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PubMed1993

Analysis of 6 VNTR loci by 'multiplex' PCR and automated fluorescent detection.

The polymerase chain reaction was used to amplify six small variable number of tandem repeat loci in two reactions (D19S20 co-amplifying with D17S5 and D1S80; D17S766 co-amplifying with D16S83 and D17S24). When coupled with fluorescent detection of the products, this provides a rapid, highly discriminating automated test. Preferential amplification of small alleles, leading to 'allelic dropout' was found to occur in D19S20 and D16S83. Population databases are presented for Caucasians and Afro-Caribbeans at loci D19S20, D16S83 and D17S24, and for Asians at D19S20.

Alleles↗

PubMedDate not supplied

The detection of microsatellite instability in blind endometrial samples--a potential novel screening tool for endometrial cancer in women from hereditary nonpolyposis colorectal cancer families?

Microsatellite instability (MSI) is the phenotypic molecular characteristic of the majority of tumors associated with the hereditary nonpolyposis colorectal cancer syndrome (HNPCC). Women in this group have an increased risk of endometrial cancer (EC). This study aimed to determine whether MSI could be demonstrated in blind endometrial samples from women with EC, HNPCC kindreds undergoing screening for EC, and women with normal endometrium. Twenty-four women with EC, 20 women from HNPCC kindreds, and 20 women undergoing gynecological surgery for benign indications underwent blind sampling. MSI analysis was performed by conventional polymerase chain reaction using fluorescent-labeled primers and automated analysis. Twelve microsatellites were studied with MSI defined as evident when novel alleles were seen in endometrial biopsy samples compared to genomic DNA. Of the 24 EC samples obtained, sufficient DNA for analysis was extracted in 17 cases. Three cases had evidence of MSI in at least 7/12 loci. None of the endometrium from the two other study groups revealed evidence of MSI. This is the first demonstration of MSI in blind endometrial biopsies. The ability to demonstrate MSI in heterogeneous endometrial samples suggests potential for the development of a novel EC screening tool for women in HNPCC kindreds.

Adult↗

PubMed2026

Difficult-to-treat resistant Gram-negative bacteria and genomic resemblances between colonization and infection among patients in an intensive care unit of a tertiary care hospital in Bangladesh.

Colonization with difficult-to-treat-resistant Gram-negative bacteria (DTR-GNB) increases the risk of subsequent infections with limited treatment options. This study aimed to assess the burden of DTR-GNB colonization in ICU patients, explore its association with clinical outcomes, and examine genomic similarities. This secondary analysis included patients enrolled within 24 h of ICU admission between July 2023 and January 2024. Rectal swabs were collected at enrollment, on days 3, 7, and weekly during ICU stay to detect colonization. Bacterial isolates grown on selective chromogenic agar media were identified and tested for antimicrobial susceptibility using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and automated broth microdilution, respectively. Blood, urine, and/or tracheal aspirate cultures were performed if clinically suspected sepsis. Whole-genome sequencing (WGS) was performed on paired colonization and infection isolates, and genomic relatedness was assessed using FastANI, core-genome single-nucleotide polymorphism (SNP) analysis, and phylogenetic reconstruction. Among 373 patients, 181 (48.5%) were colonized with DTR-GNB; 76 (20.4%) at enrollment, and 105 (53.0%) acquired during hospital stay. Among 52 (13.9%) patients evaluated for suspected infection, 30 (57.7%) had positive cultures, predominantly Acinetobacter baumannii (n = 15) and Klebsiella pneumoniae (n = 11) of DTR-phenotypes. Compared to non-colonized patients, patients colonized with DTR-GNB had higher risks of infections (risk ratio [RR]: 2.18, 95% CI: 1.27-3.76) and longer ICU stays (median 7 vs 2 days, P < 0.001). DTR-GNB-infected patients had a higher risk of death (RR: 1.57, 95% CI: 1.34-1.84) compared to patients without DTR-GNB infection. WGS revealed that 13 of 14 paired colonization-infection isolates were conspecific, with three pairs being highly clonal; whereas the remaining pairs showed greater genomic divergence, consistent with the SNP and phylogenetic analyses. While common, more than half acquired DTR-GNB colonization from the ICU. Its association with subsequent infection and prolonged ICU stays underscores the need for enhanced infection prevention and control measures to mitigate nosocomial transmission and improve patient outcomes.IMPORTANCEThis study underscores the growing threat posed by difficult-to-treat resistant Gram-negative bacteria (DTR-GNB) in intensive care units. Nearly half of critically ill patients were colonized, with a considerable proportion acquiring these multidrug-resistant organisms during their ICU stay. Colonization with these pathogens substantially increased the risk of subsequent infections, even by the same colonizing strain, prolonged ICU stays, and likely worsened clinical outcomes due to the unavailability of susceptible antibiotics. Alarmingly, more than 90% of patients infected with DTR-GNB expired in the hospital. These findings highlight the urgent need for robust infection prevention and control strategies to curb nosocomial transmission and mitigate the impact of DTR-GNB on vulnerable patient populations. Addressing this emerging resistance phenotype is critical to improving patient safety and reducing the burden on healthcare systems.

Humans↗

PubMed2004

Hepatitis B virus genotype D prevails in patients with persistently elevated or normal ALT levels in Turkey.

BACKGROUND: The clinical relevance of hepatitis B virus (HBV) genotypes are poorly understood and it is unclear if the prevalence of HBV genotypes differs with the various clinical features of HBV carriers. The aim of our study was to examine the prevalence of the HBV genotype in a group of patients with chronic hepatitis B, compared to a group with chronic inactive hepatitos B surface antigen (HbsAg) carriers. PATIENTS AND METHODS: HBV genotypes were determined in 32 patients with chronic hepatitis B and in 12 chronic inactive HBsAg carriers. 35 males and nine females with a mean age of 33.95 +/- 13.04 were studied. Serum samples were examined for the presence of HBV DNA by polymerase chain reaction (PCR). Samples negative in first round PCR were further amplified with nested PCR. The PCR product was sequenced with the Cy5/5.5 dye primer kit on a Long Read Tower automated DNA sequencer. RESULTS: HBV DNA was detectable in 29 (66%) and 44 (100%) patients by the PCR with universal primers and nested-PCR, respectively. All patients were found to be infected with HBV genotype D. Genotype D was the only detected type found in different clinical forms of chronic HBV infection, in all hepatitis B e antigen (HbeAg)-positive and negative patients, in all patients who had elevated or normal alanine transaminase (ALT) levels and in all ages. CONCLUSION: In the present study we could not find any association between genotype D and distinct clinical phenotypes. Genotype D is the predominant type among hepatitis B carriers residing in our region and is not associated with more severe liver diseases. This genotype did not influence clinical manifestations in carriers with chronic hepatitis B virus infection. However, additional large-scale longitudinal studies are needed to find the relationship of HBV genotypes to liver disease severity and clinical outcomes.

Adult↗

PubMed2005

High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris.

Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a approximately 6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use.

Amino Acid Sequence↗

PubMed2004

Genodermatoses 2003-2004.

PURPOSE OF REVIEW: Many genodermatoses have been linked in recent years to their respective genes. The underlying biology and integrative nature of these genes with other genes and organ systems is beginning to be understood. This paper reviews recent advances in neurocutaneous disorders, ectodermal dysplasias, and the phenomenon of revertant gene mosaicism. RECENT FINDINGS: In neurofibromatosis type 1, molecular assays are being developed to distinguish malignant from benign and premalignant lesions. Clinical mutation analysis for the NF1 gene has been problematic; a sensitive new assay using automated comparative sequence analysis may be helpful. Revision of clinical care guidelines is ongoing. New data for the prospective management of optic pathway gliomas is reviewed. The two genes that underlie tuberous sclerosis complex, tuberin and hamartin, lie at the center of an important signal transduction pathway with significant implications for pharmacologic treatment. Issues in genetic counseling for this highly variable disease are updated. Extensive progress has been made in understanding the basis of several forms of ectodermal dysplasia. Disorders caused by mutations in p63 and the connexin and NF-kappaB gene families will be reviewed. Finally, phenotypic in vivo amelioration of genodermatoses via revertant gene mosaicism will be discussed as a possible mechanism to be exploited in directed therapeutic approaches. SUMMARY: This paper reviews recent developments in the molecular and biologic bases of neurofibromatosis type 1, tuberous sclerosis, and ectodermal disorders related to p63 and the connexin and NF-kappaB gene families. The concept of revertant gene mosaicism is also discussed as a potential model for gene therapy.

Adolescent↗

PubMed2007

An approach to the validation of novel molecular markers of breast cancer via TMA-based FISH scanning.

Tissue microarrays (TMA) are valuable tools for validating results of array-based comparative genomic hybridization (ACGH) and other translational research applications requiring independent verification of genomic gains and losses by fluorescence in situ hybridization (FISH). However, spatial orientation and accurate manual tracking of the TMA cores is challenging and prone to error. Image analysis combined with core tracking software, implemented via an automated FISH scanning workstation, represents a new approach to FISH and TMA-based validation of novel genomic changes discovered by ACGH in breast and other cancers. Automated large-scale tissue microarray validation FISH studies of genomic gains and losses identified by ACGH for breast cancer are feasible using an automated imaging scanner and tracking/classifying software. Furthermore, by leveraging the bifunctional fluorescent and chromogenic properties of the alkaline phosphatase chromogen fast red K and combining the technology with FISH, correlative and simultaneous phenotype/genotype studies may be enabled.

Biomarkers, Tumor↗

PubMed2001

Comparison of monocyte enrichment by immuno-magnetic depletion or adherence for the clinical-scale generation of DC.

BACKGROUND: DC generated from monocytes have been used for vaccines. We have developed a monocyte enrichment procedure by depleting T and B cells with anti-CD2 and anti-CD19 Abs using the automated Isolex 300i magnetic cell selector for clinical-scale DC generation in gas permeable SteriCell culture bags. We have also compared DC function, yield and purity of DC generated from adherent monocytes using culture bags in a closed system, with DC generated in conventional tissue culture flasks. METHODS: Monocytes were enriched from normal donor apheresis products using CD2/19 depletion with experimental software on the Isolex 300i (ISO), adherence (AD) to SteriCell bags and to T175 flasks and then cultured for 7 days in serum-free X-VIVO 15 media with GM-CSF and IL-4. Phenotype and dextran uptake were analyzed by flow cytometry and allogeneic MLR was also evaluated. RESULTS: ISO-DC and AD-DC from SteriCell bags showed similar viability. Higher purity of ISO-DC than AD-DC was measured by forward- and side-scatter flow cytometry. Similar expression of CD1a, CD80, CD86 and CD83 were observed in both ISO-DC and AD-DC. Similar dextran uptake and allo MLR were also observed. DISCUSSION: These data indicated that functional DC were generated in gas permeable SteriCell culture bags from both ISO- and AD-monocytes in a closed system.

Antigens, CD19↗

PubMedDate not supplied

Collagen gel contraction serves to rapidly distinguish epithelial- and mesenchymal-derived cells irrespective of alpha-smooth muscle actin expression.

Mesenchymal-like cells in the stroma of breast cancer may arise as a consequence of plasticity within the epithelial compartment, also referred to as epithelial-mesenchymal transition, or by recruitment of genuine mesenchymal cells from the peritumoral stroma. Cells of both the epithelial compartment and the stromal compartment express alpha smooth muscle actin (alpha-sm actin) as part of a myoepithelial or a myofibroblastic differentiation program, respectively. Moreover, because both epithelial- and mesenchymal-derived cells are nontumorigenic, other means of discrimination are warranted. Here, we describe the contraction of hydrated collagen gels as a rapid functional assay for the distinction between epithelial- and mesenchymal-derived stromal-like cells irrespective of the status of alpha-sm actin expression. Three epithelial-derived cell lines and three genuine mesenchymal-derived breast cell lines were plated on top of hydrated collagen lattices. Reduction in gel height was measured every hour for 6 h and after 22 h using an x-y-z automated position table. Significantly, the epithelial-derived cells, irrespective of a high alpha-sm actin expression, had a fivefold lower contractility (10.0% reduction in gel height) than their true mesenchymal counterparts (53.1% reduction in gel height). To test whether at all force generation could be induced in the nonmesenchymal cells by alpha-sm actin, transductions were performed to obtain a tetracycline-dependent expression. Expression under these conditions did not augment contractility. It is concluded that epithelial-derived mesenchymal-like cells are functionally defective within a connective tissue environment irrespective of an apparent contractile phenotype.

Actins↗

PubMed2001

High-throughput screening for identification of small molecule inhibitors of histone acetyltransferases using scintillating microplates (FlashPlate).

The role of histone acetyltransferases (HATs) in the regulation of crucial cellular functions, e.g., gene transcription, differentiation, and proliferation, has recently been documented and there is increasing evidence that aberrant expression of these enzymes may have a role to play in the development of the malignant phenotype. The availability of potent and selective small molecule inhibitors of HATs would provide useful proof of principle probes for further validation of these enzymes as drug discovery targets and may also provide lead molecules for clinical drug development. We have developed a microplate assay for HAT activity suitable for high-throughput screening. In the assay, following incubation of histone H3, [3H]acetylCoA, and enzyme (recombinant p300/CBP-associated factor expressed as a glutathione S-transferase fusion protein), radiolabeled histone was captured onto the walls of a scintillating microplate (FlashPlate) generating a scintillation signal. The assay was reproducible, amenable to automation, and generated a wide signal to noise ratio. Although antiacetylated histone antibodies were initially used to capture the radiolabeled product, it was subsequently shown that a signal was effectively produced by histone passively binding to the walls of the FlashPlate. This resulted in a simple "mix and measure" assay that is currently being used for the identification of HAT inhibitors.

Acetyl Coenzyme A↗

PubMed2005

Use of the MicroSeq 500 16S rRNA gene-based sequencing for identification of bacterial isolates that commercial automated systems failed to identify correctly.

Reliable automated identification and susceptibility testing of clinically relevant bacteria is an essential routine for microbiology laboratories, thus improving patient care. Examples of automated identification systems include the Phoenix (Becton Dickinson) and the VITEK 2 (bioMerieux). However, more and more frequently, microbiologists must isolate "difficult" strains that automated systems often fail to identify. An alternative approach could be the genetic identification of isolates; this is based on 16S rRNA gene sequencing and analysis. The aim of the present study was to evaluate the possible use of MicroSeq 500 (Applera) for sequencing the 16S rRNA gene to identify isolates whose identification is unobtainable by conventional systems. We analyzed 83 "difficult" clinical isolates: 25 gram-positive and 58 gram-negative strains that were contemporaneously identified by both systems--VITEK 2 and Phoenix--while genetic identification was performed by using the MicroSeq 500 system. The results showed that phenotypic identifications by VITEK 2 and Phoenix were remarkably similar: 74% for gram-negative strains (43 of 58) and 80% for gram-positive strains were concordant by both systems and also concordant with genetic characterization. The exceptions were the 15 gram-negative and 9 gram-positive isolates whose phenotypic identifications were contrasting or inconclusive. For these, the use of MicroSeq 500 was fundamental to achieving species identification. In clinical microbiology the use of MicroSeq 500, particularly for strains with ambiguous biochemical profiles (including slow-growing strains), identifies strains more easily than do conventional systems. Moreover, MicroSeq 500 is easy to use and cost-effective, making it applicable also in the clinical laboratory.

Bacterial Typing Techniques↗

PubMed2000

Rapid characterisation of deep-sea actinomycetes for biotechnology screening programmes.

A continual need in natural product discovery is dereplication, that is the ability to exclude previously tested microorganisms from screening programmes. Whole-cell fingerprinting techniques offer an ideal solution to this problem because of their rapidity and reproducibility, dependence on small samples, and automation. One such technique, Curie-point pyrolysis mass spectrometry (PyMS), has been deployed for the characterisation of a unique collection of actinomycetes recovered from Pacific Ocean sediments approximately 2000 to 6500 m below sea level. This paper addresses the question: to what extent are pyrogroups, defined on the basis of PyMS fingerprinting, related to classifications derived from more conventional microbial systematics? A collection of 44 randomly chosen deep-sea rhodococci were coded and subjected to a double-blind PyMS and numerical taxonomic (NT) analysis; the latter sorted the strains into clusters (taxospecies) using large sets of equally weighted phenotypic data. At the end of the experiment the codes were disclosed and the NT classification shown to generate 6 homogeneous clusters corresponding to different deep-sea sites. The matching of these clusters with the resulting pyrogroups was very high with an overall congruence of nearly 98%. Thus, PyMS characterisation is directly ascribable to the phenotypic variation being sought for biotechnology screens. Moreover, the exquisite discriminatory power of PyMS readily revealed infraspecific diversity in these industrially important bacteria.

Actinomycetales↗

PubMed1994

p53 gene mutations in pituitary adenomas: rare events.

OBJECTIVE: Occult pituitary adenomas are said to occur in up to 20% of random autopsy examinations, yet the only oncogene known to be associated with pituitary adenomas, gsp, is found in only 40% of somatotrophinomas, a subtype that accounts for a minority of pituitary tumours. Mutations of the p53 tumour suppressor gene are thought to be involved in the pathogenesis of as many as 50% of all human cancers, including tumours of the central nervous system. The objective of this study was to determine whether p53 gene mutations are associated with pituitary adenomas. DESIGN AND PATIENTS: Fragments of pituitary adenoma tissue from 29 patients undergoing routine hypophysectomy for pituitary tumour were coated in cryostat embedding medium and frozen at -80 degrees C within 24 hours of resection. They consisted of 9 somatotroph, 4 corticotroph, 1 mammotroph and 15 endocrinologically inactive adenomas, all of the non-invasive clinical phenotype. Sequential frozen sections were subjected to in situ hybridization analysis for anterior pituitary hormone transcripts and examined histologically to ensure that the frozen sections used to generate DNA templates for polymerase chain reaction amplification were not contaminated with non-tumour tissue. MEASUREMENTS: p53 exons 7 and 8, within which 98% of substitution mutations are thought to occur, and exons 4-6 in tumours immunopositive for p53, were amplified by polymerase chain reaction and ligated into the vector pCR2. DNA from small-scale plasmid preparations of pCR2 containing cloned p53 exons from human pituitary adenomas was sequenced using an automated fluorescence-based system (DuPont Genesis 2000) and compared with wild-type sequence. Apparent mutations were confirmed or refuted by sequencing a further 2-4 clones isolated from the same template. RESULTS: Although immunocytochemical staining patterns for wild-type p53 varied markedly between different tumours, no mutations were identified in any of the exonic sequences examined. CONCLUSIONS: p53 mutations, at least within the high mutation domains of p53, occur infrequently in human pituitary adenomas. Increased steady-state levels of p53 protein identified immunocytochemically may be a consequence of binding to other cellular proteins in these tumours.

Adenoma↗

PubMed2000

Detection of Escherichia coli O157:H7 in heifers' faecal samples using an automated immunoconcentration system.

Pre-treatment of a 5-h enrichment culture with an automated immunoconcentration (ICE) system greatly improved the isolation of Escherichia coli O157:H7 from spiked heifer faecal samples. Enrichment samples plated directly onto sorbitol MacConkey agar (SMAC) and SMAC agar supplemented with cefixime and potassium tellurite (CT-SMAC) showed recovery rates of 8% and 56%, respectively. However, after ICE treatment, E. coli O157:H7 was recovered from 92% of the samples on SMAC and 100% on CT-SMAC. Immunoconcentration analysis of heifers' faecal samples collected from a slaughter-house in France, during March to June 1998, showed that 1% (three of 300) was positive for E. coli O157:H7. Phenotypic and genotypic analysis showed that all three isolates carried both the O157 and H7 antigens, did not ferment sorbitol or had beta-glucuronidase activity and carried trait virulence factors for E. coli O157:H7 (uidA allele, eaeA and pO157 plasmid). However, only one strain was toxigenic and this strain produced a single toxin, namely verotoxin 2.

Adhesins, Bacterial↗

PubMed1994

Spectrum of small length germline mutations in the RB1 gene.

A screening method based on multiplexed automated fragment length analysis of polymerase chain reaction products was used to identify germline mutations in the RB1 gene. By screening 106 unrelated patients with hereditary retinoblastoma, 20 small deletions (1-18 bp) and seven insertions (1-5 bp) were identified. When collating our data with reported mutations, recurrence of small length mutations was observed at nine sites within the RB1 gene. Most of these contained monotonic runs or direct repeats embedded in homocopolymer tracts. While the majority of mutations resulted in premature truncation, two mutations caused an in-frame loss of F755 and G540 to E545, respectively. A genotype-phenotype comparison of patients carrying different small length mutations did not reveal any consistent relation. Particularly, the two patients with in-frame mutations showed a high number of tumours consistent with regular-penetrance retinoblastoma.

Base Sequence↗

PubMed1995

Analysis of the complete coding region of the CFTR gene in a cohort of CF patients from north-eastern Italy: identification of 90% of the mutations.

A complete coding-region analysis on 225 cystic fibrosis (CF) chromosomes from a cohort that includes all the affected subjects born in two North-Eastern Italian regions over eight years was performed. In a previous study, we identified mutations on 166/225 (73.8%) CF chromosomes after screening for 62 mutations. To characterise the remaining 59 CF chromosomes, we carried out automated direct DNA sequencing (exons 9 and 13), RNA single-strand conformation polymorphism (exons 1-8 and 10-12) and denaturing gradient gel electrophoresis (exons 14a-24) of the 27 exons and flanking regions of the CF transmembrane conductance regulator gene. We identified 22 mutations, four of which are novel, viz. 711 + 5G-->A, R709X, 3132delTG and 2790-2A-->G, and we characterised 90.2% (203/225) of the CF chromosomes. Taking advantage of the homogeneity of the sample, an evaluation of the most important clinical parameters, assessed at the age of 12 years, is presented. We confirm some previously reported genotype-phenotype correlations and we report a new nonsense mutation (R709X) associated with a pancreatic sufficient phenotype.

Adolescent↗

PubMed2000

Glycogen storage disease type Ia: recent experience with mutation analysis, a summary of mutations reported in the literature and a newly developed diagnostic flow chart.

UNLABELLED: We studied the glucose-6-phosphatase (G6Pase) gene of 30 unrelated glycogen storage disease type Ia (GSD Ia) patients using single strand conformational polymorphism (SSCP) prior to automated sequencing of exons revealing an aberrant SSCP pattern. In all patients we could identify mutations on both alleles of the G6Pase gene, indicating that this method is a reliable procedure. A total of 14 different mutations were identified. R83C (16/60), 158delC (12/60), Q347X (7/60), R170X (6/60) and deltaF327 (4/60) were found most frequently. Nine other mutations accounted for the other 15 mutant alleles. Two DNA-based prenatal diagnoses were performed successfully. At present, 56 mutations in the G6Pase gene have been reported in 300 unrelated GSD Ia patients and an overview of these mutations is presented. Evidence for a clear genotype-phenotype correlation could be established neither from our data nor from those in the literature. With increased knowledge about the genetic basis of GSD Ia and GSD Ib and the high detection rate of mutations, it is our opinion that the diagnoses GSD Ia and GSD Ib can usually be based on clinical and biochemical abnormalities combined with mutation analysis instead of enzyme assays in liver tissue obtained by biopsy. A newly developed flowchart for the diagnosis of GSD I is presented. CONCLUSION: Increased knowledge of the genetic basis of glycogen storage disease type I provides a DNA-based diagnosis, prenatal DNA-based diagnosis in chorionic villus samples and carrier detection.

Chorionic Villi Sampling↗

PubMed2001

Establishment of an in vitro reporter gene assay for developmental cardiac toxicity.

This study is based on the unique potential of pluripotent embryonic stem (ES) cells to differentiate in vitro into embryoid bodies containing cell lineages representative of most cell types found in the mammalian fetus. However, the use of wild type ES cells as an in vitro assay for embryotoxicological studies is complicated by the simultaneous development of various cellular phenotypes. This prevents a quantitative assessment of drug effects on one specific cell type. Here we report the effects of 15 chemicals on cardiac differentiation as determined by various specific toxicological endpoints such as morphological inspection (contractile activity), quantitative mRNA analysis and cardiac-specific expression of green fluorescent protein (GFP), used as a quantitative reporter. The data from the different endpoints have been subjected to a statistical analysis, and a preliminary prediction model is proposed. The results demonstrate that genetically-engineered ES cells could provide a valuable tool for estimating the developmental cardiotoxic potential of compounds in vitro and form the basis for automated analysis in a high-throughput system.

Animals↗