Plasma-thrombin cell blocks: Potential source of DNA contamination.

BACKGROUND: Cell blocks are being used more frequently in cytology for ancillary testing, including molecular diagnostics. There are several different methods of processing cell blocks, with plasma-thrombin being one of the most common. Plasma is a blood-derived product and may be a source of DNA. The aim of this study was to determine whether the plasma used for the plasma-thrombin cell block method has amplifiable DNA that may potentially interfere with molecular testing results. METHODS: Expired bags of fresh frozen plasma were collected from a blood bank. From each sample, DNA was extracted from a 1-mL aliquot with the QIAsymphony MIDI kit (Qiagen). The concentration of DNA was measured on a NanoDrop instrument. The amplifiable DNA quality was assessed by polymerase chain reaction (PCR) with primers to generate amplicons of various sizes. Characterization was performed with the AmpFLSTR Identifiler Plus PCR kit with capillary electrophoresis. RESULTS: Twenty samples from 20 bags were collected. All samples showed amplifiable DNA despite low DNA concentrations in a few cases. PCR amplification revealed the presence of high-quality amplifiable DNA (up to 600 base pairs). DNA was amplified at the 16 loci interrogated in all samples tested with the AmpFLSTR Identifiler Plus PCR kit. CONCLUSIONS: The presence of genomic DNA in plasma may theoretically interfere with results of molecular testing. Particularly in clinical samples with low cellularity, the DNA in plasma may potentially either mask the presence of minute amounts of tumor-derived DNA or lead to a false-positive result.

Combination of antibody targeting and PTD-mediated intracellular toxin delivery for colorectal cancer therapy.

The bottlenecks of current chemotherapy in the treatment of colorectal cancer lie in the ineffectiveness of the existing anti-cancer small molecule drugs as well as the dose-limiting toxicity caused by the nonselective action on normal tissues by such drugs. To address these problems, we introduce a novel therapeutic strategy based on tumor targeting using a non-internalizing anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb) and intracellular delivery of the extremely potent yet cell-impermeable protein toxin gelonin via the aid of a cell-penetrating peptide (also termed as protein transduction domain; PTD). A chimeric TAT-gelonin fusion protein was genetically engineered, and it displayed remarkably enhanced anti-cancer activity against human colorectal cancer cells, with IC50 values being several orders of magnitude lower than the unmodified gelonin. On the other hand, a chemically synthesized conjugate of heparin and a murine anti-CEA mAb, T84.66 (termed T84.66-Hep) was found able to bind highly specifically to CEA over-expressing LS174T colorectal cancer cells. When mixing together, TAT-gelonin and T84.66-Hep could associate tightly and automatically through an electrostatic interaction between the cationic TAT and anionic heparin. In preliminary in vivo studies using LS174T s.c. xenograft tumor bearing mouse, selective and significantly augmented (58-fold) delivery of TAT-gelonin to the tumor target was observed, when compared with administration of TAT-gelonin alone. More importantly, efficacy studies also revealed that only the TAT-gelonin/T84.66-Hep complex yielded a significant inhibition of tumor growth (46%) without causing gelonin-induced systemic toxicity. Overall, this study suggested a generic strategy to effectively yet safely deliver potent PTD-modified protein toxins to the tumor.

[Construction for detecting of human papillomaviruses types by using a multiplexed Luminex assay].

AIM: To develop multiplex human papillomavirus genotyping, a quick and sensitive high-throughput procedure for the identification of multiple HPV genotypes by using a multiplexed Luminex array. METHODS: Standard plasmids containing HPV genomic DNA were developed a standard multiplexed suspension array for detection 13 HPV types on Luminex analysis platform. the primer and probes were selected, HPV type-specific oligonucleotide probes were coupled to fluorescence-labeled polystyrene beads. RESULTS: The one or two of mixed HPV standard plasmids were identified HPV type by using 13 HPV genetypes multiplexed suspension array, different HPV types can be correctly detected simultaneously. The positives of HPV clinical samples were detected by using this method, and the genetypes of samples by detecting were the same results with using BLAST in NCBI. CONCLUSION: The proposed method allowed a high through-out, special, simple, rapid and economical identification of HPV DNA genotypes, It is expected to be an extremely useful tool in the identification of HPV genetypes for a lot of clinical samples.

DNA prime protein boost strategies protect cattle from bovine viral diarrhea virus type 2 challenge.

At present, infections with bovine viral diarrhea virus (BVDV) type 2 occur nearly as frequently as those with BVDV type 1, so development of vaccines that protect cattle from both type 1 and type 2 BVDV has become critical. In this study, we compared various DNA prime-protein boost vaccination strategies to protect cattle from challenge with BVDV-2 using the major protective antigen of BVDV, glycoprotein E2. Calves were immunized with a plasmid encoding either type 1 E2 (E2.1) or type 2 E2 (E2.2) or with both plasmids (E2.1+E2.2). This was followed by a heterologous boost with E2.1, E2.2 or E2.1 and E2.2 protein formulated with Emulsigen and a CpG oligodeoxynucleotide. Subsequently, the calves were challenged with BVDV-2 strain 1373. All vaccinated calves developed both humoral and cell-mediated immune responses, including virus-neutralizing antibodies and IFN-gamma-secreting cells in the peripheral blood. Depletion studies showed that CD4+ T cells were responsible for IFN-gamma production. Furthermore, the calves vaccinated with either the E2.2 or the E2.1+E2.2 vaccines were very well protected from challenge with BVDV-2, having little leukopenia and showing no weight loss or temperature response. In addition, the animals vaccinated with the E2.1 vaccine were partially protected, so there was a certain level of cross-protection. These data demonstrate that a vaccination strategy consisting of priming with E2.2 or E2.1+E2.2 DNA and boosting with E2.2 or E2.1+E2.2 protein fully protects cattle from BVDV-2 challenge.

Random amplified polymorphic DNA typing and phylogeny of Pythium insidiosum clinical isolates in Thailand.

Forty-three Pythium insidiosum clinical isolates recovered from human pythiosis cases in Thailand were characterized by random amplified polymorphic DNA (RAPD) analysis. Three random oligonucleotide primers, OPW11, OPW12 and OPX13 generated 39, 34 and 35 DNA patterns with high value of typeability (100%), reproducibility (98.5, 88.8 and 93.3%) and discriminatory power (0.83, 0.82 and 0.77), respectively. Using GelCompar software based on band similarity, the 43 clinical isolates of P. insidiosum could be arranged into 9, 13 and 11 clades using OPW11, OPW12 and OPX13, respectively and the combination of all three primers revealed 36 RAPD patterns. Members in each RAPD pattern varied in both clinical forms and/or geographical locations. RAPD pattern 15 was found in 6 isolates, half of which were found in central region of Thailand. Isolates MCC15 and MCC16 isolated from different patients exhibited identical pattern with all three primers. Our results revealed high genetic heterogeneity among Pythium insidiosum isolates in Thailand. RAPD method should be appropriate for future epidemiological studies of P. insidiosum strains from patients and from natural habitats.