cellular immunontherapies, such as chimeric antigen receptor (CAR) T cell therapy, have transformed cancer treatment, yet challenges such as resistance, relapse, and high costs limit their efficacy and accessibility. A comprehensive understanding of cellular heterogeneity and molecular profiles is essential to improve these therapies. Advanced single-cell multiomics technologies have the power to analyze the complex interactions between CAR-engineered cells, immune cells, and tumor cells. However, standardized single-cell multiomics computational pipelines specifically tailored to CAR-engineered cell products are lacking. Due to the synthetic nature of CAR transgenes, additional steps for reliable identification and characterization of CAR-positive cells are required but not included in existing data-processing workflows. To address this, we present CERTOMICS, a Nextflow-based, CAR-aware pipeline offering enhanced CERTainty in immunophenotyping and data interpretation, tailored for single-cell multiOMICSprofiling of adoptive cellular immunotherapies. The pipeline standardizes processing 10x Genomics single-cell multiomics data and integrates CAR-specific identification and quality control. Additionally, a curated repository of CAR construct sequences and annotation data is provided, serving as an extensible resource to support the analysis and development of CAR T cell therapies. AVAILABILITY AND IMPLEMENTATION: Detailed documentation of this pipeline, along with a resource on latest FDA-approved CAR therapies is available on our website: https://fraunhofer-izi.github.io/Living-Drugs-Wiki/. The data underlying this article are available on GitHub at https://github.com/fraunhofer-izi/CERTOMICS. The code is also published on Zenodo at https://doi.org/10.5281/zenodo.18709693.

Multiomics↗
PubMed2003

DNA integrity determination in marine invertebrates by Fast Micromethod.

This study was focused toward the adaptation of the previously developed Fast Micromethod for DNA damage determination to marine invertebrates for the establishment of biomonitoring assessment. The Fast Micromethod detects DNA damage (strand breaks, alkali-labile sites and incomplete excision repair) and determines DNA integrity in cell suspensions or tissue homogenates in single microplates. The procedure is based on the ability of the specific fluorochrome dye PicoGreen to preferentially interact with high integrity DNA molecules, dsDNA, in the presence of ssDNA and proteins in high alkaline medium, thereby allowing direct fluorometric measurements of dsDNA denaturation without sample handling and stepwise DNA separations. The results presented herein describe the influence of the DNA amount and the pH of the denaturation media on slopes of the kinetic denaturation curves and calculated strand scission factors (SSFs). The optimal amount of DNA in Mytilus galloprovincialis gills homogenate was found to be 100 ng ml(-1) and the greatest differences in DNA unwinding kinetics (slopes and SSF values) were reached at pH 11.5. The induction of DNA damage and loss of DNA integrity was measured in native DNA isolated from cotton-spinner Holothuria tubulosa, marine sponge Suberites domuncula cells and mussel M. galloprovincialis gills homogenate. DNA damage and loss of DNA integrity were detected after induction by different doses of (gamma-rays, generated by 137Cs 1800 Ci; 0-500 rad in marine sponge S. domuncula cells up to SSFx(-1) values 0.082 +/- 0.012 for the highest radiation dose). Analysis by chemical xenobiotics based on the in vitro action of bleomycin (bleomycin-Fe(II) complex 0-50 or 0-83 microg ml(-1) (microM)) with native DNA from cotton-spinner H. tubulosa and mussel M. galloprovincialis gills homogenate yielded values of 0.537 +/- 0.072 and 0.130 +/- 0.018, respectively. In vivo experiments with mussel M. galloprovincialis gills homogenate by 4-nitroquinoline-N-oxide (NQO; 0-1 microg g(-1) NQO mussel) and benzo[a]pyrene (B[a]P; 0-20 microg g(-1) B[a]P mussel) indicated SSFx(-1) values of 0.121 +/- 0.016 and 0.090 +/- 0.007, respectively, for the highest applied doses of chemical xenobiotics. The analytical technique described here allows simple and fast analysis of DNA integrity, requires very short time for multiple analyses (less than 3 h) and even less than 100 ng DNA per single well (50 ng DNA isolated from cotton-spinner, 12,500 sponge cells or about 10 mg of mussel gills homogenate) in a microplate. This makes the Fast Micromethod applicable for the measurement of DNA integrity of small samples for genotoxicity assessment (biomonitoring), the effects of genotoxins on lower marine taxa or sessile invertebrates in marine environment (e.g. sponges, mussels) and the estimation of directional changes and harmful effects in the ecosystem.

Animals↗
PubMed2026

Monocytes Defined by Platelet Interactions and Oxidative Stress Signaling Underlie HIV-Associated Atherosclerosis.

BACKGROUND: Monocytes contribute to atherosclerosis by migrating into inflamed endothelium and differentiating into lipid-laden macrophages. In people living with HIV, chronic inflammation increases atherosclerosis risk, yet the role of specific monocyte subsets remains unclear. We investigated how distinct monocyte populations contribute to vascular pathology in early HIV-associated atherosclerosis. METHODS: We profiled 123 965 circulating monocytes using single-cell RNA sequencing and integrated plasma microparticle proteomics in 32 individuals stratified by HIV and atherosclerosis status. Supervised learning identified cluster- and disease-specific signatures, validated by platelet-monocyte cocultures, reverse transcription-quantitative polymerase chain reaction, bulk RNA sequencing, flow cytometry, and ELISA. RESULTS: Seven monocyte clusters were identified, including a subset characterized by platelet-monocyte complexes. Bulk RNA sequencing of platelet-monocyte cocultures revealed platelet-driven upregulation of genes involved in inflammation, lipid metabolism, oxidative stress, and endothelial adhesion. Platelet-monocyte complex-derived macrophages secreted higher levels of TGF-β (transforming growth factor-β) and IL-10 (interleukin-10), displayed decreased CD14 and increased CD80/CD86 while retaining CD36, and promoted endothelial-to-mesenchymal transition (decreased expression of CDH5 and PECAM1; increased expression of S100A4 and markers of vascular inflammation (ICAM1), VCAM), and IL-6 (interleukin-6), and CCL2 (C-C motif chemokine ligand 2) secretion). Additionally, CD14+ monocytes from HIV+ atherosclerosis-negative and HIV+ atherosclerosis-positive groups showed enhanced ROS-NRF2 (reactive oxygen species-nuclear factor erythroid 2-related factor 2) pathway activities, supported by increased basal and H2O2-induced p90RSK phosphorylation, indicating oxidative stress priming. CONCLUSIONS: Two monocyte clusters contribute independently to vascular immune dysregulation in people living with HIV. Platelet-monocyte complex-derived macrophages promote endothelial dysfunction while adopting a profibrotic cytokine profile. CD14+ monocytes show heightened oxidative signaling and stress responses, consistent with vascular activation. Together, these mechanisms may accelerate atherosclerosis development in HIV, even in the absence of traditional cardiovascular risk factors.

Humans↗
PubMed2004

Functional integration of embryonic stem cell-derived neurons in vivo.

Pluripotency and the potential for continuous self-renewal make embryonic stem (ES) cells an attractive donor source for neuronal cell replacement. Despite recent encouraging results in this field, little is known about the functional integration of transplanted ES cell-derived neurons on the single-cell level. To address this issue, ES cell-derived neural precursors exhibiting neuron-specific enhanced green fluorescent protein (EGFP) expression were introduced into the developing brain. Donor cells implanted into the cerebral ventricles of embryonic rats migrated as single cells into a variety of brain regions, where they acquired complex morphologies and adopted excitatory and inhibitory neurotransmitter phenotypes. Synaptic integration was suggested by the expression of PSD-95 (postsynaptic density-95) on donor cell dendrites, which in turn were approached by multiple synaptophysin-positive host axon terminals. Ultrastructural and electrophysiological data confirmed the formation of synapses between host and donor cells. Ten to 21 d after birth, all EGFP-positive donor cells examined displayed active membrane properties and received glutamatergic and GABAergic synaptic input from host neurons. These data demonstrate that, at the single-cell level, grafted ES cell-derived neurons undergo morphological and functional integration into the host brain circuitry. Antibodies to the region-specific transcription factors Bf1, Dlx, En1, and Pax6 were used to explore whether functional donor cell integration depends on the acquisition of a regional phenotype. Our data show that incorporated neurons frequently exhibit a lacking or ectopic expression of these transcription factors. Thus, the lack of an appropriate regional "code" does not preclude morphological and synaptic integration of ES cell-derived neurons.

Animals↗
PubMed2005

Standardized detection of Simian virus 40 by real-time quantitative polymerase chain reaction in pediatric malignancies.

BACKGROUND AND OBJECTIVES: Recent studies have detected Simian virus 40 (SV40) DNA in specific human tumors albeit with significant discrepancies in frequency. Possible inefficiency of DNA isolation and different detection methods do not allow comparable and precise quantification of SV40 in human samples. A standardized and common detection method is, therefore, essential for further routine SV40 analysis. DESIGN AND METHODS: We established a real time quantitative (RQ-) polymerase chain reaction (PCR) based TaqMan assay on the LightCycler system for reproducible detection and quantification of SV40 DNA. We used 500 ng of the COS-1 cell line, containing one single integrated copy of SV40 DNA, as the quantification standard. Amplification of b-globin served as the quality control for DNA integrity. For the extraction of the episomal form of SV40 we compared a column and a precipitation-based DNA extraction method. DNA samples from 149 healthy controls, from 26 fresh frozen childhood cases of acute lymphoblastic leukemia (ALL) (B-, BCP- and T-ALL) and from 12 paraffin-embedded osteosarcomas were investigated. RESULTS: The RQ-PCR assay had a linear amplification rate from 10 to 100,000 copies of SV40 in 500 ng genomic DNA. Very low copy numbers of SV40 DNA were detectable in 2/149 (1,3%) blood samples from healthy German controls. Various amounts of SV40 were detectable in 20/26 (77%) childhood ALL samples of German origin and, in part, high amounts were visible in 11/12 (92%) paraffin embedded Hungarian osteosarcomas. The column-based DNA isolation method allowed the detection of both, the integrated and the episomal forms of SV40. INTERPRETATION AND CONCLUSIONS: Our assay provides a standardized and reproducible quantification of SV40 DNA in a wide spectrum of specimens. Exact quantification strongly depends on the source, as well as on the quality, of the DNA used. Quantification of paraffin-embedded DNA generally leads to lower sensitivity of SV40 DNA detection. We strongly recommend this RQ-PCR assay for standardized detection of SV40.

Animals↗