Oligonucleotide Probe Sequences Research Articles

Quadruplex real-time PCR for rapid detection of human alphaherpesviruses

Infections with the herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) as well as with the varicella-zoster virus (VZV) may take a serious course. Thus, rapid and reliable detection of these alphaherpesviruses is urgently needed. For this, we established a qualitative quadruplex real-time polymerase chain reaction (PCR) covering HSV-1, HSV-2, VZV and endogenous human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The PCR was validated with quality assessment samples and pre-characterized clinical samples including swabs, blood and cerebrospinal as well as respiratory fluids. For comparison, nucleic acids (NA) of selected samples were extracted manually and automatically. The protocol takes approx. 90min, starting with the preparation of NA until the report of results. The oligonucleotide and hydrolysis probe sequences specifically detect and distinguish HSV-1 (530nm), HSV-2 (705nm) and VZV (560nm) DNA. The detection limit was estimated with 100-500 copies/ml HSV-1 and HSV-2/VZV, respectively. All quality assessment samples as well as all the patient samples were classified correctly. Parallel detection of GAPDH (670nm) DNA was implemented to demonstrate correct sampling, but was uncertain in case of swabs. To this end, alphaherpesvirus-free human DNA was also added directly into the mastermix to exclude PCR inhibition. The established protocol for parallel detection and differentiation of alphaherpesviruses is fast, highly specific as well as rather sensitive. It will facilitate HSV-1/2 and VZV diagnostics and may be further improved by opening the 670nm channel for a combined extraction and PCR inhibition control.

Diagnosis of Tuberculosis Using Colorimetric Gold Nanoparticles on a Paper-Based Analytical Device.

We have developed a colorimetric sensing strategy employing gold nanoparticles and a paper-based analytical platform for the diagnosis of tuberculosis (TB). By utilizing the surface plasmon resonance effect, we were able to monitor changes in the color of a gold nanoparticle colloid based on the effects of single-stranded DNA probe molecules hybridizing with targeted double-stranded TB DNA. The hybridization event changes the surface charge density of the nanoparticles, causing them to aggregate to various degrees, which modifies the color of the solution in a manner that can be readily measured to determine the concentration of the targeted DNA analyte. In order to adapt this TB diagnosis method to resource-limited settings, we extended this label-free oligonucleotide and unmodified gold nanoparticle solution-based technique to a paper-based system that can be measured using a smartphone to obtain rapid parallel colorimetric results with low reagent consumption and without the need for sophisticated analytical equipment. In this study, we investigated various assay conditions, including the denaturing temperature and time, different oligonucleotide probe sequences, as well as the ratio of single stranded probe and double stranded target DNA. After optimizing these variables, we were able to achieve a detection limit of 1.95 × 10-2 ng/mL for TB DNA. Furthermore, multiple tests could be performed simultaneously with a 60 min turnaround time.

Controlled deposition of functionalized silica coated zinc oxide nano-assemblies at the air/water interface for blood cancer detection

We report results of the studies relating to controlled deposition of the amino-functionalized silica-coated zinc oxide (Am-Si@ZnO) nano-assemblies onto an indium tin oxide (ITO) coated glass substrate using Langmuir-Blodgett (LB) technique. The monolayers have been deposited by transferring the spread solution of Am-Si@ZnO stearic acid prepared in chloroform at the air-water interface, at optimized pressure (16 mN/m), concentration (10 mg/ml) and temperature (23 °C). The high-resolution transmission electron microscopic studies of the Am-Si@ZnO nanocomposite reveal that the nanoparticles have a microscopic structure comprising of hexagonal assemblies of ZnO with typical dimensions of 30 nm. The surface morphology of the LB multilayer observed by scanning electron microscopy shows uniform surface of the Am-Si@ZnO film in the nanometer range (<80 nm). These electrodes have been utilized for chronic myelogenous leukemia (CML) detection by covalently immobilizing the amino-terminated oligonucleotide probe sequence via glutaraldehyde as a crosslinker. The response studies of these fabricated electrodes carried out using electrochemical impedance spectroscopy show that this Am-Si@ZnO LB film based nucleic acid sensor exhibits a linear response to complementary DNA (10−6–10−16 M) with a detection limit of 1 × 10−16 M. This fabricated platform is validated with clinical samples of CML positive patients and the results demonstrate its immense potential for clinical diagnosis.

On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1.

Fabrication of oligonucleotide and protein arrays on rigid and flexible substrates coated with reactive polymer multilayers.

We report a top-down approach to the fabrication of oligonucleotide and protein arrays on surfaces coated with ultrathin, amine-reactive polymer multilayers fabricated by the covalent "layer-by-layer" (LbL) assembly of polyethyleneimine (PEI) and the amine-reactive, azlactone-functionalized polymer poly(2-vinyl-4,4-dimethylazlactone) (PVDMA). Manual spotting of amine-terminated oligonucleotide probe sequences on planar glass slides coated with PEI/PVDMA multilayers (~35 nm thick) yielded arrays of immobilized probes that hybridized fluorescently labeled complementary sequences with high signal intensities, high signal-to-noise ratios, and high sequence specificity. Treatment of residual azlactone functionality with the nonfouling small-molecule amine d-glucamine resulted in regions between the features of these arrays that resisted adsorption of protein and permitted hybridization in complex media containing up to 10 mg/mL protein. The residual azlactone groups in these films were also exploited to immobilize proteins on film-coated surfaces and fabricate functional arrays of proteins and enzymes. The ability to deposit PEI/PVDMA multilayers on substrates of arbitrary size, shape, and composition permitted the fabrication of arrays of oligonucleotides on the surfaces of multilayer-coated sheets of poly(ethylene terephthalate) and heat-shrinkable polymer film. Arrays fabricated on these flexible plastic substrates can be bent, cut, resized, and manipulated physically in ways that are difficult using more conventional rigid substrates. This approach could thus contribute to the development of new assay formats and new applications of biomolecule arrays. The methods described here are straightforward to implement, do not require access to specialized equipment, and should also be compatible with automated liquid-handling methods used to fabricate higher-density arrays of oligonucleotides and proteins on more traditional surfaces.

Chitosan encapsulated quantum dots platform for leukemia detection

We report results of the studies relating to electrophoretic deposition of nanostructured composite of chitosan (CS)–cadmium-telluride quantum dots (CdTe-QDs) onto indium-tin-oxide coated glass substrate. The high resolution transmission electron microscopic studies of the nanocomposite reveal molecular level coating of the CdTe-QDs with CS molecules in the colloidal dispersion medium. This novel composite platform has been explored to fabricate an electrochemical DNA biosensor for detection of chronic myelogenous leukemia (CML) by immobilizing amine terminated oligonucleotide probe sequence containing 22 base pairs, identified from BCR–ABL fusion gene. The results of differential pulse voltammetry reveal that this nucleic acid sensor can detect as low as 2.56pM concentration of complementary target DNA with a response time of 60s. Further, the response characteristics show that this fabricated bioelectrode has a shelf life of about 6 weeks and can be used for about 5–6 times. The results of experiments conducted using clinical patient samples reveal that this sensor can be used to distinguish CML positive and the negative control samples.

Porcine reproductive and respiratory syndrome virus: Antigenic and molecular diversity of British isolates and implications for diagnosis

Porcine reproductive and respiratory syndrome (PRRS) is an endemic disease of pigs, caused by PRRS virus, a member of the Arteriviridae family. First seen in Britain in 1991, the disease continues to be a significant economic and welfare problem for pig producers.To date, only PRRSV genotype 1 has been found in Britain. At the genetic level, a considerable increase has been reported in the diversity of PRRS viruses isolated in Britain between 2003 and 2007, versus the early 1990s. In this study, the diversity has been shown to extend to the antigenic level too, with potential consequences for diagnostic methods. Antigenic diversity was assessed using a panel of twelve monoclonal antibodies, only one of which reacted with all isolates tested. Nine diverse viruses were compared as potential antigens in immunoperoxidase monolayer assays, where each one produced quite different results for a common panel of sera. As a single virus is used in each diagnostic assay, results must therefore be interpreted cautiously.For a real-time RT-PCR assay, published oligonucleotide primer and probe sequences were evaluated against available genetic sequences of British and European viruses, and were re-designed where considerable mismatches were found. The multiplex assay incorporating these modified primers to detect genotype 1 and 2 PRRS viruses was then validated for use with diagnostic sera and tissues.As the increasing degree of diversity exhibited by British strains is mirrored in other countries, PRRSV will continue to provide an ongoing challenge to diagnosis at a global, as well as national level.

Nanopatterned Cadmium Selenide Langmuir–Blodgett Platform for Leukemia Detection

We present results of the studies relating to preparation of Langmuir-Blodgett (LB) monolayers of tri-n-octylphosphine oxide-capped cadmium selenide quantum dots (QCdSe) onto indium-tin oxide (ITO) coated glass substrate. The monolayer behavior has been studied at the air-water interface under various subphase conditions. This nanopatterned platform has been explored to fabricate an electrochemical DNA biosensor for detection of chronic myelogenous leukemia (CML) by covalently immobilizing the thiol-terminated oligonucleotide probe sequence via a displacement reaction. The results of electrochemical response studies reveal that this biosensor can detect target DNA in the range of 10(-6) to 10(-14) M within 120 s, has a shelf life of 2 months, and can be used about 8 times. Further, this nucleic acid sensor has been found to distinguish the CML-positive and the control negative clinical patient samples.

On-Chip Transduction of Nucleic Acid Hybridization Using Spatial Profiles of Immobilized Quantum Dots and Fluorescence Resonance Energy Transfer

The glass surface of a glass-polydimethylsiloxane (PDMS) microfluidic channel was modified to develop a solid-phase assay for quantitative determination of nucleic acids. Electroosmotic flow (EOF) within channels was used to deliver and immobilize semiconductor quantum dots (QDs), and electrophoresis was used to decorate the QDs with oligonucleotide probe sequences. These processes took only minutes to complete. The QDs served as energy donors in fluorescence resonance energy transfer (FRET) for transduction of nucleic acid hybridization. Electrokinetic injection of fluorescent dye (Cy3) labeled oligonucleotide target into a microfluidic channel and subsequent hybridization (within minutes) provided the proximity for FRET, with emission from Cy3 being the analytical signal. The quantification of target concentration was achieved by measurement of the spatial length of coverage by target along a channel. Detection of femtomole quantities of target was possible with a dynamic range spanning an order of magnitude. The assay provided excellent resistance to nonspecific interactions of DNA. Further selectivity of the assay was achieved using 20% formamide, which allowed discrimination between a fully complementary target and a 3 base pair mismatch target at a contrast ratio of 4:1.

Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs

Hybrid organisms may fail to develop, be sterile or they may be more vigorous than either of the parents. Examples of hybrid vigour or hybrid necrosis in the F1 are often not inherited stably in subsequent generations if they are associated with overdominance. There can also be transgressive phenotypes that are inherited stably in these later generations, but the underlying mechanisms are not well understood. Here we have investigated the possibility that stable transgressive phenotypes in the progeny of crosses between cultivated tomato (Solanum lycopersicum cv. M82) and a wild relative (Solanum pennellii, accession LA716) are associated with micro or small interfering(si) RNAs. We identified loci from which these small(s)RNAs were more abundant in hybrids than in either parent and we show that accumulation of such transgressive sRNAs correlated with suppression of the corresponding target genes. In one instance this effect was associated with hypermethylation of the corresponding genomic DNA. Our results illustrate a potential role of transgressive sRNAs in plant breeding and in natural evolution with wild plants.

Probe Design for DNA Chips

DNA chips are a promising fundamental technology for the post-genome research [1]. A large number of DNA oligonucleotide probes integrated in a small area of the chip surface facilitate speedy simultaneous detection of many target sequences. So, expression profile analysis of thousands of genes can be finished in a few days by using DNA chip technology although it takes years to finish the analysis by using conventional methods such as northern hybridization and SAGE. High-density DNA chips are made by light-directed combinatorial oligonucleotide synthesis technology, which is based on photolithography technology for semiconductor micro-fabrication [4, 5]. Recently, high-density DNA oligonucleotide chips have been commercially available [2]. Those chips can detect thousands of mRNA transcripts simultaneously. However, as many as 20 pairs of perfect match and center-mismatch oligonucleotide probes are needed to identify each transcript. This is due to low specificity of probe sequences designed by unsatisfactory method [3]. The necessity of many probes not only decreases the detection capacity of DNA chips but also makes DNA chip development difficult and expensive. Here, we describe a more rational method for designing oligonucleotide probe sequences of DNA chips for gene expression profile analysis, which can reduce the number of DNA probes needed for identification of target sequences. Each transcript is identified with a pair of two oligonucleotide probes, which can hybridize to target transcript specifically under the same hybridization condition. In addition, they can be used as a PCR primer pair, which helps experiments to examine the specificity of designed probe sequences.

Exploiting sequence similarity to validate the sensitivity of SNP arrays in detecting fine-scaled copy number variations

High-density single nucleotide polymorphism (SNP) genotyping arrays are efficient and cost effective platforms for the detection of copy number variation (CNV). To ensure accuracy in probe synthesis and to minimize production costs, short oligonucleotide probe sequences are used. The use of short probe sequences limits the specificity of binding targets in the human genome. The specificity of these short probeset sequences has yet to be fully analysed against a normal reference human genome. Sequence similarity can artificially elevate or suppress copy number measurements, and hence reduce the reliability of affected probe readings. For the purpose of detecting narrow CNVs reliably down to the width of a single probeset, sequence similarity is an important issue that needs to be addressed. We surveyed the Affymetrix Human Mapping SNP arrays for probeset sequence similarity against the reference human genome. Utilizing sequence similarity results, we identified a collection of fine-scaled putative CNVs between gender from autosomal probesets whose sequence matches various loci on the sex chromosomes. To detect these variations, we utilized our statistical approach, Detecting REcurrent Copy number change using rank-order Statistics (DRECS), and showed that its performance was superior and more stable than the t-test in detecting CNVs. Through the application of DRECS on the HapMap population datasets with multi-matching probesets filtered, we identified biologically relevant SNPs in aberrant regions across populations with known association to physical traits, such as height, covered by the span of a single probe. This provided empirical confirmation of the existence of naturally occurring narrow CNVs as well as the sensitivity of the Affymetrix SNP array technology in detecting them. The MATLAB implementation of DRECS is available at http://ww2.cs.mu.oz.au/ approximately gwong/DRECS/index.html.

Expression of tissue factor pathway inhibitor (TFPI) in human breast and colon cancer tissue

Activation of blood coagulation, a phenomenon frequently observed in breast and colon cancer patients, contributes to tumour progression. The principal initiator of blood coagulation activation in cancer patients is tissue factor (TF), while tissue factor pathway inhibitor (TFPI) is the main inhibitor of the TF-dependent pathway of blood coagulation. Previous immunohistochemical studies revealed no expression of TFPI in human cancer cells. The aim of the study was to evaluate the expression of TFPI protein and mRNA in breast and colon cancer tissues. A total of 108 cancer tissues (from primary tumours and metastatic lymph nodes) were obtained from 87 patients during surgical treatment. Immunohistochemical studies using a polyclonal anti-TFPI antibody were performed including a semiquantitative analysis. The in situ hybridisation method employed single-stranded DNA oligonucleotide (probe sequence: 5'Biotin-CCACCATACTTGAAACGTTCACACT-Biotin3') directed against TFPI mRNA. Strong or medium expression of TFPI protein was observed in cancer cell bodies in all breast cancers and in most (39/66 cases) colon cancers examined. Weaker expression of TFPI was detected in cancer cells localised in lymph node metastatic foci of breast cancer. Endothelial cells were also TFPI-positive. TFPI mRNA was demonstrated in all cases of breast and in approximately 80% cases of colon cancer cells. TFPI mRNA and protein are present in association with colon and breast cancer cells, suggesting that the protein may play a role in cancer biology. The presence of TFPI in association with breast cancer cells localised in regional lymph nodes may indicate its role in lymphatic spread.

Development and Application of Bovine and Porcine Oligonucleotide Arrays with Protein-Based Annotation

The design of oligonucleotide sequences for the detection of gene expression in species with disparate volumes of genome and EST sequence information has been broadly studied. However, a congruous strategy has yet to emerge to allow the design of sensitive and specific gene expression detection probes. This study explores the use of a phylogenomic approach to align transcribed sequences to vertebrate protein sequences for the detection of gene families to design genomewide 70-mer oligonucleotide probe sequences for bovine and porcine. The bovine array contains 23,580 probes that target the transcripts of 16,341 genes, about 72% of the total number of bovine genes. The porcine array contains 19,980 probes targeting 15,204 genes, about 76% of the genes in the Ensembl annotation of the pig genome. An initial experiment using the bovine array demonstrates the specificity and sensitivity of the array.

Homogeneous DNA Detection Based on Fluorescence Quenching by Nanoparticles in Single‐step Format: Target‐Induced Configuration Transform

AbstractA new strategy for homogeneous detection of DNA hybridization in single‐step format was developed based on fluorescence quenching by gold nanoparticles. The gold nanoparticle is functionalized with 5′‐thiolated 48‐base oligonucleotide (probe sequence), whose 3′‐terminus is labeled with fluorescein (FAM), a negatively charged fluorescence dye. The oligonucleotide adopts an extended configuration due to the electrostatic repulsion between negatively charged gold nanoparticle and the FAM‐attached probe sequence. After addition of the complementary target sequence, specific DNA hybridization induces a conformation change of the probe from an extended structure to an arch‐like configuration, which brings the fluorophore and the gold nanoparticle in close proximity. The fluorescence is efficiently quenched by gold nanoparticles. The fluorescence quenching efficiency is related to the target concentration, which allows the quantitative detection for target sequence in a sample. A linear detection range from 1.6 to 209.4 nmol/L was obtained under the optimized experimental conditions with a detection limit of 0.1 nmol/L. In the assay system, the gold nanoparticles act as both nanoscaffolds and nanoquenchers. Furthermore, the proposed strategy, in which only two DNA sequences are involved, is not only different from the traditional molecular beacons or reverse molecular beacons but also different from the commonly used sandwich hybridization methods. In addition, the DNA hybridization detection was achieved in homogenous solution in a single‐step format, which allows real‐time detection and quantification with other advantages such as easy operation and elimination of washing steps.

Streptococcus thermophilus core genome: comparative genome hybridization study of 47 strains.

A DNA microarray platform based on 2,200 genes from publicly available sequences was designed for Streptococcus thermophilus. We determined how single-nucleotide polymorphisms in the 65- to 75-mer oligonucleotide probe sequences affect the hybridization signals. The microarrays were then used for comparative genome hybridization (CGH) of 47 dairy S. thermophilus strains. An analysis of the exopolysaccharide genes in each strain confirmed previous findings that this class of genes is indeed highly variable. A phylogenetic tree based on the CGH data showed similar distances for most strains, indicating frequent recombination or gene transfer within S. thermophilus. By comparing genome sizes estimated from the microarrays and pulsed-field gel electrophoresis, the amount of unknown DNA in each strain was estimated. A core genome comprised of 1,271 genes detected in all 47 strains was identified. Likewise, a set of noncore genes detected in only some strains was identified. The concept of an industrial core genome is proposed. This is comprised of the genes in the core genome plus genes that are necessary in an applied industrial context.

Application of cadmium sulfide nanoparticles as oligonucleotide labels for the electrochemical detection of NOS terminator gene sequences

A mercaptoacetic acid (MAA)-modified cadmium sulfide (CdS) nanoparticle was synthesized in aqueous solution and used as an oligonucleotide label for the electrochemical detection of nopaline synthase (NOS) terminator gene sequence. The carboxyl groups on the surface of the CdS nanoparticle can be easily covalently linked with NH2-modified NOS oligonucleotide probe sequences. The target ssDNA sequence was fixed onto the electrode surface by covalently linking to a mercaptoethanol self-assembled gold electrode, and the DNA hybridization of target ssDNA with probe ssDNA was accomplished on the electrode surface. The CdS nanoparticles anchored on the hybrids were dissolved in the solution by the oxidation with HNO3 and further detected by a sensitive differential pulse anodic stripping voltammetric method. The detection results can be used for monitoring the hybridization, and the NOS target sequence was satisfactorily detected in the approximate range from 8.0 x 10(-12) to 4.0 x 10(-9) mol L(-1) with a detection limit of 2.75 x 10(-12) mol L(-1) (3sigma). The established method extended the nanoparticle-labeled electrochemical DNA analysis to genetically modified organisms (GMOs) specific sequence samples with higher sensitivity and selectivity.

Development of a base stacking hybridization-based microarray method for rapid identification of clinical isolates

A base stacking hybridization-based microarray method was developed for rapid identification of clinical isolates within 2 h. The oligonucleotide probe sequences for species or genus-level identification were targeted against ribosomal RNA. Isolates were lysed and directly hybridized to the microarray-bound capture probes without conventional DNA or RNA isolation and prior polymerase chain reaction amplification. Five bacterial species encountered frequently in the clinical setting, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae, and one genus Enterococcus, could be discriminated by the microarray-based assay. Identification by this method matched biochemical identification for 150 of 152 clinical strains. This base-stacking hybridization microarray offers a simple, fast (≤2 h), and accurate identification of bacterial cultures and is a potential tool for routine clinical diagnosis.

Regulation of Pancreas Duodenum Homeobox-1 Expression by Early Growth Response-1

The homeodomain transcription factor pancreas duodenum homeobox-1 (PDX-1) is a key regulator of pancreatic beta-cell development, function, and survival. Deficits in PDX-1 expression result in insulin deficiency and hyperglycemia. We previously found that the glucose-responsive transcription factor early growth response-1 (Egr-1) activates the insulin promoter in part by increasing expression levels of PDX-1. We now report that Egr-1 binds and activates multiple regulatory sites within the pdx-1 promoter. We identified consensus Egr-1 recognition sequences within proximal and distal regions of the mouse pdx-1 promoter and demonstrated specific binding of Egr-1 by chromatin immunoprecipitation and electrophoretic mobility shift assays. Overexpression of Egr-1 increased transcriptional activation of the -4500 proximal pdx-1 promoter and of the highly conserved regulatory Areas I, II, and III. Mutagenesis of a specific Egr-1 binding site within Area III substantially decreased Egr-1-mediated activation. Egr-1 increased the transcriptional activation of Areas I and II, despite the absence of Egr-1 recognition sequences within this promoter segment, suggesting that Egr-1 also can regulate the pdx-1 promoter indirectly. Egr-1 increased, and a dominant-negative Egr-1 mutant repressed, the transcriptional activation of distal pdx-1 promoter sequences. Mutagenesis of a specific Egr-1 binding site within regulatory Area IV reduced basal and Egr-1-mediated transcriptional activation. Our data indicate that Egr-1 regulates expression of PDX-1 in pancreatic beta-cells by both direct and indirect activation of the pdx-1 promoter. We propose that Egr-1 expression levels may act as a sensor in pancreatic beta-cells to translate extracellular signals into changes in PDX-1 expression levels and pancreatic beta-cell function.

TFPI (Tissue Factor Pathway Inhibitor) Is Present in Breast Cancer Tumor Cells.

Abnormalities in hemostatic system are common in breast cancer patients. The principal initiator of blood coagulation activation in cancer patients is tissue factor (TF). Tissue factor plays an important role in the processes of angiogenesis, invasion and metastatic dissemination. Tissue factor pathway inhibitor (TFPI) is the main inhibitor of TF-dependent pathway of blood coagulation. The catalytic activity of the TF/VIIa complex triggers activation of factor X, consequently thrombin generation and finally fibrin formation. Thrombin and fibrin facilitate cancer progression. Previous immunohistochemical studies revealed no expression of TFPI in cancer cells of different malignant tumor types (Werling et al, Thromb Haemost 1993; 69: 366–369) The aim of the study was to evaluate the expression of TFPI protein and mRNA in tumor cells of breast cancer.Breast cancer tissues were obtained during surgical treatment of 35 women. The patients undergoing surgery had not received any previous anticancer therapy. Tumor fragments were preserved in 4% paraformaldehyde/phosphate buffered saline and than embedded in paraffin. Immunohistochemical studies (avidin-biotin complex-ABC - technique) were performed using polyclonal antibodies against TFPI. In situ hybridization method employed single-stranded DNA oligonucleotide (probe sequence: 5′Biotin-CATCGCCCAGTGCAGCCTCCGTCAG-Biotin3′) directed against locus NM 006287 of TFPI mRNA.Strong expression of TFPI protein was observed in cancer cell bodies in examined specimens. Weak expression of the above protein was observed in tumor infiltrating macrophages. Normal cells revealed much weaker staining for TFPI than cancer cells. Similarly, mRNA for TFPI was observed in cancer cells of all breast cancer tissues.The above data indicate that TFPI is present in neoplastic cells of breast cancer and may play a role in neutralizing TF procoagulant and signaling functions.