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Rikki A Cannioto, Lara E Sucheston-Campbell, Shalaka Hampras, Ellen L Goode, Keith Knutson, Roberta Ness, Francesmary Modugno, Paul K Wallace, J Brian Szender, Paul Mayor, Chi-Chen Hong, Janine M Joseph, Grace Friel, Warren Davis, Mary Nesline, Kevin H Eng, Robert P Edwards, Bridget Kruszka, Kristina Schmitt, Kunle Odunsi, Kirsten B Moysich
OBJECTIVE: There is a mounting body of evidence demonstrating higher percentages of regulatory T (Treg) cells in the peripheral blood of patients with cancer in comparison to healthy controls, but there is a paucity of epidemiological literature characterizing circulating Treg cells among patients with epithelial ovarian cancer (EOC). To investigate the role of peripheral Treg cells in ovarian neoplasms, we conducted a case-control study to characterize circulating concentrations of Treg cells among patients with EOC, women with benign ovarian conditions, and healthy controls without a history of cancer...
October 18, 2016: International Journal of Gynecological Cancer
Afshin Shirkani, Mohammad Shahrooei, Gholamreza Azizi, Hassan Rokni-Zadeh, Hassan Abolhassani, Shokrollah Farrokhi, Glynis Frans, Xavier Bossuyt, Asghar Aghamohammadi
ZAP-70 deficiency is a rare autosomal recessive form of combined immunodeficiency (CID) characterized by selective absence of circulating CD8 T cells with low, normal, or increased CD4 T cells in peripheral blood. Up to now, 14 unique mutations in the ZAP70 gene have been identified in patients with ZAP-70-related CID. We present a 3-year-old boy with a history of recurrent bacterial infections and autoimmunity. Initial laboratory findings showed a normal total lymphocyte count, but low levels of CD8 and CD4 T cells and an abnormal lymphocyte proliferation response...
October 19, 2016: Immunological Investigations
Katarzyna Sznurkowska, Anton Żawrocki, Jacek Sznurkowski, Maciej Zieliński, Piotr Landowski, Katarzyna Plata-Nazar, Ewa Iżycka-Świeszewska, Piotr Trzonkowski, Agnieszka Szlagatys-Sidorkiewicz, Barbara Kamińska
BACKGROUND/AIMS: To determine the proportion of T-regulatory cells (CD4(+)CD25(high)FOXP3(+) cells) in peripheral blood and the number of FOXP3(+) cells in intestinal mucosa of children with inflammatory bowel disease (IBD), and to verify whether these parameters correlate with the activity of the disease. MATERIAL AND METHODS: 24 patients newly diagnosed for IBD were included in the study: ulcerative colitis (UC; n = 13) and Crohn's disease (CD; n = 11). Seventeen healthy controls (HC) and 16 patients with irritable bowel syndrome (IBS) served as a control group for peripheral and intestinal Tregs assessment, respectively...
October 19, 2016: Immunological Investigations
Alexander Wolf, Katharina Beller, Sebastian Groemminger, Wera Hofmann, Matthias Sachse, Jana Fassunke
Increasing sample numbers for screening and diagnostics using circulating cell-free DNA (ccfDNA) as analyte demands an automated solution for ccfDNA extraction. The efficiency of a new, automated, large volume ccfDNA extraction method was evaluated against a manual reference method. The new kit for automated ccfDNA extraction on the QIAsymphony showed a comparable yield of total ccfDNA from healthy donors as well as a comparable recovery of circulating cancer and fetal DNA. In conclusion, a new kit for automated ccfDNA extraction was established successfully...
2016: Advances in Experimental Medicine and Biology
Jurgen Distler, Reimo Tetzner, Gunter Weiss, Thomas König, Anne Schlegel, Michal Bagrowski
For the subsequent analysis of the methylated mSEPT9 colorectal cancer screening marker in plasma, different blood collection tubes and blood storage conditions were investigated. The study demonstrated that methylated Septin 9 ((m)SEPT9) can be consistently detected in plasma samples derived from whole blood samples collected with S-Monovette® K3E and BD Vacutainer ® K2EDTA tubes stored at 2-8 °C for a maximum of 24 h and for samples collected in S-Monovette CPDA tubes stored at 18-25 °C for up to 48 h...
2016: Advances in Experimental Medicine and Biology
Vipulkumar Patel, Peter Celec, Magdalena Grunt, Heidi Schwarzenbach, Ingo Jenneckens, Timo Hillebrand
Circulating cell-free DNA (ccfDNA) is a promising diagnostic tool and its size fractionation is of interest. However, kits for isolation of ccfDNA available on the market are designed for small volumes hence processing large sample volumes is laborious. We have tested a new method that enables enrichment of ccfDNA from large volumes of plasma and subsequently allows size-fractionation of isolated ccfDNA into two fractions with individually established cut-off levels of ccfDNA length. This method allows isolation of low-abundant DNA as well as separation of long and short DNA molecules...
2016: Advances in Experimental Medicine and Biology
Jelena Belic, Marina Koch, Peter Ulz, Martina Auer, Teresa Gerhalter, Sumitra Mohan, Katja Fischereder, Edgar Petru, Thomas Bauernhofer, Jochen B Geigl, Michael R Speicher, Ellen Heitzer
Recent progress in the analysis of cell-free DNA fragments (cell-free circulating tumor DNA, ctDNA) now allows monitoring of tumor genomes by non-invasive means. However, previous studies with plasma DNA from patients with cancer demonstrated highly variable allele frequencies of ctDNA. Comprehensive genome-wide analysis of tumor genomes is greatly facilitated when plasma DNA has increased amounts of ctDNA. In order to develop a fast and cost-effective pre-screening method for the identification of plasma samples suitable for further extensive qualitative analysis, we adapted the recently described FAST-SeqS method...
2016: Advances in Experimental Medicine and Biology
Hongdo Do, Daniel Cameron, Ramyar Molania, Bibhusal Thapa, Gareth Rivalland, Paul L Mitchell, Carmel Murone, Thomas John, Anthony Papenfuss, Alexander Dobrovic
Identifying circulating tumour DNA (ctDNA) for monitoring of cancer therapy is dependent on the development of readily designed, sensitive cancer-specific DNA markers. Genomic rearrangements that are present in the vast majority of cancers provide such markers.Tumour DNA isolated from two fresh-frozen lung tumours underwent whole genome sequencing. Genomic rearrangements were detected using a new computational algorithm, GRIDSS. Four genomic rearrangements from each tumour were chosen for further study using rearrangement-specific primers...
2016: Advances in Experimental Medicine and Biology
Svetlana N Tamkovich, Oleg S Tutanov, Danil S Serdukov, Maxim S Belenikin, Anatoliy G Shlikht, Natalia A Kirushina, Vladimir E Voytsitskiy, Yuri P Tsentalovich, Vsevolod A Tkachuk, Pavel P Laktionov
In the current study we have investigated the protein content of blood plasma deoxyribonucleoprotein complexes. The complexes were isolated using affinity chromatography with immobilized polyclonal anti-histone antibodies. Proteins were separated by SDS PAAGE and identified by MALDI-TOF mass-spectrometry. 111 and 56 proteins (excluding histones), respectively, were identified with a good score in deoxyribonucleoprotein complexes of healthy females and breast cancer patients. However, only four of these proteins were found in 30 % of all samples...
2016: Advances in Experimental Medicine and Biology
Anna Yu Alekseeva, Larisa V Kameneva, Svetlana V Kostyuk, Natalia N Veiko
Oxidized cell-free DNA acts as a stress signal molecule and triggers an adaptive response in human cells. Various membrane DNA recognizing receptors are known as potential sensors for such DNA fragments. In order to clarify which of these sensors are able to interact with cfDNA fragments, circulating in human blood flow in heath and disease, we studied the influence of various cfDNA types on endothelial cells. We incubated these fragments at a physiologically optimal concentration with HUVEC cells for 3-24 h and detected the expression of either TLR9 or AIM2, RIG1 and STING receptors at mRNA and protein levels...
2016: Advances in Experimental Medicine and Biology
Grigory A Stepanov, Julia A Filippova, Anna A Nushtaeva, Elena V Kuligina, Olga A Koval, Vladimir A Richter, Dmitriy V Semenov
Fragments of small nucleolar RNAs (snoRNAs) were found among various non-coding RNAs (ncRNAs) circulating in human blood. Currently, the function of such cell-free sno-derived-RNAs is not clearly defined. This work is aimed at identifying regulatory pathways controlled by extracellular snoRNAs. In order to determine the molecular targets and pathways affected by artificial snoRNAs, we performed Illumina array analysis of MCF-7 human adenocarcinoma cells transfected with box C/D RNAs. The genes related to the innate immune response and apoptotic cascades were found to be activated in transfected cells compared with control cells...
2016: Advances in Experimental Medicine and Biology
Anna V Savelyeva, Dmitriy N Baryakin, Elena D Chikova, Elena V Kuligina, Vladimir A Richter, Dmitriy V Semenov
Human blood contains a great variety of membrane-covered RNA carrying vesicles which are spherical or tubular particles enclosed by a phospholipid bilayer. Circulating vesicles are thought to mediate cell-to-cell communication and their RNA cargo can act as regulatory molecules. In this work, we separated blood plasma of healthy donors by centrifugation and determined that vesicles precipitated at 16,000 g were enriched with CD41a, marker of platelets. At 160,000 g, the pellets were enriched with CD3 marker of T cells...
2016: Advances in Experimental Medicine and Biology
Hada C Macher, G Suárez-Artacho, Pilar Jiménez-Arriscado, S Álvarez-Gómez, N García-Fernández, Juan M Guerrero, Patrocini Molinero, Elena Trujillo-Arribas, M A Gómez-Bravo, Amalia Rubio
The evaluation of the transplanted liver health by non-invasive approaches may offer an improvement in early clinical intervention. As transplanted organs have genomes that are distinct from the host's genome, the quantification of the specific DNA of the donated liver in the patient serum will allow us to obtain information about its damage. We evaluated the state of transplanted liver health by monitoring the RH gene in serum circulating DNA (cirDNA) from 17 recipient and donor mismatched for this gene. cirDNA RH gene was quantified by RT- PCR before, at the moment of transplantation (day 0) and during the stay at the intensive care unit...
2016: Advances in Experimental Medicine and Biology
Vasilina A Sergeeva, Svetlana V Kostyuk, Elizaveta S Ershova, Elena M Malinovskaya, Tatiana D Smirnova, Larisa V Kameneva, Natalia N Veiko
It has been established that cell-free DNA circulating in the bloodstream affects cells. The characteristics of cfDNA depend on the physiological state of the organism. As we showed previously, diseases can cause either GC-enrichment of the cell-free DNA pool or its oxidation. Thus, in cases of cerebral atherosclerosis, heart attack and rheumatic arthritis the cell-free DNA pool is GC-enriched and, in the case of cancer, both GC-enriched and oxidized. Herein we investigated the time-dependent effect of oxidized and GC-rich cell-free DNA on NF-kB and NRF2 signaling pathways in human mesenchymal stem cells and showed that they affect cells in different ways...
2016: Advances in Experimental Medicine and Biology
Anna V Cherepanova, Zhanna K Nazarkina, Pavel P Laktionov
Oligodeoxynucleotide (ODN) analogues of cell-surface-bound circulating DNA inhibit the dsRNA-induced production of pro-inflammatory interleukin 6, interferon beta and antibacterial peptide beta-defensin 2 not only in human gingival fibroblasts, but also in human primary endothelial and transformed cells (Hela and A431). ODN analogues do not effect dendritic cells activation by poly(I:C). The data obtained indicate that the early stages of the signal transduction cascade are violated by ODN analogues and the effects depend on the cell type...
2016: Advances in Experimental Medicine and Biology
Eva Pazourkova, Sarka Pospisilova, Iveta Svobodova, Ales Horinek, Antonin Brisuda, Viktor Soukup, Jan Hrbacek, Otakar Capoun, Jaroslav Mares, Tomas Hanus, Marek Babjuk, Marie Korabecna
MicroRNAs (miRNAs) in urine are examined as potential biomarkers. We examined the urine samples from 70 individuals (45 males, 25 females, mean age 65 years, range 20-84 years). Of the urine donors, 15 were healthy volunteers, 5 were patients with non-cancer diseases, 50 were patients with different stages of bladder cancer. To examine the spectrum of miRNAs in the cell-free fraction of urine, TaqMan Human miRNA Array Card A v.2.1 was used. A set of 30 miRNAs were found that are constantly present in urine supernatants independently of sex, age and health status of the subjects...
2016: Advances in Experimental Medicine and Biology
Janine Aucamp, Abel Jacobus Bronkhorst, Piet J Pretorius
The quantitative and qualitative differences of circulating nucleic acids (cirNAs) between healthy and diseased individuals have motivated researchers to utilize these differences in the diagnosis and prognosis of various pathologies. The position maintained here is that reviewing the rather neglected early work associated with cirNAs and extracellular vesicles (EVs) is required to fully describe the nature of cirNAs. This review consists of an empirically up-to-date schematic summary of the major events that developed and integrated the concepts of heredity, genetic information and cirNAs...
2016: Advances in Experimental Medicine and Biology
Stefan Holdenrieder
Liquid profiling is a traditional concept in laboratory diagnostics using patterns of blood-derived biochemical molecules for disease detection and characterization. Beyond protein and cellular parameters, molecular biomarkers at the DNA, RNA and miRNA level have been developed as promising diagnostic tools in metabolic and malignant diseases as new technologies for ultrasensitive profiling of nucleic acids in blood and body fluids became available. In cancer disease, they are successfully applied for the stratification of patients for individually tailored therapies, treatment monitoring and the sensitive detection of minimal residual disease...
2016: Advances in Experimental Medicine and Biology
Svetlana N Tamkovich, Natalia A Kirushina, Vladimir E Voytsitskiy, Vsevolod A Tkachuk, Pavel P Laktionov
Size and termini of cell-free DNA molecules circulating in blood plasma and being bound with blood cell surface of healthy females and untreated breast cancer patients were investigated. The size and concentration of circulating blood DNA were analyzed by Agilent 2100 Bioanalyser (TM) and TaqMan PCR. The termini of circulating DNA were examined by ligation using biotinylated double-stranded oligonucleotide adapters with random 1-3 b overhangs of both chains and subsequent quantification by PCR. Short (180 bp) and longer (>8 kbp) DNA fragments were found in cell free DNA from both groups, but short were less represented in primary breast cancer patient plasma...
2016: Advances in Experimental Medicine and Biology
Elena Trujillo-Arribas, Hada C Macher, Pilar Jiménez-Arriscado, Fernando de la Portilla, Patrocinio Molinero, Juan M Guerrero, Amalia Rubio
Genomic characterization of cell-free circulating tumour DNA (ctDNA) may offer an opportunity to assess clonal dynamics throughout the course of a patient's illness. The existence of KRAS driver mutations in colon cancer patients is determinant to decide their treatment and to predict their outcome. DNA is extracted automatically from 400 μL of serum using the MagNa Pure Compact with the Nucleic Acid Isolation Kit I. DNA amplification, COLD-PCR and HRM were performed in the same run in the Light Cycler 480...
2016: Advances in Experimental Medicine and Biology
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